text:
The role of the Criminal Justice System in Combating Organised Crime in South Africa.
"No man is above the law and no man is below it; nor do we ask any man's permission when we require him to obey it. Obedience to the law is demanded as a right; not asked as a favour" (1)
Until fairly recently organised crime was a rather foreign phenomenon in the South African legal system. Although gangs have been known to be involved in criminal activities, these activities were usually limited to crimes which the criminal justice system was well capable of dealing with. However, since South Africa's re-integration into the international (2) not only have the country's borders been opened for foreign investment, tourism, etc, but also for international criminals. Crimes such as transnational drug trafficking, smuggling in stolen and robbed motor vehicles, arms smuggling, money laundering and corruption became the order of the day. Lawlessness increased and violent crimes against all people became so rife that more people than ever before either emigrated or temporarily left the country. Factors such as a shortage of human and other resources among the police and prosecutors also play no small role in promoting criminal conduct.
Criminal liability is based on the principle of nullum crimen nulla poena nisi mens sit rea. (3) To be held criminally liable the crime must firstly be clearly defined. (4) Criminal liability is also very personal it depends on the perpetrator's unlawful and blameworthy conduct. Because of this rather restricted approach many criminals escaped the net of justice. To counter this, concepts such as attempt, conspiracy, incitement, common purpose, vicarious liability and others were incorporated in the law. (5) Despite the incorporation of these concepts into the legal system, it remains difficult to beyond a reasonable doubt prove the guilt of a participant in certain criminal activities. Based on the English rule of common purpose, a court may infer criminal liability on the part of a person despite that person not having physically committed the crime. An accused person may, for instance, be convicted of murder despite his personally not having caused the death of the deceased. It is, however, necessary for t he state to prove that the accused was present where the violence was being committed; was aware of the assault on the victim; intended making a common cause with those perpetrating the assault; performed some act of association with the conduct of the other perpetrators thereby manifesting his/her sharing of a common purpose; and had the necessary mens rea. (6)
A conspiracy is often even more difficult to prove. Usually the only witness the state has is a co-conspirator or a police agent who has successfully infiltrated the criminal syndicate or gang. If it is the former, the co-conspirator's evidence must be evaluated with caution as he/she has a particular interest and it could easily be said that he/she is biased. (7) Corroboration for the co-conspirator's evidence or that of the police agent is, fortunately, often found in audio and video recordings of the conversations and actions of the members of the criminal syndicate.
South African law also provides that a corporation, directors and servants thereof and members of certain associations may under certain circumstances be held criminally liable. (8) Before the intervention by the Constitutional Court, (9) the state's case was strengthened by the operation of a presumption in terms of which the accused (a director or servant of the corporation) was expected to prove that he did not take part in the commission of the offence and that he could not have prevented it. (10) Although the impression is prima facie created that the association referred to in section 332(7) of the Criminal Procedure Act, 1977 (Act 51 of 1977), must be a legitimate body, the reference to the provisions of subsections (8) and (9) (11) in the decision of S v Twala (12) means that it may be applicable to a criminal association as well.
In the South African law a conspirator, accomplice, inciter or person convicted of an attempt to commit a crime may receive the same sentence as the principal offender, although such persons are usually dealt with somewhat more leniently.
There is an international concern about organised crime and criminal organisations. At the 51st session of the General Assembly of the United Nations (UN) the Chairman submitted a draft resolution urging "Member States (to) seek to protect the security and well-being of their citizens and all persons within their jurisdictions by taking effective national measures to combat serious transnational crime, including organised crime, ... and shall pledge their mutual cooperation in those efforts". (13)
That "organised crime" is difficult to define is evident from the fact that the proposed Prevention of Organised Crime Bill does not contain a definition as such. (*) It seems that the Council of Europe also have difficulty with the same term and for that reason they have resorted to defining a "criminal organisation" as "a lasting, structured association of more that two persons, acting in concert with a view to committing crimes or other offences which are punishable by deprivation of liberty or a detention order ..., whether such crimes or offences are an end in themselves or a means of obtaining material benefits and, if necessary, of improperly influencing the operation of public authorities". (14) This elimination method is apparently also to be used in the proposed South African legislation. (15) The Polish government has also made a specific recommendation regarding organised crime, namely that "... 'organised crime' means group activities of three or more persons, with hierarchical links or personal relationships, which permit their leaders to earn profits or control territories or markets, internal or foreign, by means of violence, intimidation or corruption, both in furtherance of criminal activity and to infiltrate the legitimate economy", in particular to commit the following offences: illicit trafficking in drugs; trafficking in persons; counterfeiting currency; illicit stealing of or dealing in cultural objects; stealing of nuclear material; terrorism; arms smuggling; motor vehicle smuggling; and corruption. (16)
In the absence of a definition for either 'organised crime' or 'criminal activities', prosecuting authorities are presently being led by the facts of every criminal investigation. Should the facts indicate that a number of persons are being suspected of involvement in committing serious crimes in an organised manner, it is dealt with as such. The practical effect is that the Attorney-General gets involved in the investigation phase and special investigation techniques are used.
The traditional approach that it is the task of the police service to ensure law and order, was reiterated by the Constitution where it states that: "The objects of the police service are to prevent, combat and investigate crime, to maintain public order, to protect and secure the inhabitants of the Republic and their property, and to uphold and enforce the law". (17)
Although the purpose of the prosecuting authority is to institute and conduct criminal prosecutions, (18) provision is also made for Investigating Directorates in the office of the National Director of Public Prosecutions. (19) These Investigating Directorates may be established in respect of specific offences or specified categories of offences.
Despite a lack of authority in any law, experience has taught that one way of effectively addressing organised crime is by way of a joint task team consisting of members of the police service, a prosecutor and, where necessary, an accountant or other forensic experts. The benefit of using a task team is that the prosecutor can give direction to the investigation and the investigating officers are immediately in a position to execute his requests. Once the whole task team is satisfied that sufficient evidence exists to arrest all the role-players, the operation can be terminated and the matter may be set down for trial with the minimum delay.
Since the investigation of organised criminal activities is very dependent on the infiltration of syndicates by police agents, information supplied by informants, observation of suspects by the police and interception of telephone and mail communications, (20) the Legislature recognised the importance of the involvement of the Attorney-General. The situation supra in fact received statutory sanctioning when section 252A was inserted in the Criminal Procedure Act. (21)
Section 252A of the Criminal Procedure Act now provides for specific guidelines regarding undercover operations and traps. Briefly it provides for the following:
(a) Law enforcement agencies may utilise the services of traps or do undercover investigations provided the conduct of the trap does not go beyond providing an opportunity to commit an offence.
(b) Various factors must be considered when the court rules on whether or not the conduct of the trap, the police agent or the police investigator has gone beyond providing an opportunity to commit an offence. These are: (22)
-- whether prior approval was obtained from the relevant Attorney-General;
-- the nature of the offence(s) under investigation, having regard to the security of the state, the safety of the public, the maintenance of public order, the national economy, the prevalence of the offence in the particular area; and the seriousness of the offence;
-- the availability of other investigating techniques;
-- the degree of persistence and the number of attempts made before the accused succumbed and committed the offence;
-- the type of inducement, including the degree of deceit, trickery, misrepresentation or reward;
-- the timing of the conduct, in particular whether the police instigated the offence or became involved in illegal activities;
-- the exploitation of human characteristics;
-- whether any threats were used against the accused;
-- whether, prior to the setting of the trap, there existed suspicion based on reasonable grounds that the accused was involved in criminal activities;
-- whether the agent or other official acted in good or bad faith;
-- the proportionality between the crimes allegedly committed by the accused and the conduct of the agent; and
-- any other factor which in the court's opinion is relevant to the issue.
(c) If the court finds that the conduct of the agent or trap has gone beyond providing an opportunity to commit an offence, such evidence may be refused if it was obtained in an improper or unfair manner and if the evidence so obtained would render the trial unfair.
(d) An Attorney-General may issue general or specific guidelines regarding supervision and control of traps and undercover operations.
(e) Criminal liability for the agent will not arise for any act constituting a crime if the agent acted in good faith. No prosecution of an agent may be instituted without the written authority of the Attorney-General.
(f) The onus of proving on a balance of probabilities that evidence under subsection (3) should be allowed rests on the state. The accused is, however, obliged to furnish the grounds on which the evidence is challenged.
(g) The question of whether evidence under subsection (3) should be allowed or not may be adjudicated as a separate issue.
The objective of the Legislature was to statutorily provide for entrapment as a defence and to prescribe guidelines for the use of undercover operations and traps. In this regard the crux of the legislation is found in the following:
-- Subsection (1) "that the conduct does not go beyond providing an opportunity to commit an offence";
-- Subsection (3) "If a court ... finds that ... the conduct goes beyond providing an opportunity ..., the court may refuse to allow such evidence ..."; and
-- Subsection (6) "the burden of proof ... that the evidence is admissible, shall rest on the prosecution ...".
The Constitution of the Republic of South Africa, 1996, (23) being the supreme law of the land, (24) is the law against which all other laws must be compared. In this regard section 35(5) provides as follows: "Evidence obtained in a manner that violates any right in the Bill of Rights must be excluded if the admission of that evidence would render the trial unfair or otherwise detrimental to the administration of justice".
It is evident from the provisions of section 252A that the emphasis is on fairness of the trial, (25) thus in conformity with the Constitution. This is contrary to the some of the court-rulings in the United States (US) in terms of which the evidence must be excluded as inadmissible and that the trial would therefore probably be unfair. Regarding fairness, the Constitutional Court ruled as follows: "What the Constitution demands is that the accused be given a fair trial. (F)airness is an issue which has to be decided upon the facts of each case, and the trial judge is the person best placed to take that decision. At times fairness might require that evidence unconstitutionally obtained be excluded. But there will also be times when fairness will require that evidence, albeit obtained unconstitutionally, nevertheless be admitted" (26)
The situation regarding entrapment in a number of countries, including South Africa, is discussed below.
7.1 United States of America
Legislation of the State of Arkansas pertaining to entrapment reads as follows:
"(a) It is an affirmative defence that the defendant was entrapped into committing an offence. (b) Entrapment occurs when a law enforcement officer or any person acting in cooperation with him induces the commission of an offence by using persuasion or other means likely to cause normally law-abiding persons to commit the offence. Conduct merely affording a person an opportunity to commit an offence does not constitute entrapment"(own emphasis).
In Sherman v US (27) the court ruled as follows: "The function of law enforcement is the prevention of crime and the apprehension of criminals. Manifestly, that function does not include the manufacture of crime. Criminal activity is such that stealth and strategy are necessary weapons in the arsenal of the police officer. However, a different question is presented when criminal design originates with the officials of the Government, and they implant in the mind of an innocent person the disposition to commit the alleged offence and induce its commission in order that they may prosecute". (28) According to this dictum: "Entrapment occurs only when the criminal conduct was the product of creative activity of law enforcement officials".
7.2 Canada
In Canada entrapment rests on the basis that such conduct brings the justice system into disrepute. (29) The position in Canada is emphasised in the very important decision of R v Mack (30) where the court ruled as follows: "It is the belief that the administration of justice must be kept free from disrepute that compels recognition of the doctrine of entrapment. In the context of the Charter, the court has stated that disrepute may arise from judicial condonation of unacceptable conduct by the investigatory and prosecutional agencies", and "the basis on which entrapment is recognised lies in the need to preserve the purity of the administration of justice".
Entrapment is present when: "(a) The authorities provide a person with an opportunity to commit an offence without acting on reasonable suspicion that this person is already engaged in criminal activity or pursuant to a bona fide inquiry; (b) although having a reasonable suspicion or acting in the course of a bona fide inquiry, they go beyond providing an opportunity and induce the commission of an offence". (31) According to the judgement, to be acting bona fide and having reasonable suspicion, there must be sufficient connection between the previous conduct of the accused and the provision of an opportunity to the commission of an offence.
7.3 Europe
In Ludi v Switzerland (32) the European Court of Human Rights was requested to consider the constitutionality of the use of undercover agents and traps and the interception of telephone conversations. Regarding the latter the Court ruled that: "The telephone surveillance ordered in this case on a basis of serious suspicion of crime was certainly not an abuse of the law".
Regarding the use of undercover agents the Court remarked as follows: (33) "(I) would not be permissible if the undercover agent were to take the initiative, as it were, and provoke the criminal activity which would otherwise not have come about at all; for the prosecuting authorities must not provoke criminality ... Covert investigation does not encroach on a basic right protected by the Federal Constitution. The person concerned id free as regards his decisions and his behaviour towards the undercover agent; he is, however, deceived as to the identity of his negotiating partner and the latter's connection with the police. A criminal is not protected by constitutional law against being observed in the course of his illegal conduct by a police officer who is not recognisable to him as such. Nor can any protection of a criminal against covert investigation be derived from the European Convention".
The Court also ruled that the use of undercover operations is allowed despite no specific provision being made in statutory law. It was, however, emphasised that the trap, acting as a buyer, should not instigate the crime. The Court also expressed an opinion on the protection of investigative methods and ruled that the protection of the identity of the agent does not infringe the accused's constitutional rights. (34)
7.4 United Kingdom
The United Kingdom (UK) does not have a written constitution. The British depend on convention, statute and court judgements. Their criminal justice system is in part regulated by the Police and Criminal Evidence Act (35) which also provides for the use of traps but which does not create the defence of entrapment. A court may exclude such evidence if found to be unfair or not to be in the interest of justice. (36)
7.5 South Africa
A trap has been defined as "a person who, with a view of securing the conviction of another, proposes certain criminal conduct to him, and himself ostensibly takes part therein. In other words he creates the occasion for someone else to commit the offense". (37)
Traps have, even prior to the introduction of section 252A, been allowed in the South African system, albeit as "a necessary evil". (38) Because traps (and though not explicitly said so, also undercover agents) are seen as accomplices, conspirators and instigators, (39) their evidence must be examined with caution and courts would usually want some reliable corroboration for their evidence. Although traps have been exonerated on the basis of absence of wrongfulness or mens rea, one could infer from the judge's remarks in S v Ohlenschlager (40) that he does not agree with this method of crime investigation. (41) In S v Hassen (42) the court held as follows: "The courts are best placed to judge which type of investigation techniques may bring the administration of justice into disrepute ... and ... (a) The police must have independent and reliable information that the person to be trapped was an existing offender. The reason for this is that the state should not artificially create crime ... (b) verbal persuasi on should not be used ... (c) If methods of enticement are used they should simulate the ordinary situation as it normally exists in the community ... (e) The police must verify the information received from the informers ... (f) The person sent to negotiate the trap must be of good character, preferably a policeman ... (g) ... where the trap is executed, does not lend itself to the planting of evidence, etc". (43) Regarding the defence of entrapment the judge said: "To start with, there is no basis in the Constitution for such a defence. Trapping in general is in any event not so abhorrent as to justify a substantive defence of entrapment ...". (44)
Regarding the interception and monitoring of telephone conversations the following was inter elia said in Protea Technology v Weiner: (45) "Uncovering the truth and exposing the ungodly are not thereby relegated to unimportance ... Privacy is not an absolute right under the Constitution".
Traps, undercover operations and the interception and monitoring of telephone conversations and mail are valuable weapons in the armoury of the police and prosecutors. Although these methods are necessary to gather sufficient evidence against criminals involved in organised criminal activities, they must be used with circumspection and restraint while at all times be subservient to the Constitution.
The courts must ensure that accused persons receive fair trials -- this is what the Constitution demands. Courts therefore carefully scrutinise all evidence obtained from traps arid undercover agents against the background of the various rules of evidence and the provisions of section 252A. Therefore, to avoid a finding of unfair treatment of an accused followed by a possible acquittal, police should not do more than to providing an opportunity to commit an offence, subject to a bona fide and reasonable suspicion that the accused was or is involved in criminal activities. Any instigation, incitement or provocation to commit an offence may be regarded as unfair practice.
Keep in mind what Blaise Pascal said: "Justice without strength is helpless, strength without justice is tyrannical. Unable to make what is just strong, we have made what is strong just".
(*.) Since 1 November 1998 the Deputy Director of Public Prosecutions: Transvaal.
(*.) The Prevention of Organized Crime Act, 1998 (Act 121 of 1998) subsequently came into operation on 21 January 1999.
(1.) Theodore Roosevelt.
(2.) Provisionally in 1992 upon the release of Mr. Nelson Mandela and finally on 27 April 1994 when Mr. Mandela was sworn in as President.
(3.) No crime and no punishment unless one is blamed (according to the laws of the land).
(4.) Section 35(3)(I) of the Constitution of the Republic of South Africa, 1996 (Act 108 of 1996) - "Every accused person has a right ... not to be convicted for an act or omission that was not an offence under either national or international law at the time it was committed or omitted;" Also see S v Friedman (1) 1996(1) SACR 181 (W).
(5.) See section 18 of the Riotous Assemblies Act, 1956 (Act 17 of 1956) and Burchell, South African Criminal Law and Procedure, Volume 1 (Third Edition), Chapters 38 to 40.
(6.) See S v Sefatsa 1988 (1) SA 868 (A) and S v Mgedezi l989 (1) SA 687 (A).
(7.) See Hoffmann & Zeffertt, The South African Law of Evidence, Fourth Edition, 575 et seq.
(8.) Section 332 of the Criminal Procedure Act, 1977 (Act 51 of 1977).
(9.) Sv Coetzee 1997 (1) SACR 379 (CC).
(10.) Section 332(5) of Act 51 of 1977.
(11.) Of section 332.
(13.) Article 1 of UN document A/C.3/51/L. 11 dated 4 November 1996.
(14.) Article 1 of Communique 2075, Council, Justice and Home Affairs, 19 March 1998.
(15.) This appears to be the case regarding Act 121 of 1998. See the definitions of "criminal gang"; "pattern of illegal conduct"; and "pattern of criminal gang activity".
(16.) United Nations (General Assembly) document A/C.3/51/7 dated 1 October 1996.
(17.) Section 205(3) of Act 108 of 1996.
(18.) Section 20 of the National Prosecuting Authority Act, 1998 (Act 32 of 1998).
(19.) Section 7 of Act 32 of 1998.
(20.) The Interception and Monitoring Prohibition Act, 1992 (Act 127 of 1992).
(21.) By Act 85 of 1996.
(22.) Section 252A(2).
(23.) Act 108 of 1996.
(24.) See section 2.
(25.) Key v Attorney-General, Cape 1996 (4) SA 187 (CC).
(26.) Key v Attorney-General (supra) at 196A-B.
(28.) At 851. See also Sorrells v US, (1932) 287 US 435.
(29.) Amato v The Queen, (1982) 69 CCC (2d) 31: "the successful application of the doctrine of entrapment results in the staying of prosecution, the court, in the exercise of its inherent powers, withholding its process from the prosecution on the basis that such would bring the administration of justice into disrepute".
(30.) 4 CCC 3d 1989 and confirmed in R v Elzein, 82 CCC (3d) 455 Quebec Court of Appeal.
(31.) Mack (supra) at 559.
(32.) Publications of the European Court of Human Rights, Series A: Judgements and Decisions, Volume 238, judgement of 15 June 1992.
(33.) At page 17.
(34.) "If it is recognised that the use of undercover agents is justified in the public interest in fighting as effectively as possible against drug dealing, it follows that the identity and the investigative methods of such agents are not lightly to be given away in criminal proceedings; for their continued use would thereby effectively be made largely impossible. Preserving the secrecy of undercover agents does not in itself infringe principles of criminal procedure or constitutional rights."
(35.) 1984.
(36.) Sections 76 and 78.
(37.) See S v Malinga 1963 (1) SA 692 (A) and S v Tsochlas 1974 (1) SA 565 (A).
(38.) S v Kramer 1991 (1) SACR 25 (Nm).
(39.) S v Kellner 1963 (2) SA 435 (A); R v Mary Pound (1882) 2 SC 2; S v Ohlenschlager 1992 (1) SACR 695 (T).
(40.) Supra (footnote 36).
(41.) At 705: "If members of the police or their representatives commit offences while they are acting as traps, such conduct remains punishable ... It will only be a defence if his conduct is authorised by a statutory provision or if knowledge of unlawfulness on his part cannot, as a result of the police instructions, be proved."
(43.) At 248.
(44.) At 250 Also see Mendes v Kitchen 1995 (2) SACR 634 (E).
(45.) All SA LR 1997 Case no. 97/06338 (WLD).
Reader Opinion
|Printer friendly Cite/link Email Feedback
|Welch, John I.
|Institute for Strategic Studies
|Article Type:
|Topic Overview
|Geographic Code:
|May 1, 1999
|Previous Article:
|Prevention and Combating of Organised Crime in South Africa.
|Next Article:
|Doctrine development in the South African armed forces up to the 1980s. (Chapter One).


text:
This is an efficient hotel for business people. No one would call it cosy but it does its job well. - Fred
very good facilities, clean and comfortable. Wifi working perfectly, good location, about 10 minutes by bus 11 or 4 to the center of Stavanger. Staffs are friendly. I would stay here again if I have another chance to visit Stavanger - Anonymous
Central position. Cosy and clean. - Anonymous
Free tea, coffee, cereals and juice all day. Very good location just a 7
minute walk from Stavanger centre. Staff was nice and very helpful
Wireless signal very strong inside the room which is often rare Very
good value for money, it was actually cheap for Norwegian standards - Anonymous
The owners were lovely, very friendly, helpful and we felt very
welcomed. A unique hotel developed from a well appointed ex Hydro
electric generating company staff accommodation. Breakfasts were
typical continental but very good. The decor and furnishings are
minimal and in keeping with the style of the hotel. The basic double
room was compact but room enough, we were offered an upgrade to junior
suite which was very welcomed and a great space. Views from all
bedrooms, lounge and dining area al - Anonymous
Centralt läge med hyfsat pris. Bra frukost och trevlig personal - Dag
Sköna sängar
Mysigt och smakfullt inredd
Tyst på nätterna
Kanonlägenhet - Anonymous
Usful location for go out of town(near of road that through the town to Stavanger) - Anonymous
The room was very small but that was expected. It was fine for solo traveler - not sure if it would have worked for two with bags and all. The hotel was clean and the staff were helpful. The breakfast was extra but had a good variety and since not to much around hotel for breakfast options worth the price. I would stay at the hotel again if on my own. There is a bus (2) which is about three blocks away that goes to Stavanger centrum in about 20 min. The Kvadrat shopping center is a 5 min. - Anonymous
Friendly staff, comfortable rooms, beautiful location but you will need a car. Good breakfast. - Anonymous
Oyvind was a gentleman and a perfect host. - Anonymous

subset: commoncrawl


text:
For more information about the AAVMC-AVMA DiVersity Matters Climate Survey, please contact Lisa Greenhill at email@example.com.
For information on the Employee College Climate Survey, please click here.
Iowa State University CVM - Informed Consent Form
Iowa State University CVM Climate Survey - Paper Version
Western University of Health Sciences Student Earns AAVMC Diversity Leader Scholarship
Colorado State University Veterinary Dean Emeritus Earns AAVMC Recognition Lecture Award
Texas A&M University Professor Earns AAVMC Leadership in Public Policy Award
AAVMCp: 202.371.9195
f: 202.842.0773
VMCASp: 617.612.2884
f: 617.612.2051


text:
Thin with a slight spongy texture to it, lavash bread appears as a simple staple to accompany Armenian dishes, whether its used to mop up stews, wrap grilled meats or eat with smoky cheeses, you'd think at first it's just like any local bread.
But lately, the humble lavash has hit the headlines for being one of UNESCO's newest cultural additions, and the international backlash it received. (more…)


text:
1978 Chevrolet C10 S10This Pickup is with its amazing flames real eye-candy and typical American. The paint is in a good condition and shines... More
1965 Chevrolet C10 StepsideA very nice and strong Pickup Truck which will make your heart skip a beat.The engine of the Pickup runs very... More
1977 Chevrolet C10 NL kentekenA very beautiful Pickup! It has a view signs of use, but this car will definitely make your heart skip... More
1966 Chevrolet C10 Pickup TruckThis beautiful C10 is in excellent condition. The paint, chrome, interior, cargo area, just everything is in fabulous condition. The... More
1964 Chevrolet C10 High Performance EngineThis pickup is in excellent condition.It comes with a very stylish two-tone paint and looks fabulous. The chrome shines... More
A very solid truck that would make a great project. It runs and drives down the road very good. Appears to have the original... More
This terrific C-10 is just waiting to bring the right owner lots of joy and happiness with years of trouble-free use! Dare to compare!... More
dreaming about for a super deal on a reliable Vehicle? Well, we've got it. It doesn't stop showing off once you get inside* ATTENTION!!!... More
Tired of the same dull drive? Well change up things with this big league Truck* Awesome! At Bogue and Oceanside Auto Sales, you are... More
1981 Chevrolet C10 Hot Rod Northwest Classic Auto Mall advertises this vehicle for the owner. If you are interested in this vehicle or have any questions,... More
1975 Chevy C-10 HOT ROD (California smog exempt) Northwest Classic Auto Mall advertises this vehicle for the owner. If you are interested in this vehicle or... More
1972 Chevrolet C10 1/2 Ton 2 Door Fleetside Short Bed Pickup. 350 V-8, TH350 trans, very fast pickup!... More
1967 Chevrolet C10 Pickup VIN: CS147S151444 This gorgeous 1967 Chevy C-10 Short bed, Fleet side Pickup was fully restored less than 5 years ago.... More
Now in our Las Vegas Indoor Showroom! Great For Cruisin' OR a Real Nice Daily Driver. 1964 Chevrolet C10 Fleetside Pickup. ... More
To view more pictures or for more information, please visit our website at www.classiccarliquidators.com then give us a call 903-891-0000.... More


text:
Little Turkey is a populated place located in Chickasaw County at latitude 43.13 and longitude -92.101.
The elevation is 1,102 feet. Little Turkey appears on the Protivin U.S. Geological Survey Map. Chickasaw County is in the Central Time Zone (UTC -6 hours).
Chickasaw County: Cities & Towns | ZIP Codes | Schools | Features (airports, churches, hospitals, etc.)
BIG Map |
Driving Directions |
Data Source - USGS GNIS FID: 464624
BOUNDARY MAPS: Township of Utica | Chickasaw County
Little Turkey, IA
(< 0.1 mile)
Jackson Junction, IA (3.6 miles ESE)
Reilly Settlement, IA (4.3 miles WNW)
Lawler, IA (4.7 miles SSW)
Jacksonville, IA (5.5 miles W)
Saude, IA
(5.6 miles NNW)
Protivin, IA (6 miles N)
Waucoma, IA (6.1 miles SE)
Bethany Hall, IA (7.7 miles WNW)
Fort Atkinson, IA (8.5 miles E)
Navan, IA
(3.1 miles SE)
Nansen, IA (7 miles W)
Twin Springs, IA (12.2 miles E)
Douglass, IA (14.4 miles SE)


text:
Check out this exclusive interview with Tony Porcellini from the Walt Disney World Swan and Dolphin Resort! He reveals the menus and details about the upcoming first annual Swan and Dolphin Food and Wine Classic!
Posts Tagged 'Swan and Dolphin Food and Wine Classic'
Disney Food Post Round-Up: August 22, 2010
Today's Disney Food Post round-up has some great posts about character meals, the mara at Disney's Animal Kingdom Lodge, Club 33, and more! Also, check out news on the 2010 Epcot Food and Wine Festival, La Hacienda de San Angel, and free dining packages in Disney World for 2011!
Swan and Dolphin Introduce the Food and Wine Classic, October 8-9
Head over to the Swan and Dolphin on October 8-9 to take part in their brand new Food and Wine Classic!


text:
Latest Headlines
BT's rivals are pressuring Ofcom to break up the former incumbent as part of the regulator's latest review into the UK's digital communications market, which Ofcom opened on Thursday (Mar. 12).
The FCC has delivered its long-awaited net neutrality order that reclassifies ISPs under Title II of the Communications Act. The FCC posted the 400-page order to its website two weeks after it was approved in a 3-2 vote by the Democratic-controlled commission.
The FCC has delivered its long-awaited net neutrality order that reclassifies ISPs under Title II of the Communications Act, one that will open a host of legal challenges from major telcos like AT&T and Verizon.
The Federal Trade Commission has charged DirecTV with deceptive advertising, alleging that the satellite TV operator has been luring prospective customers with 12-month promotional rates but not properly explaining to them the implications of the two-year contract these deals entail.
The FCC published its full net neutrality rules and gave wireless carriers insight into three key elements of the regulations: what constitutes "reasonable network management;" how future wireless data practices will be evaluated to make sure they comply with the rules; and why wireless networks are covered in the first place.
In the first Senate Commerce Committee hearing on its progress toward establishing a common nationwide broadband public-safety network, the First Responder Network Authority (FirstNet) was queried on everything from how it will incorporate rural areas into its broadband network to the excruciatingly long periods it takes FirstNet to hire people.
In a highly anticipated demonstration of its technology before the FCC, Globalstar showed that Terrestrial Low Power Service (TLPS) operations on Channel 14 had no negative impact on Wi-Fi operations within the public Wi-Fi channels in the ISM band.
The cable industry has a new hedge in its battle to control spiraling program costs: Hook the struggle to the federal government's effort to expand high-speed Internet deployment.
Verizon Wireless is not that worried that cable MSOs will start eating into the wireless business with Wi-Fi-first business models, according to a senior Verizon executive. At an investor conference today, Verizon Communications CFO Fran Shammo touched on several hot-button issues for Verizon and the wider industry, including new FCC regulations on net neutrality and data roaming.
Verizon Wireless came out against joint bidding arrangements for future spectrum auctions, allying itself with AT&T Mobility and setting up a battle with Sprint and T-Mobile US ahead of next year's 600 MHz incentive auction.
Press Releases
- Sling TV to launch HBO in time for 'Game of Thrones' premiere
- DIRECTV Tunes Up Its Swing For 2015 Masters Tournament Coverage
- WOW! Wholesale and iFiber Networks Team to Deliver High-Speed Internet Service in Chicago-Area Community
- Frontier Communications Delivers Symmetrical Broadband Speeds to Washington
- Bandwidth's API Portal Accelerates HTC, Inc.'s Out-of-Network VoIP Expansion
- More Press Releases
Sponsored Links


text:
An easy 30-minute meal recipe for you today, Cheesy Sloppy Joe Bread Bowls with LAND O LAKES® Sharp Cheddar American Blend! A fun twist on sloppy joe sandwiches with ground beef, an easy homemade sloppy joe sauce, pretzel rolls, and Sharp Cheddar American Blend! Delicious and ready in 30 minutes, making this recipe perfect for… [read more]
Cheesy Sloppy Joe Bread Bowls
French Onion Beer Burgers
Burger time! I love grilling, love beef, and love burgers! This FRENCH ONION BEER BURGERS recipe is delicious! This easy burger recipe is perfect for summer BBQs, parties, and even quick weeknight meals. And did I mention there's beer in these burgers??? But don't worry, I have a substitute option for the non-beer fans.
Easy Caribbean Tacos
I have a delicious and easy Caribbean Tacos recipe for you today! My kids love "Taco Tuesday"! These Caribbean Tacos are a quick and easy dinner that you can make in less than 30 minutes! They are full of fresh flavors including mango, lime, and cilantro and also a bit sweet and a bit spicy!… [read more]


text:
It's Time for Change!
A famous musician once sang, "these times are a-changing' and it is absolutely true for the name of American Women's Club of the Taunus!
In the past years, the Board has looked for ways to better reflect the demographics of our membership. We are a club of women from over 40 countries around the world and we wanted a name to reflect this diversity and inclusion. The newly named AIWC invites you to welcome our new name and branding, the American International Women's Club (AIWC), here on the website, in our office and throughout the many Club activities that are offered each month.
Over the next weeks, this website will be experiencing graphic and administrative changes to reflect the new name. We ask for your patience as these changes take place and may disrupt access to the site. In return, we will do our best to make down-time minimal. Check back often to see our new logo take shape and to read about other important and interesting info and events offered to our members.
What's Up This Month and Next . . .
Tickets on Sale Now!!!
* * *
Take a look at some of the hot items available for our Annual Charity Event:
2 VIP tickets to Borussia Dortmund (donated by Opel)
Martone Custom Bike (donated by Wertheim Village) VALUE: 1200€
JW Marriott Hotel & Spa Venice: 2 night stay (including breakfast) in brand new hotel, junior suite VALUE: 1300€
Pera Palace Hotel Jumeriah Istanbul: 2 night stay in room that Agatha Christie stayed in while writing 'Murder on the Orient Express' VALUE 750€
Ritz Carlton Berlin: 2 night stay including breakfast, with tickets to WYLD, VALUE: 600€
Lithography from Sarit Lichenstein, VALUE 300€
Rainer Brenner Optics: Men's Trussardi Sunglasses VALUE: 315€
Susanna Strech Busch: Silver Necklace with mother of pearl and smokey quartz VALUE: 80€
This is only a teaser -- there is so much more (hotels, theater tickets, signed books, etc.! Come to the Silver Soiree to bid on these and many more items and support Frauen Helfen Frauen.
* * *
21 March: Tickets still available! Our annual Spring Charity Event will take place on 21 March to benefit FRAUEN Helfen FRAUEN, an Oberursel-based NGO giving shelter to abused women and their children in addition to counseling. The event will take place in the evening and is open to members and their spouses/partners. Contact the office to buy your tickets for this fun annual event.
* * *
17 April: Art Exhibition: A group of members will hold a showing of acrylic paintings at the Clubhouse starting on 15 April. All members and visitors are free to stop by the Clubhouse to see the collection
* * *
Visit our Facebook page for photos and posting from our members!


text:
The present invention provides a family of BAG-1 related proteins from humans (BAG-1L, BAG-1, BAG-2, BAG-3, BAG-4 and BAG-5), the invertebrate C. elegans (BAG-1, BAG-2) and the fission yeast S. pombe (BAG-1A, BAG-1B) and the nucleic acid molecules that encode them.
1. A substantially purified nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 5.
2. A substantially purified nucleic acid molecule having a nucleotide sequence of at least 25 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 5.
3. A substantially purified nucleic acid molecule having a nucleotide sequence fully complementary to at least 25 nucleotides of the nucleotide sequence of SEQ ID NO:5.
4. The nucleic acid of
5. The nucleic acid of
6. A composition comprising a nucleic acid molecule of
This application is a continuation of U.S. Ser. No. 11/879,274, filed Jul. 16, 2007, which is a divisional of U.S. Ser. No. 10/782,627, filed Feb. 18, 2004, now U.S. Pat. No. 7,615,623, which is a continuation of U.S. Ser. No. 09/394,142, filed Sep. 9, 1999, now U.S. Pat. No. 6,696,558, which claims the benefit of U.S. Provisional Application No. 60/155,212, filed Sep. 9, 1998, which was converted from U.S. Ser. No. 09/150,489, each of which the entire contents are incorporated herein by reference.
This invention was made with government support under grant number CA-67329 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.
1. Field of the Invention
This invention relates generally to the fields of molecular biology and molecular medicine and more specifically to a novel family of proteins that can regulate protein folding. The functions of these proteins are potentially diverse, including promoting tumor cell growth and metastasis.
2. Background Information
The Hsc70/Hsp70-family of molecular chaperones participate in protein folding reactions, controlling protein bioactivity, degradation, complex assembly/disassembly, and translocation across membranes. These proteins interact with hydrophobic regions within target proteins via a carboxyl (C)-terminal peptide binding domain, with substrate binding and release being controlled by the N-terminal ATP-binding domain of Hsc70/Hsp70. Hsc70/Hsp70-assisted folding reactions are accomplished by repeated cycles of peptide binding, refolding, and release, which are coupled to ATP hydrolysis by the ATP-binding domain (ATPase) of Hsc70/Hsp70 and by subsequent nucleotide exchange. The chaperone activity of mammalian Hsc70/Hsp70 is regulated by partner proteins that either modulate the peptide binding cycle or that target the actions of these chaperones to specific proteins and subcellular compartments. DnaJ-family proteins (Hdj-1/Hsp40; Hdj-2; Hdj-3) stimulate the ATPase activity of Hsc70/Hsp70, resulting in the ADP-bound state which binds tightly to peptide substrates. The Hip protein collaborates with Hsc70/Hsp70 and DnaJ homologues in stimulating ATP hydrolysis, and thus also stabilize Hsc70/Hsp70 complexes with substrate polypeptides, whereas the Hop protein may provide co-chaperone functions through interactions with the C-terminal peptide binding domain.
The Bcl-2 associated athanogene-1 (bag-1) is named from the Greek word athanos, which refers to anti-cell death. BAG-1 was previously referred to as Bcl-2-associated protein-1 (BAP-1) in U.S. Pat. No. 5,539,094 issued Jul. 23, 1996, which is incorporated herein by reference. In this earlier patent, BAG-1 is described as a portion of the human BAG-1 protein, absent the N-terminal amino acids 1 to 85. In addition, a human protein essentially identical to human BAG-1 was described by Zeiner and Gehring, (Proc. Natl. Acad. Sci., USA 92:11465-11469 (1995)). Subsequent to the issuance of U.S. Pat. No. 5,539,094 the N-terminal amino acid sequence from 1 to 85 of human BAG-1 was reported.
BAG-1 and its longer isoforms BAG-1M (Rap46) and BAG-1L are recently described Hsc70/Hsp70-regulating proteins. BAG-1 competes with Hip for binding to the Hsc70/Hsp70 ATPase domain and promotes substrate release. BAG-1 also reportedly stimulates Hsc70-mediated ATP hydrolysis by accelerating ADP/ATP exchange, analogous to the prokaryotic GrpE nucleotide exchange protein of the bacterial Hsc70 homologue, DnaK. Gene transfection studies indicate that BAG-1 proteins can influence a wide variety of cellular phenotypes through their interactions with Hsc70/Hsp70, including increasing resistance to apoptosis, promoting cell proliferation, enhancing tumor cell migration and metastasis, and altering transcriptional activity of steroid hormone receptors.
Despite the notable progress in the art, there remains an unmet need for the further identification and isolation of additional homologous BAG protein species, and the nucleic acid molecules and/or nucleotide sequences that encode them. Such species would provide additional means by which the identity and composition of the BAG domain, that is, the portion of the protein that is influencing or modulating protein folding, could be identified. In addition, such species would be useful for identifying agents that modulate apoptosis as candidates for therapeutic agents, in particular, anticancer agents. The present invention satisfies these need, as well as providing substantial related advantages.
The present invention provides a family of BAG-1 related proteins from humans [BAG-1L (SEQ ID NO:2), BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO: 4), BAG-3 (SEQ ID NO:6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22) and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24)], the invertebrate C. elegans [BAG-1 (SEQ ID NO:12), BAG-2 (SEQ ID NO:14)] and the fission yeast S. pombe [BAG-1A (SEQ ID NO:16), BAG-1B (SEQ ID NO:18)] and the nucleic acid molecules that encode them.
Another aspect of the present invention provides an amino acid sequence present in the family of BAG-1 related proteins, that modulates Hsc70/Hsp70 chaperone activity, that is, the BAG domain.
Another aspect of the present invention provides novel polypeptide and nucleic acid compositions and methods useful in modulating Hsc70/Hsp70 chaperone activity.
Another aspect of the present invention is directed to methods for detecting agents that modulate the binding of the BAG family of proteins, such as BAG-1 (beginning at residue 116 of SEQ ID NO:2), and related proteins with the Hsc70/Hsp70 Family of proteins or with other proteins that may interact with the BAG-Family proteins.
Still another aspect of the present invention is directed to methods for detecting agents that induce the dissociation of a bound complex formed by the association of BAG-Family proteins with Hsc70/Hsp70 Family molecule chaperones or other proteins.
The term ōapoptosisö, as used herein, refers to the process of programmed cell death, although not all programmed cell deaths occur through apoptosis, as used herein, ōapoptosisö and ōprogrammed cell deathö are used interchangeably.
The term ōtumor cell proliferationö, as used herein refers to the ability of tumor cells to grow and thus expand a tumor mass.
The term ōcell migrationö, as used herein refers to the role cell motility plays in the invasion and potentially metastasis by tumor cells.
The term ōmetastasisö, as used herein refers to the spread of a disease process from one part of the body to another, as in the appearance of neoplasms in parts of the body remote from the site of the primary tumor; results in dissemination of tumor cells by the lymphatics or blood vessels or by direct extension through serious cavities or subarachnoid or other spaces.
The term ōsteroid hormone receptor functionö, as used herein refers to physiological, cellular and molecular functioning of receptors sites that bind with steroid hormones.
The term ōsubstantially purifiedö, as used herein, refers to nucleic acid or amino acid sequence that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
ōNucleic acid moleculeö as used herein refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single or double stranded, and represent the sense or antisense strand.
ōHybridizationö, as used herein, refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
The terms ōcomplementaryö or ōcomplementarityö, as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence ōA-G-T binds to the complementary sequence ōT-C-Aö.
The term ōhomologyö, as used herein, refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid and is referred to using the functional term ōsubstantially homologous.ö The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency.
The term ōantisenseö, as used herein, refers to nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term ōantisense strandö is used in reference to a nucleic acid strand that is complementary to the ōsenseö strand. Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation. In this manner, mutant phenotypes may be generated. The designation ōnegativeö is sometimes used in reference to the antisense, and ōpositiveö is sometimes used in reference to the sense strand.
ōAmino acid sequenceö as used herein refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. Where ōamino acid sequenceö is recited herein this term excludes an amino acid sequence of a naturally occurring protein. ōAmino acid sequenceö, ōpolypeptideö or ōproteinö are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
The term ōfunctional fragmentsö or ōfragmentsö, as used herein, with regard to a protein refers to portions of that protein that are capable of exhibiting or carrying out the activity exhibited by the protein as a whole. The portions may range in size from three amino acid residues to the entire amino acid sequence minus one amino acid. For example, a protein ōcomprising at least a functional fragment of the amino acid sequence of SEQ ID NO:1ö, encompasses the full-length of the protein of SEQ ID NO:1 and portions thereof.
A ōderivativeö of a BAG protein, as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The derivative may have ōconservativeö changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., substitution of an apolar amino acid with another apolar amino acid (such as replacement of leucine with isoleucine). The derivative may also have ōnonconservativeö changes, wherein a substituted amino acid has different but sufficiently similar structural or chemical properties that permits such a substitution without adversely effecting the desired biological activity, e.g., replacement of an amino acid with an uncharged polar R group with an amino acid with an apolar R group (such as replacement of glycine with tryptophan), or alternatively replacement of an amino acid with a charged R group with an amino acid with an uncharged Polar R group (such as replacement of lysine with asparagine).
Amino AcidsŚApolar R Groups
Similar minor modifications may also include amino acids deletions or insertions or both. Guidance in determining which amino acid residues may be modified as indicated above without abolishing the desired biological functionality may be determined using computer programs well known in the art, for example, DNASTAR software. In addition, the derivative may also result from chemical modifications to the encoded polypeptide, including but not limited to the following, replacement of hydrogen by an alkyl, acyl, or amino group; esterification of a carboxyl group with a suitable alkyl or aryl moiety; alkylation of a hydroxyl group to form an ether derivative. Further a derivative may also result from the substitution of a L-configuration amino acid with its corresponding D-configuration counterpart.
The term ōmimeticö, as used herein, refers to a molecule, the structure of which is developed from knowledge of the structure of a protein/polypeptide or portions thereof (such as BAG-1) and, as such, is able to effect some or all of the actions of BAG-1 protein.
ōPeptide nucleic acidö, as used herein, refers to a molecule which comprises an oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid (Nielsen, P. E. et al., Anticancer Drug Des. 8:53-63 (1993)).
The present invention provides a family of BAG-1 related proteins from humans [BAG-1L (SEQ ID NO:2), BAG-1S beginning at residue 116 of SEQ ID NO:2, BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO: 8) and (SEQ ID NO:22) and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24)], the invertebrate C. elegans [BAG-1 (SEQ ID NO:12), BAG-2 (SEQ ID NO:14)] and the fission yeast S. pombe [BAG-1A (SEQ ID NO:16), BAG-1B (SEQ ID NO:18)], specifically the full length amino acid sequences comprising human BAG-1L (SEQ ID NO:2), BAG-1 (beginning at residue 116 of SEQ ID NO:2), and BAG-2 (SEQ ID NO:4) C. elegans BAG-1 (SEQ ID NO:12), and BAG-2 (SEQ ID NO:14), and S. pombe BAG-1A (SEQ ID NO:16) and BAG-1B (SEQ ID NO:18); and partial sequences comprising human BAG-3 (SEQ ID NO: 6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22), and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24) and functional fragments thereof. In particular, the invention provides the amino acid sequences comprising human BAG-2 (SEQ ID NO:4), BAG-3 (SEQ ID NO:6) and (SEQ ID NO:20), BAG-4 (SEQ ID NO:8) and (SEQ ID NO:22), and BAG-5 (SEQ ID NO:10) and (SEQ ID NO:24) proteins.
Another aspect of the present invention provides the nucleic molecule and nucleotide sequences that encode the family of BAG-1 related proteins from humans [BAG-1 (SEQ ID NO:1), BAG-2 (SEQ ID NO:3), BAG-3 (SEQ ID NO:5) and (SEQ ID NO:19), BAG-4 (SEQ ID NO:7) and (SEQ ID NO:21) and BAG-5 (SEQ ID NO:9) and (SEQ ID NO:23)], the invertebrate C. elegans [BAG-1 (SEQ ID NO:11), BAG-2 (SEQ ID NO:13)] and the fission yeast S. pombe [BAG-1A (SEQ ID NO:15), BAG-1B (SEQ ID NO:17)].
BAG-1L (SEQ ID NO:2) is a multifunctional protein that blocks apoptosis, promotes tumor cell metastasis, and contributes to factor-independent and p53-resistant cell growth. BAG-1L (SEQ ID NO:2) interacts with several types of proteins, including Bcl-2, some tyrosine kinase growth factor receptors, steroid hormone receptors, and the p53-induced cell cycle regulator Siah-1A.
BAG-1 is a regulator of Hsc70/Hsp70 family molecular chaperones. A carboxyl-terminal domain in this protein binds tightly to the ATPase domains of Hsc70 and Hsp70 (KD=1 nM) (Zeiner, M., Gebauer, M., and Gehring, U., EMBO J. 16: 5483-5490, (1997)). BAG-1 modulates the activity of these molecular chaperones, acting as an apparent functional antagonist of the Hsp70/Hsc70-associated protein Hip (3-5)(H÷hfeld, J. and Jentsch, S., EMBO J. 16: 6209-6216, (1997); Takayama, S., Bimston, D. N., Matsuzawa, S., Freeman, B. C., Aime-Sempe, C., Xie, Z., Morimoto, R. J., and Reed, J. C., EMBO J. 16: 4887-96, (1997); Zeiner, M., Gebauer, M., and Gehring, U., EMBO J. 16: 5483-5490, (1997)). In general, protein refolding is accomplished by Hsp70/Hsc70 through repeated cycles of target peptide binding and release, coupled to ATP hydrolysis (Ellis, R., Curr Biol. 7: R531-R533, (1997)). BAG-1 appears to promote substrate release, whereas Hip stabilizes Hsp70/Hsc70 complex formation with target peptides (H÷hfeld, J., Minami, Y., and Hartl, F.-U., Cell. 83: 589-598, (1995)). Since each substrate interaction with Hsc70/Hsp70 is unique in terms of the optimal length of time the protein target should remain complexed with Hsc70/Hsp70 for achieving new conformations, the net effect of BAG-1 can be either enhancement or inhibition of the refolding reaction.
The 70 kd heat shock family proteins (Hsp70/Hsc70) are essential to a variety of cellular processes and have been implicated in cancer, yet it is unclear how these proteins are regulated in vivo. A variety of co-chaperones have been identified which may target Hsp70/Hsc70 to different subcellular compartments or promote their interactions with specific protein or protein complexes. BAG-1 appears to represent a novel Hsp70/Hsc70 regulator which differs functionally from all other mammalian co-chaperones identified to date, such as members of the DnaJ-, Hip-, Hop-, and cyclophilin-families of proteins.
Another aspect of the present invention provides the amino acid sequence of a binding domain of about 40 to 55 amino acids that bind the a Hsc70/Hsp70 ATPase domain. The BAG domain is situated near the C-terminus, and the ubiquitin-like domains are situated near the N-terminus.
The BAG family of proteins of the present invention contain a common conserved C-terminal domain (the ōBAGö domain) that facilitates binding to the ATPase domain of Hsp70/Hsc70. The carboxyl-terminal domain of BAG-1 binds to the ATPase domain of Hsc70/Hsp70 and regulates its chaperone function by acting as a ADP-ATP exchange factor. Other domains of BAG-1 mediate interactions with proteins such as Bcl-2 and retinoic acid receptors (RARs), allowing BAG-1 to target Hsc70/Hsp70 to other proteins, presumably modulating their function by changing their conformations.
Human BAG-1 was previously shown to inhibit Hsc70/Hsp70 dependent refolding of denatured protein substrates in vitro (S. Takayama, et al., EMBO J. 16, 4887-96 (1997); M. Zeiner, M. Gebauer, U. Gehring, EMBO J. 16, 5483-5490 (1997); and J. H÷hfeld, S. Jentsch, EMBO J. 16, 6209-6216 (1997)). In Example III, Part A the effects of recombinant human BAG-1, BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) were compared using in vitro protein refolding assays similar to those employed previously for assessing BAG-1. The study showed that addition of equimolar amounts of each of the recombinant proteins to Hsc70 resulted in significant inhibition of luciferase refolding, with BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) showing somewhat greater inhibitor activity than BAG-1 (
Additional follow on studies using the same experimental protocols as the previous studies, as taught in Example IIA, have shown that BAG-4 (SEQ ID NO:22) also undergoes association with Hsc70/ATPase.
Yet another aspect of the present invention provides a nucleotide sequence having at least about 15 nucleotides and, generally, about 25 nucleotides, preferably about 35 nucleotides, more preferably about 45 nucleotides, and most preferably about 55 nucleotides that can hybridize or is complementary under relatively stringent conditions to a portion of the nucleic acid sequences shown in
Yet another aspect of the present invention provides a compound of the formula,
RN is a group of 1 to 552 independently selected amino acids;
R1 is a group of 3 independently selected amino acids;
X1 is an amino acid with a charged or uncharged R group, such as aspartic acid, glutamic acid, asparagine, or glutamine;
R2 is a group of 7 independently selected amino acids;
X2 is an amino acid with a charged R group, such as glutamic acid;
R3 is a group of 5 independently selected amino acids;
X3 is an amino acid with an apolar R group, such as leucine, methionine, or isoleucine;
R4 is a group of 3 independently selected amino acids;
X4 is an amino acid with charged R group, such as aspartic acid or glutamine acid;
R5 is a single independently selected amino acid;
X5 is an amino acid with apolar or uncharged R group, such as leucine, valine, methionine, alanine or threonine;
R6 is a group of 15 independently selected amino acids;
X6 is an amino acid with a charged or uncharged R group, such as arginine, lysine, glutamine or aspartic acid;
R7 is a group of 2 independently selected amino acids;
X7 is an amino acid with a charged R group, such as arginine;
X8 is an amino acid with a charged R group, such as arginine or lysine;
R9 is a group of 2 independently selected amino acids;
X9 is an amino acid with an apolar R group, such as valine;
R10 is a group of 3 independently selected amino acids;
X10 is an amino acid with an uncharged R group, such as glutamine;
R11 is a group of 2 independently selected amino acids;
X11 is an amino acid with an apolar R group, such as leucine; and
RC is a group of 1 to 100 independently selected amino acids.
A nucleotide sequence of at least about 15 nucleotides and, generally, about 25 nucleotides, preferably about 35 nucleotides, more preferably about 45 nucleotides, and most preferably about 55 nucleotides can be useful, for example, as a primer for the polymerase chain reaction (PCR) or other similar reaction mediated by a polymerase such as a DNA or RNA polymerase (see PCR Protocols: A guide to methods and applications, ed. Innis et al. (Academic Press, Inc., 1990), which is incorporated herein by reference; see, for example, pages 40-41). In addition, such a nucleotide sequence of the invention can be useful as a probe in a hybridization reaction such as Southern or northern blot analysis or in a binding assay such as a gel shift assay.
A nucleotide sequence of the invention can be particularly useful as an antisense molecule, which can be DNA or RNA and can be targeted to all or a portion of the 5′-untranslated region or of the 5′-translated region of a bag-1 nucleic acid sequence in a cell. For example, an antisense molecule can be directed to at least a portion of the sequence shown as the BAG domain in
A nucleotide sequence of the invention also can be useful as a probe to identify a genetic defect due a mutation of a gene encoding a BAG protein in a cell. Such a genetic defect can lead to aberrant expression of a BAG protein in the cell or to expression of an aberrant BAG protein, which does not properly associate with a Bcl-2-related protein or Hsc70/Hsp70 protein in the cell. As a result, a genetic defect in a gene encoding, for example, human BAG-1 can result in a pathology characterized by increased or decreased levels in protein folding.
Further a nucleotide compound or composition as taught in the present invention can be synthesized using routine methods or can be purchased from a commercial source. In addition, a population of such nucleotide sequences can be prepared by restriction endonuclease or mild DNAse digestion of a nucleic acid molecule that contains nucleotides as shown in the nucleotide sequences shown in
A particular nucleotide sequence can be designed based, for example, on a comparison of the nucleic acid molecules encoding any one of the BAG family proteins, as shown in
If desired, a nucleotide sequence of the invention can incorporate a detectable moiety such as a radiolabel, a fluorochrome, a ferromagnetic substance, a luminescent tag or a detectable binding agent such as biotin. These and other detectable moieties and methods of incorporating such moieties into a nucleotide sequence are well known in the art and are commercially available. A population of labelled nucleotide sequences can be prepared, for example, by nick translation of a nucleic acid molecule of the invention (Sambrook et al., supra, 1989; Ausubel et al., supra, 1989).
One skilled in the art would know that a method involving hybridization of a nucleotide sequence of the invention can require that hybridization be performed under relatively stringent conditions such that nonspecific background hybridization is minimized. Such hybridization conditions can be determined empirically or can be estimated based, for example, on the relative GC content of a sequence and the number of mismatches, if known, between the probe and the target sequence (see, for example, Sambrook et al., supra, 1989).
The invention further provides antibodies specific for human BAG family protein. As used herein, the term ōantibodyö includes polyclonal and monoclonal antibodies, as well as polypeptide fragments of antibodies that retain a specific binding activity for human BAG-1 of at least about 1ū105 M−1. One skilled in the art would know that anti-BAG-1 antibody fragments such as Fab, F(ab′)2 and Fv fragments can retain specific binding activity for human BAG-1 (beginning at residue 116 of SEQ ID NO:2) and, thus, are included within the definition of an antibody. In addition, the term ōantibodyö as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies and fragments that retain binding activity such as chimeric antibodies or humanized antibodies. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al., Science 246:1275-1281 (1989), which is incorporated herein by reference.
One skilled in the art would know that purified BAG family protein, which can be prepared from natural sources or synthesized chemically or produced recombinantly, or portions of a BAG family protein, including a portion of human BAG family protein such as a synthetic peptide as described above, can be used as an immunogen. Such peptides useful for raising an antibody include, for example, peptide portions of the N-terminal 85 amino acids or the BAG domain of any of the human BAG proteins (see
Non-immunogenic fragments or synthetic peptides of BAG proteins can be made immunogenic by coupling the hapten to a carrier molecule such bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), as described in Example IV, below. In addition, various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art and described, for example, by Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988), which is incorporated herein by reference.
The following examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.
This example describes methods for isolating and characterizing of BAG-family cDNA sequences from human, nematode and yeast.
A. Cloning of Human BAG cDNA Sequences
Yeast two-hybrid library screening of a human Jurkat cell cDNA library was performed as described by Takayama et al., EMBO J., 16:4887-96 (1997); Matsuzawa et al., EMBO J., 17:2736-2747 (1998), which are incorporated herein by reference) using EGY48 strain yeast transformed with pGilda-Hsc70/ATPase (67-377 amino acids) and the lacZ reporter plasmid pSH18-34. Of the resulting ś5ū106 transformants, 112 Leu+ colonies were obtained after 1 week incubation at 30░ C. Assay of β-galactosidase (β-gal) activity of these colonies resulted in 96 clones. Mating tests were then performed using RFY206 yeast strain transformed with pGilda, pGilda mBAG-1 (1-219), or pGilda Hsc70/ATPase. Of these, 66 displayed specific interactions with Hsc70/ATPase. The pJG4-5 cDNAs were recovered using KC8 E. coli strain which is auxotrophic for tryptophan (Trp). DNA sequencing revealed 3 partially overlapping human BAG-1, 4 identical and one overlapping cDNAs encoding BAG-2, and 2 partially overlapping BAG-3 clones.
Using the above described yeast two-hybrid screen with the ATPase domain of Hsc70 as ōbaitö, several human cDNAs were cloned which encode portions of BAG-1 or of two other BAG-1-like proteins which are termed BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6). The longest of the cDNAs for BAG-2 (SEQ ID NO:3) and BAG-3 (SEQ ID NO:5) contained open reading frames (ORFs) of 207 and 162 amino acids, respectively, followed by stop codons. All BAG-1 (SEQ ID NO:1), BAG-2 (SEQ ID NO:3) and BAG-3 (SEQ ID NO:5) cDNAs obtained by two-hybrid library screening with Hsc70/ATPase contained a conserved domain of about 40-50 amino acids which are termed the ōBAGö domain and are shown in
B. Identification of Additional BAG-Family Proteins
A search of the translated Genbank database using the bBLAST and FASTA search programs also identified human ESTs that provided sequences for further investigation of BAG-family proteins. The putative BAG-4 (SEQ ID NO:8) and BAG-5 (SEQ ID NO:10) proteins contain BAG-domains that share the greatest sequence similarity with the BAG-domain of BAG-3 (SEQ ID NO:6). These were designated BAG-4 (Accession number AA693697, N74588) and BAG-5 (Accession number AA456862, N34101). BAG-4 has 62% identity and ś81% similarity to BAG-3, and BAG-5 has 51% identity and ś75% similarity to BAG-3.
Additional BAG-family orthologues or homologues were also identified using computer-based searches and resulted in BAG-family homologue in the nematode C. elegans and the fission yeast S. pombe. The C. elegans genome encodes two apparent BAG-family proteins, which are most similar in their overall sequences to the human BAG-1 (Afo39713, gi:2773211) (SEQ ID NO:12) and BAG-2 (SEQ ID NO:14) (Afo68719, gi:3168927). The S. pombe contains two BAG-family proteins that share the greatest overall sequence similarity with human BAG-1 (Alo23S54, gi/3133105 and Alo23634, gi/3150250). The human and C. elegans BAG-1 proteins as well as S. pombe BAG-1A all have ubiquitin-like domains near their N-termini (see
The overall predicted amino acid sequences of the C. elegans BAG-1 (SEQ ID NO:12) and S. pombe BAG-1A (SEQ ID NO:16) proteins are ś18% identical (ś61% similar) and ś17% identical (ś64% similar), respectively, to human BAG-1, implying origin from a common ancestral gene. The C. elegans BAG-1 protein (SEQ ID NO:12), however, contains a 5 to 7 amino acid insert in its BAG-domain as compared to the human, murine, and yeast BAG-1 homologues (see
C. Yeast Two-Hybrid Assay of BAG Binding to Hsc70/ATPase
The longest of the cDNAs obtained for the BAG-2 and BAG-3 proteins were expressed with N-terminal transactivation (TA) domains in yeast and tested by yeast two-hybrid assay for interactions with fusion proteins consisting of Hsp70/ATPase or a variety of unrelated proteins (Fas, Siah, Fadd) containing N-terminal LexA DNA-binding domains. TA-BAG-2 and TA-BAG-3 demonstrated positive interactions with LexA-Hsc70/ATPase, resulting in transactivation of a lacZ reporter gene that was under the control of LexA operators (
In order to determine whether the BAG proteins are capable of forming heterodimers, coexpression of BAG-2 and BAG-3 in the yeast two-hybrid assay was also performed. Coexpression of BAG-2 and BAG-3 failed to show interaction with BAG-1 or a deletion mutant of BAG-1 (ΔC) which is missing part of its C-terminal domain required for Hsp70/Hsc70 binding suggest that these proteins do not form heterdimers.
D. Isolation and Characterization of the Complete Open Reading Frame Sequences of BAG-2 and BAG-3
In order to deduce the complete ORFs of BAG-2 and BAG-3, a λ-phage cDNA library was screened as follows, using hybridization probes derived from the two-hybrid screening. A human jurkat T-cell λ-ZapII library cDNA library (Stratagene) was screened by hybridization using 32P-labeled purified insert DNA from the longest of the human BAG-2 (clone #11) and human BAG-3 (clone #28) cDNA clones. From about one million clones screened, 38 BAG-2 and 23 BAG-3 clones were identified, cloned, and their cDNA inserts recovered as pSKII plasmids using a helper phage method (Stratagene). DNA sequencing of λ-phage derived human BAG-2 cDNA clones revealed an ORF encoding a predicted 211 amino acid protein, preceded by an in-frame stop codon. The longest human BAG-3 λ-phage cDNA clone contains a continuous ORF of 682 amino acids followed by a stop codon, but without an identifiable start codon (see
Although BAG-1L (SEQ ID NO:2), BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) all contain a homologous BAG domain near their C-terminus, the N-terminal regions of these proteins are dissimilar. Using a combination of search tools (Prosite Search: PP search, using the Prosite pattern database, BCM Search Launcher, Baylor College of Medicine, and Blocks Search), it was determined that the BAG-2 N-terminal region contains potential kinase phosphorylation sites but otherwise shares no apparent similarity with other proteins or known functional domains.
In contrast, the predicted N-terminal region BAG-3 contains a WW domain as shown in
This example demonstrates that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) bind Hsc70/ATPase in various in vitro assays.
A. Solution Binding Assay of BAG-2 and BAG-3 to Hsc70/ATPase
Association of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) with Hsc70/ATPase was determine by an in vitro protein binding assay where Hsc70/ATPase or BAG-family proteins were expressed in bacteria as Glutathione S-Transferase (GST) fusion proteins. Purified cDNA sequences encoding residues 5 to 211 of human BAG-2 (clone #11) and the C-terminal 135 amino acids of human BAG-3 (clone #28) (see
Cells were recovered and incubated with 0.5 mg/ml lysozyme in 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Tween-20, 0.1% 2-mercaptoethanol, 5 mM EDTA, 1 mM PMSF and a mixture of other protease inhibitors obtained from Boehringer Mannheim (1697498) at room temperature for 0.5 h, followed by sonication. Cellular debris were pelleted by centrifugation at 27,500 g for 10 min and the resulting supernatants were incubated with 30 ml of glutathionine-Sepharose (Pharmacia) at 4░ C. overnight. The resin was then washed with 20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% Tween-20, and 0.1% 2-mercaptoethanol until the OD 280 nm reached <0.01. For removal of GST, the resin with immobilized GST-fusion protein was incubated with 10 U of thrombin (Boehringer, Inc.) at 4░ C. in 20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% Tween-20, 0.1% 2-Mercaptoethanol, and 2.5 mM CaCl2 overnight. Released proteins were then purified on Mono Q (HR10/10, Pharmacia) by FPLC using a linear gradient of 0.5M NaCl at pH 8.0 and dialyzed into chaperone assay buffer.
The ability of BAG-2 (SEQ ID NO:4) or BAG-3 (SEQ ID NO:6) to bind Hsc70/ATPase in solution was then examined. GST control or GST-BAG proteins were immobilized on glutathione-Sepharose and tested for binding to 35S-labeled in vitro translated (IVT) proteins. Immunoprecipitation and in vitro GST-protein binding assays were performed as described by Takayama et al., supra (1997), using pCI-Neo flag or pcDNA3-HA into which human Bag-2 (clone #11) or human BAG-3 (clone #28) had been subcloned for in vitro translation of 35S-L-methionine labeled proteins or expression in 293T cells. As shown in
B. Binding of BAG Proteins to Hsc70 In Vivo
The ability of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins to interact in cells with Hsc70 was tested by expressing these proteins with N-terminal Flag epitope tags in 293T human epithelial cells using co-immunoprecipitation assays as described previously (Takayama et al., supra (1997)). cDNAs encoding the λ-phage cloned regions of BAG-2 and BAG-3 were subcloned in-frame into pcDNA3-Flag. Anti-Flag immune complexes prepared from 293T cells after transfection with plasmids encoding Flag-BAG-1, Flag-BAG-2, or Flag-BAG-3 were analyzed by SDS-PAGE/immunoblot assay. As shown in
C. BIAcore Assay of BAG Protein Binding to the ATPase Domain of Hsc70
BAG-1 (beginning at residue 116 of SEQ ID NO:2) is known to bind tightly to the ATPase domain of Hsc70 (Stuart et al., J. Biol. Chem., In Press (1998)). BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins were therefore, examined for their ability to bind to Hsc70/ATPase. The affinity and binding kinetics of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) to Hsc70/ATPase was also compared to that of BAG-1 (beginning at residue 116 of SEQ ID NO:2) for Hsc70/ATPase, using a surface plasmon resonance technique (BIAcore) which has been described previously (Stuart et al., supra, (1998) which is incorporated herein by reference).
BAG-family proteins were produced in bacteria and purified to near homogeneity as shown in
The rates of Hsc70 binding to BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), and BAG-3 (SEQ ID NO:6) proteins were similar, following pseudo first-order kinetics with estimated association rate constants (κa) of 2.1, 2.1 and 2.4ū105 M−1 sec−1, respectively. After allowing binding of Hsc70 to immobilized BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) to reach plateau levels, the chaperone was removed from the flow solution and the dissociation rate was monitored. BAG-1 (beginning at residue 116 at SEQ ID NO:2) and BAG-2 (SEQ ID NO:4) exhibited similar dissociation rates, with relatively slow loss of Hsc70 from the chip surface, resulting in estimated dissociation rate constants (κd) of 3.0 and 5.0ū10−4 sec−1, respectively (see
This example demonstrates that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) proteins inhibit Hsp70/Hsc70-dependent refolding of denatured proteins similarly to a BAG-1 (beginning at residue 116 of SEQ ID NO:2) protein.
The effects of BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) protein on Hsp70/Hsc70-dependent protein refolding was determined using in vitro protein refolding assays similar to those described previously by Takayama et al., supra, 1998; Terada et al., J. Cell Biol., 139:1089-1095, 1997, which are incorporated herein by reference. Briefly, luciferase (20 μM) was denatured in 25 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 5 mM DTT, 6M guanidine hydrochloride at ś25░ C. for 1 h. Denatured luciferase was diluted 1:40 into 25 mM Hepes-KOH, pH 7.2, 50 mM potassium acetate, 5 mM DTT. Hsc70 (1.8 μM), DnaJ (StressGen, Inc.) (0.9 μM), and various purified recombinant proteins as indicated were added to refolding buffer (30 mM Hepes-KOH, pH 7.6, 120 mM potassium acetate, 3 mM magnesium acetate, 2 mM DTT, 2.5 mM ATP) with 0.2 volume of diluted denatured luciferase to a final concentration of 0.1 μM. Luciferase activity was measured after 1.5 hr incubation at 35░ C.
The combination of Hsc70 and DnaJ resulted in ATP-dependent refolding of chemically denatured firefly luciferase, with function of over half the denatured enzyme restored in a 90 minute reaction, as monitored by a chemiluminescence assay. In contrast, neither Hsc70 nor DnaJ alone were able to induce substantial refolding of denatured luciferase. Furthermore, little spontaneous restoration of luciferase activity was observed with control proteins, BSA, GST or Bcl-XL (see
Addition of recombinant purified BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) to the above assays in amounts equimolar to Hsc70 (1.8 μM) resulted in striking inhibition of luciferase refolding. BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) displayed somewhat greater inhibitory activity than BAG-1 (beginning at residue 116 of SEQ ID NO:2) as shown in
In an additional refolding assay, described previously by Minami et al., J. Biol. Chem. 271:19617-24, 1996), purified Hsc70 and human DnaJ homolog Hdj-1 (Hsp 40) were used with additional cofactors provided in reticulocyte lysates (5% v:v) to produce a system capable of refolding denatured luciferase. Briefly, additional cofactors included, recombinant Luciferase (Promega: QuantiLumÖ), that had been heat denatured at 42░ C. for 10 min, 1.8 μM Hsc70 (Sigma; purified from bovine brain), 0.9 μM Hsp40, and various recombinant purified proteins. Luciferase activity was measured (Promega luciferase assay kit) using a luminometer (EG&G Berthold, MicroLumat luminometer, Model #LB96P). All results were normalized relative to non-denatured luciferase that had been subjected to the same conditions. Control reactions lacking ATP, Hsc70, or Hsp40 resulted in negligible luciferase refolding.
Various amounts of purified BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6), relative to amounts of Hsc70 were used in the above-described protein refolding assay. Addition of BAG-family proteins resulted in a concentration-dependent inhibition of Hsc70 chaperone activity. Furthermore, the BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) inhibition of Hsc70 chaperone activity was demonstrated to be as potent as that observed for BAG-1 (beginning at residue 116 of SEQ ID NO:2). In contrast, the BAG-1 (ΔC) mutant as well as other control proteins did not suppress Hsc70-mediated refolding of denatured luciferase. These results indicate that BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) can inhibit Hsc70/Hsp70 dependent protein refolding activity to the same extent as BAG-1 (beginning at residue 116 of SEQ ID NO:2).
B. BAG Competes with Hip for Binding to Hsc70.
It is known that BAG-1 competes with Hip for binding to Hsc70, with these proteins exerting opposite effects on Hsc70-mediated protein refolding (Hohfeld, J., and Jentsch, S., Embo J., 16:6209-6216, 1997, which is incorporated herein by reference). In order to determine whether BAG-2 (SEQ ID NO:4) and BAG-3 (SEQ ID NO:6) also compete with Hip for binding to Hsc70, refolding assays were performed as described above in the presence of Hip protein.
Hip was purified as His6-protein. The fusion protein was induced from pET28-Hip (V. Prapapanich et al., Mol Cell Biol., 18:944-952, 1998, which is incorporated herein by reference) with 0.1 mM IPTG at 25░ C. for 6 h in BL21 cells. Cells from 1 L of culture were resuspended into 50 ml of 50 mM Phosphate buffer (pH 6.8), 150 mM NaCl, and 1% (v/v) Tween-20 and then incubated with 0.5 mg/ml lysozyme at 25░ C. for 0.5 h, followed by sonication. After centrifugation at 27,500 g for 10 min, the resulting supernatant was mixed with 15 ml nickel resin (Qiagen, Inc.) at 4░ C. for 3 h with 25 mM imidazol. The resin was then washed with 50 mM phosphate buffer (pH 6.8), 25 mM imidazol, 150 mM NaCl and 0.1% Tween-20 until the OD280 nm reached a value of <0.01. His6-Hip protein was eluted with 250 mM imidazol in washing buffer (Qiagene, Inc.) and purified on Mono Q (HR10/10 Pharmacia) by FPLC using a linear gradient of 0.5M NaCl at pH 8.0, followed by dialysis in chaperone assay buffer.
In the refolding assay reactions, addition of purified Hip at equimolar concentrations relative to BAG-1 (beginning at residue 116 of SEQ ID NO:2), BAG-2 (SEQ ID NO:4), or BAG-3 (SEQ ID NO:6) (1.8 μM) completely negated the inhibitory effects of the BAG-family proteins on refolding of denatured luciferase (see
In summary, these results demonstrate that BAG-family proteins all contain a conserved BAG domain near their C-terminus that binds Hsc70/Hsp70, and that human BAG-family proteins can bind with high affinity to the ATPase domain of Hsc70 and inhibit its chaperone activity through a Hip-repressable mechanism.
Following the procedures disclosed herein, the nucleic acid and amino acids sequences to human BAG-3, BAG-4 and BAG-5 were further expanded. The expanded sequences for BAG-3, BAG-4 and BAG-5 are shown in
Patent Citations
Non-Patent Citations
Legal Events
Original Image


text:
It's still not too late to de-nobble Greenbury
City & Business
Sunday 12 November 1995
This was not some abstruse actuarial requirement. It goes to the heart of the debate about disclosure of executive pay. As every fat cat knows, their annual salary - as revealed in the annual report - tells only half the story about their total remuneration package.
With each year that passes, they also earn a growing pension entitlement. The true value of this entitlement is never revealed. Companies are only obliged to disclose a figure when the company pension fund is in deficit. Then when they do, the figure they quote is highly misleading because it is based on an average scheme contribution rate. As the whole sorry saga of 1990s boardroom excess has illustrated, directors are anything but average.
Canny directors have learnt that a hefty pay rise in their last year before retirement makes a vast difference to their take from the pension scheme. The actuaries' normal rule of thumb here is that a pounds 1,000 pay rise in the final year leads to an extra pounds 10,000 of total pension income during the remaining lifetime of the executive and his or her spouse. The chief executive who gets a pounds 100,000 pay rise in the pre-retirement year - and there are such people - receives an extra pounds 1m in due course from the pension fund. This is paid for either out of pension contributions made on behalf of less elevated employees, or from additional company contributions. Either way, it is the shareholders who bankroll this largess. They at least have the right to know that it is happening.
It is not too late for them to fight a rearguard action. Business has something to learn from the Commons here. Against all the odds, MPs are after all to be forced to disclose their outside earnings. If Nolan can escape being nobbled, so can Greenbury.
Pricing the railways
SUDDENLY, railway privatisation is upon us. Last week the three rolling- stock companies formed out of the broken-up British Rail were sold. As we report on page 7, Railtrack is busy grooming itself for a flotation. Preferred bidders for the first three train-operating franchises will be announced in the next fortnight, I understand. And there is keen interest from the construction industry in the 13 infrastructure service companies.
Already, however, there is a suspicion that some rail assets are being sold off too cheaply. Opposition politicians have been quick to point out that the rolling stock, which was valued at pounds 3bn last year, only fetched pounds 1.8bn.
The truth is that it is impossible to value any part of the broken-up railway in isolation. Each of the 70 companies formed from the old British Rail is dependent on others. Each company's worth is determined not so much by its physical assets, but by the long-term contracts it has signed with other parts of the railway. Thus the price fetched by the rolling- stock companies is determined not by some arbitrary valuation placed on their locomotives and carriages, but by the fact that the train operators are committed to paying them leasing charges long into the future.
Underpinning the entire artificial market-place are the subsidies the Government has promised to pay the train operators. The privatisation proceeds, the ongoing subsidies and the colossal fees paid to the myriad advisers will all have to be taken into account in any calculation of whether taxpayers are getting value for money. The National Audit Office faces a Herculean project.
Welsh mountain
WELSH Water's fresh tilt at Swalec has got off to a poor start. At the best of times the idea of Wales's monopoly water supplier taking over Wales's monopoly electricity supplier would get a cool reception. Matters were made worse for Welsh when it was forced by the Takeover Panel to rush out a statement. It now looks caught with its pants down, not yet ready to back up its merger ambitions with hard arguments. Swalec's management have not unreasonably asked to talk: the earliest Welsh will now see them is Friday - nine days after its announcement.
The City is less than thrilled by Welsh's previous diversifications - into hotels, rubbish collection and international consultancy. And it has already had to persuade Welsh to sell off a previously accumulated stake in Swalec.
Then there is finance. Swalec shareholders would almost certainly demand cash. A bid at pounds 10.20 would cost pounds 960m. A bid capable of winning would cost well over pounds 1bn. Even after stripping out Swalec's pounds 200m National Grid stake, Welsh Water would still have to find pounds 800m. That would mean a rights issue.
Welsh management have a mountain to climb if they are to pull off a bid. But I would not write them off. They are determined to capture Swalec, and were persuaded of the merits of a combined power and water utility long before Sir Des Pitcher created the first such beast in the North West.
Marshall's dilemma
INCHCAPE has done well to persuade Sir Colin Marshall to come to the rescue as chairman. But there is a fly in the ointment, according to at least one of Inchcape's big shareholders.
The arrival of Marshall is all very well. He is highly regarded as one of the team who turned round British Airways.
But though the chairman has changed, the chief executive remains the same. Investors believe that Charles Mackay has to take some responsibility for Inchcape's dismal performance in recent years. "He's not out of the woods yet," one told me last week.
Marshall, of course, could always edge him out. He's a tough businessman perfectly capable of swinging the axe when he thinks it necessary. But here's the potential problem.
Marshall and Mackay are old business friends, indeed Marshall in 1993 invited Mackay on to the BA board (a post he is now quitting). They also both sit on the board of HSBC, the banking group. All very awkward for Marshall if he has to be brutal.
- 1 Finland schools: Subjects scrapped and replaced with 'topics' as country reforms its education system
- 2 The West has it totally wrong on Lee Kuan Yew
- 3 #FreeTheNipple: Women in Iceland bare breasts in solidarity with trolled student
- 4 Scientists have discovered a simple way to cook rice that dramatically cuts the calories
- 5 Zayn Malik quits One Direction: Hundreds of workers request compassionate leave following band member's exit
Germanwings captain Patrick Sondenheimer tried to break into locked cockpit door 'with an axe' as plane was descending
Amanda Knox murder conviction: Italian court overturns verdict for US student and Raffaele Sollecito in the killing of Meredith Kercher
Saudi Arabia says it won't rule out building nuclear weapons
The battle for the Middle East's future begins in Yemen as Saudi Arabia jumps into the abyss
#FreeTheNipple: Women in Iceland bare breasts in solidarity with trolled student
Nigel Farage brands LGBT activists 'filth' and 'scum' and accuses them of scaring away his children after they invade his local pub
Ukip supporters are 55 or older, white and socially conservative, finds British Social Attitudes Report
JK Rowling responds to fan tweeting she 'can't see' Dumbledore being gay
Russia threatens Denmark with nuclear weapons if it tries to join Nato defence shield
Jeremy Clarkson sacked live: Alan Yentob 'wouldn't rule out' ex Top Gear host's BBC return
Germanwings plane crash live: Co-pilot Andreas Lubitz wanted to 'do something people would remember him for'
Win a trip to Tobago for two
Discover Tobago with our competition for one lucky reader to have the chance to win a seven night self-catering holiday for two. Enter here.
How researchers are set to leave their marks on solving crime
The technology, which is now being trialled by West Yorkshire Police, could lead to the biggest change in forensic fingerprinting in its 100-year history.
Taking steps to tackle the housing crisis
Whatever the result of the forthcoming General Election, tackling Britain's housing crisis should be at the top of a new government's to-do list.
Making informed decisions when borrowing money
Borrowing money is not a decision which should be taken lightly. Read this guide to help get the right information.
Crowdfunding: The end-end solution for business success.
Calling all entrepreneurs! See how crowdfunding can provide you with a full-circle, end to end solution for your business.
Enter the latest Independent competitions
Win anything from gadgets to five-star holidays on our competitions and offers page.
Business videos from commercial thought leaders
Watch the best in the business world give their insights into the world of business.
Independent Dating
iJobs Money & Business
Recruitment Genius: Retirement Coordinator - Financial Services
Negotiable: Recruitment Genius: To provide a prompt, friendly and efficient se...
Recruitment Genius: Annuities / Pensions Administrator
Negotiable: Recruitment Genius: You will be the first point of contact for all...
Ashdown Group: HR, Payroll & Benefits Officer - Altrincham - up to £24,000.
£18000 - £24000 per annum + benefits: Ashdown Group: HR, Payroll & Benefits Of...
Ashdown Group: Learning and Development Programme Manager
£35000 - £38000 per annum + benefits : Ashdown Group: A highly successful, int...


text:
Audrina Michelle Boe
Audrina Michelle Boe was born on July 7, 2013, to Brittney and Jesse Boe of Fargo, N.D. Audrina Michelle weighed 7 lbs. 11 oz. and was 20 3/4" long. Grandparents are Randy and Karla Schmiesing of Chokio and Randy and Cathy Boe of Morris.
Are you a newspaper subscriber but you don't have a Digital Access account yet? You can activate your account here: Activate All Access.
You will need your subscription account number and phone number. Not sure if you have an account? Email us at email@example.com and we can help you.


text:
I've wanted a 458 SOCOM ever since they came out,but always had other things that came first.
I went to a gun show today looking to trade my Ruger Blackhawk Bisley 45 Colt for a 40 S&W carry pistol.That didn't happen,but I did finally decide to buy a RRA 458 SOCOM upper to go on the spare lower in the safe.
The lower is a DPMS with a RRA 2 stage NM trigger and parts kit,with a Hogue grip and an Ace skeleton stock.
I changed out the buffer spring,and then put on the upper.Nice tight fit between the two.I dug out an old scope mount,along with a spare scope-a Redfield Partner 3-12x44,and set them up.
Now I'm just waiting for the Starline brass,Lee dies,and bullets to show up.
Join FirearmsTalk.com Today - It's Free!
Are you a firearms enthusiast? Then we hope you will join the community. You will gain access to post, create threads, private message, upload images, join groups and more.
Firearms Talk is owned and operated by fellow firearms enthusiasts. We strive to offer a non-commercial community to learn and share information.
Join FirearmsTalk.com Today! - Click Here
That's a stout looking stick ya' got there.
Congrats Tx, she looks beautiful!
You will like that RRA .458 SOCOM upper. I have been pleased with mine. Wait till ya shoot her!
congrats!! looks like mine!! when i was looking at dies for the socom i think i remember mixed reviews about the lee dies. i use redding dies for the 458. i bought two sets since i fkd up the first set by not enough case lube and stuck a case in one so badly i broke a case extractor on it.
be sure you dont crimp and seat at the same time. this is super critical for the socom. crimping has to be a seperate function. i broke a few cases trying it.
if you need load data i got the files marty terweem sent me or you can get it from him. i havent found any data in a current manual yet.
"Gun control: The theory that a woman found dead in an alley, raped and strangled with her panty hose, is somehow morally superior to a woman explaining to police how her attacker got that fatal bullet wound." L. Neil Smith
The problem with being stupid is you cannot simply decide to stop doing dumb things...
"I crapped my pants to avoid the draft!!" -Ted Nugent
Thanks Jon,I was reading up on all of the different dies.I think everyone had some problems with getting things right when this caliber came out.
According to Lee,they have fixed their problems,and I've also read about over lubing the cases,no matter what brand of dies are used.
I'll try the Lee dies,and if I have problems I'll probably buy some of the C&H Tool dies.Those suckers are expensive-$128,but they are supposed to be the best out there.
I'm going to load some 300gr bullets to start with,and then I'll try some a little bigger.I know that a bunch of people shoot Barnes bullets,but you could'nt give me any of their stuff.I have never had any of their bullets shoot worth a damn.


text:
Publisher's Description+
Fieldrunners is a game developed by Subatomic Studios, exclusively for the iPhone and iPod Touch platform. Descend into the world of Fieldrunners, the most anticipated and visually stunning game in mobile Tower Defense history. Defend and control the field using a diverse selection of upgradeable towers in a limitless adventure, using a wide array of tactics and strategies against countless waves of unique land and air combatants.
Version 1.2.3 had made the extended and endless game play modes less difficult.
Note: The "Download Now" link directs you to the iTunes App Store, where you must continue the download process. You must have iTunes installed in order to open the link, and you must have an active iTunes account to download the application. This download may not be available in some countries."
What's new in this version:
Version 1.2.3 had made the extended and endless game play modes less difficult.


text:
Hey everyone, just posting again to let you all know that the game is coming along great and we launched a Kickstarter campaign for the game today. If you visit the page, you can learn a little more about me and my co-developer, and about the game. Please check it out and tell your friends if you think they'd be interested.Fiz: The Brewery Management Game Kickstarter.
If you're unfamiliar with Kickstarter, it's a way for project creators to raise funding from fans of the project. It gives donors (known as backers) the chance to take part in creating something, usually in exchange for rewards of some type. In our case, we have t shirts, drink coasters, and special in game items. If you're feeling really spendy, you can even design recipes or marketplaces to be included in the game for all to see. The best part about Kickstarter is that if the funding goal for a project is not met within its timeline, then no funding is received, which means you don't get charged anything. This is nice because you can "pledge" funding to a project and not have to worry about throwing your money away if the project creator doesn't get the minimum amount of funds they needed to go forward.
Great looking game so far, I am really interested to see how it goes. Yup, unh-unh, sure... If my wife knew there was something else beer related for me to spend my time on, I would be sleeping in the garage. Just because she at present does not have any hobbies, doesn't meam she gets to play kill joy with mine. I hope she doesn't read this
So close! Do it do it do it!http://www.kickstarter.com/projects/...nagement-game/