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|Jan 17, 1995
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|Sep 22, 1993
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|Aug 19, 1991
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|Byrant Benson
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|Arizona Board Of Regents
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NH2 -Ser-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-COOH,
The present invention was made in the course of work conducted under Research Grant 5 RO1 HD 19521, from the National Institute of Child Health and Human Development, Department of Health and Human Services, United States Public Health Service.
This application is a continuation of application Ser. No. 07/747,782 filed Aug. 19, 1991 now abandoned.
The present invention relates to neurotropic peptides which effect increased turnover and release of the catecholamine dopamine, thereby regulating the release of gonadotropins and prolactin by the pituitary gland in mammals.
Peptides are compounds which contain two or more amino acids in which the carboxyl group of one acid is linked in peptide bond to the amino group of the other acid. Conventional representations of peptides are constructed such that the amino group appears to the left and the carboxyl group to the right; the position of the amino acid residues, usually identified by trivial names, are numbered from left to right as indicated in the example of a tripeptide below where R=the ionizing group of a specific amino acid ##STR1##
The pituitary gland is attached by a stalk to the region in the base of the brain known as the hypothalamus. The anterior portion of the pituitary secretes a number of protein hormones that travel through the bloodstream to reach other organs of internal secretion. In particular, the reproductive hormones follicle stimulating hormone (FSH) and luteinizing hormone (LH), collectively called gonadotropins, as well as prolactin (PRL), are released by the pituitary gland and stimulate the production of gametes and steroid hormones by the gonads.
Pituitary gonadotropins and prolactin are necessary for reproduction, and their secretion requires prior synthesis and release of hypothalamic factors such as gonadotropin hormone releasing hormone (GnRH) and catecholamines. The catecholamine norepinephrine (NE) stimulates GnRH release from the hypothalamus; in contrast, a second catecholamine dopamine (DA) generally inhibits GnRH release consequently reducing gonadotropin, prolactin and gonadal steroid secretion. In mammals, hypothalamic control of reproduction is modulated by yet another part of the brain, the epiphysis cerebri or pineal gland, which plays primarily an inhibitory or antigonadotropic role in several species.
Since the first reports of abnormal reproductive development in humans with pineal gland tumors (Huebner, O., Tsch. Med. Wschr. 24:214-222, 1898; Kitay, J. I. and M. D. Altschule, The Pineal Gland, Cambridge:Harvard Press, 1954), questions have been posed about the chemical nature of pineal hormones and particularly about the nature of the hormonal mediators of pineal antigonadotropic function. It is clear from modern investigations that the pineal gland regulates reproduction in most mammals through modulation of the hypothalamic-pituitary-gonadal axis in response to a number of environmental factors.
The pineal hormonal factors mediating this function vary among species. For example, in the hamster and sheep, indolic compounds derived from serotonin may convey the pineal's hormonal message to the hypothalamus, whereas this is not the case in the more commonly employed laboratory rat. Early work in this area suggested that in the human, rat and mouse the pineal antigonadotropic factor is proteinaceous in character (for review see: Benson, B. and I. Ebels, In: The Pineal Gland, Ed. by R. J. Reiter, CRC Press, Boca Raton, pp.165-187, 1981); more recent investigations suggest that this antigonadotropin acts by stimulation of hypothalamic dopamine synthesis (Benson, B. et al., in: Role of Peptides and Proteins in Control of Reproduction, Ed. by S. M. McCann and D. S. Dhindsa, Elsevier Sci. Pub. Co., N. Holland, pp. 111-130, 1933; Benson, B. et al., in: Advances In Pineal Research, Vol. 4, Ed. by R. J. Reiter and A. Lukaszyk, Libbey & Co., Ltd., pp. 99-111, 1990).
For many years investigators have searched for potent antagonists of GnRH, the hypothalamic decapeptide that regulates pituitary secretion of gonadotropins (M. Karten and Je. E. Rivier, Endocrine Reviews 7:44-66, 1986). The high degree of interest in such antagonists is due to their usefulness in the fields of reproductive endocrinology, gynecology, contraception and oncology. In general most prior art GnRH antagonists are GnRH analogs in which the GnRH amino acid sequence has been modified by the deletion and/or replacement of key amino acids, particularly those at positions 2 or 6. See, for example, the United States patents listed below.
______________________________________U.S. PAT. NO. INVENTOR ISSUE DATE______________________________________3,933,782 Yardley 01/20/763,941,763 Sarantakis 03/02/764,253,997 Sarantakis 03/03/814,307,083 Rivier et al. 12/22/814,619,914 Vale et al. 10/28/864,800,191 Schally et al. 01/24/894,801,577 Nestor et al. 01/31/894,866,160 Coy et al. 09/12/89______________________________________
The desired GnRH analogs are those that bind to the GnRH receptor and block endogenous GnRH access to the receptor, thus rendering endogenous GnRH ineffective.
In contrast to the prior art GnRH antogonists, the pineal antigonadotropin of the present invention effects GnRH antagonism via a different mechanism, viz. by inhibition the release of GnRH from the hypothalamus. The distinction between the prior art and the present invention is illustrated in FIG. 1, which is a diagram of the hormonal control of reproduction. Because the inhibition is accomplished by increased turnover and release of catecholamines including the potent prolactin inhibiting factor dopamine, pituitary prolactin secretion is concomitantly reduced by the pineal antigonadotropin. The potential utility of the pineal antigonadotropic peptide of the present invention extends therefore beyond the uses in reproductive endocrinology mentioned above to other areas of neuroscience, including neuropathological states such as Parkinson's and Alzheimer' disease which may be caused by deficits in catecholaminergic neurotransmitters.
The present invention relates in one embodiment to a novel and highly potent deceptide which inhibits the release of prolactin and luteinizing hormone by effecting increased turnover and release of the potent catecholamine and prolactin inhibiting factor dopamine. The invention is also directed to various methods of use of the novel compound and to pharmaceutical compositions and methods of use therefor.
More specifically, the present invention relates to compounds of the formula:
NH2 -Ser-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-COOH
or a synthetic analogue or pharmaceutically acceptable salt thereof.
It is an object of the present invention to provide compounds which effect increased turnover and release of dopamine.
It is an object of the present invention to provide a peptide which inhibits the release of prolactin & luteinizing hormone.
It is another object of the present invention to provide a method of inhibiting the release of hormones necessary to the reproductive cycle, substantially free of the deleterious side effects commonly associated with steroid-based methods of reproductive inhibition.
It is another object of the present invention to provide a method of treating neuropathogical states caused by deficiencies in catecholaminergic neurotransmitters.
These and other objects of the present invention will become more apparent from the following detailed description.
FIG. 1 is a simplified diagram of the hormonal control of reproduction.
For convenience in describing the present invention, the conventional abbreviations for the nomenclature used to define peptides as specified by the IUPAC-IUB Commission on Biochemical Nomenclature (European J. Biochem. 138:9-37, 1984) will be used. In accordance with conventional representation, the amino groups at the N-terminus appears to the left and the carboxyl group at the C-terminus to the right. The abbreviations for the amino acid residues found in the pineal antigonadotropin are based on the trivial names of the amino acids and are: Lys, lysine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; and Tyr, tyrosine. The systemic names are given below. All amino acids discussed are of the L-series unless stated otherwise.
Other abbreviations used are:
ACN=acetonitrile; a common solvent in peptide HPLC
C=carbon atom
CO2 =carbon dioxide
COOH=a monovalent radicle called a carboxyl group
Cys=cysteic acid
FSH=follicle stimulating hormone; a glycoprotein hormone secreted by the pituitary which stimulates follicle and ovum development, estrogen production in females and spermatogenesis in males.
GnRH=a gonadotropin releasing decapeptide produced in the hypothalamus which stimulates pituitary release of LH and FSH; synonyms used are LHRH, LRF and LRH
HPLC=high performance liquid chromatography
LH=luteinizing hormone; a pituitary glycoprotein which stimulates ovulation, ovarian luteinization and progesterone production in females, and testosterone production and spermatogenesis in males.
Lys=lysine; 2-6-diaminohexanoic acid
M=mole or molar
min=minute or minutes
Mr =molecular weight
NH2 =monovalent amino group
NH2 C2 H3 O2 =ammonium acetate, used for ion pairing in HPLC
PAG=pineal antigonadotropin or antigonadtropic decapeptide isolated from pineal glands
Phe=phenylalanine; 2-amino-3-phenylpropanoic acid
Pro=proline; pyrrolidine-2-carboxylic acid
PRL=prolactin; a glycoprotein produced by the pituitary gland that stimulates breast development and lactation, and augments gonadal steroid secretion.
Ser=serine; 2-amino-3-hydroxypropanoic acid
TFA=trifluoroacetic acid; a common HPLC solvent
Thr=threonine; 2-amino-3-hydroxybutanoic acid
Tyr=tyrosine; 2-amino-3-(4-hydroxyphenyl)propanoic acid
v/v=volume per volume
As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like: and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; (b) base addition salts formed with polyvalent metals cations such as zinc, calcium, bismuth, barium magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an organic cation formed from N,N' -dibenzylethylene-diamine or ethylenediamine; or (c) combinations, of (a) and (b), e.g., a zinc tannate salt and the like.
In one embodiment, the present invention is a substantially pure decapeptide having the following amino acid sequence: NH2 -Ser-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-COOH, and synthetic analogues and nontoxic salts thereof.
The non-indolic antigonadotropic peptide of the present invention was found in human, bovine, ovine and rat pineal glands. For partial purification of the antigonadotropin on a large scale, fresh bovine or ovine pineal glands were collected fresh at the abattoir, frozen on solid CO2 and maintained between -60� and -80� C. before extraction. The extraction and partial purification of the pineal antigonaodtropin followed standard methods for peptide isolation: Benson, et al., Int. J. Peptide Protein Res. 36:109-121,1990. Purification and structure elucidation steps follow:
1. 150 g batches of frozen glands were freeze-dried and the lyophilizate pulverized and ground to a fine powder in a mechanical blender. The powdered glands were defatted by stirring in 500 mL of acetone at room temperature for 30 min. The acetone was removed by vacuum filtration through Whatman #1 paper with recovery of the residue. This procedure was repeated twice with acetone and twice again with methylene chloride. The defatted residue was thoroughly desiccated in vacuo.
2. The dried, defatted residue was homogenized with a Tekmar Tissumizer at 1.0� C. in 250 mL of 0.2N acetic acid containing 10-6 M Pepstatin A. Homogenates were centrifuged at 10,000�G at 4� C. for 20 min and the pellets rehomogenized three additional times with the same quantities of 0.2N acetic acid/Pepstatin A and centrifuged as before. The supernatant fractions were combined, stirred overnight at 4� C. and centrifuged at 10,000�G for 20 min. After vacuum filtration through Whatman #41 paper fitted in a Buchner Flask, the supernatant fractions served as starting material for subsequent ultrafiltration.
The filtered extracts were serially ultrafiltered in 0.2N acetic acid through Amicon membranes XM300 and YM30 (Mr cutoffs at 300,000 and 30,000, respectively) at 4� C. under low N2 pressure (<20 psi). YM30 filtrates containing the peptides of interest (Mr <30,000) were divided into two portions, concentrated by lyophilization and stored at -80� C. before further fractionation by gel filtration chromatography on Sephadex G-25.
1. Concentrated ultrafiltration YM30 filtrates, representing the residues from 75 g wet weight equivalents of pineal glands, were chromatographed at 4� on 5�95 cm Sephadex G-25 (fine) columns equilibrated and eluted with 0.2N acetic acid.
2. Fractions were collected over a ten min period per tube at a flow rate of 20 drops/min. Column eluents were monitored at 280 nm, and fractions pooled according to observed peaks. Molecular size markers included Dextran Blue (Mr 2�103), ribonuclease (Mr 14�103) and Bacitracin (M2 1.4�103).
3. Under the conditions described above the pineal antigonadotropic peptide was located by association with two peaks of 280 nm absorbance: one with a retention time between 1.6 and 1.8�103 min (in association with proteins of approximate 10,000-15,000 Mr) and a second peak that had a retention time between 2.6 and 2.8�102 min (in mixture with other compounds of Mr between 500 and 1500). The individual fractions collected in association with these two absorbance peaks were pooled and concentrated by lyophilization.
The lyophilized, pooled fractions from Sephadex chromatography containing the antigonadotropic peptide were taken up in 5% formic acid and further fractionated by serial ultrafiltration at 4� through Amicon membranes YM10, YM2 and UM05. In this manner peptides bound to larger proteins were dissociated, passed through the YM10 and YM2 membranes and localized to the UM05 retentate in mixture with compounds of Mr >500. In addition to further purification, this step effectively removed salts and other low Mr materials. The UM05 residue containing the antigonadotropin was lyophilized and stored at -80� C.
A chromatographic system consisting of a Spectra-Physics model SP8800-010 ternary gradient pump with semipreparative heads, a Rheodyn model 7125, 1.0 mL volume loop injector and a model SP4290-010 computing integrator was used with a Waters model 450 variable wavelength UV detector. Peptides in column eluents were detected at 210 nm; fractions were collected with a Buchler Fractomette model 400 fraction collector.
Lyophilized UM05 retentates from Sephadex G-15 fractions containing the antigonadotropin were further fractionated by sequential semipreparative HPLC. Several chromatographic procedures utilized ternary gradients with the aqueous buffer coded "A", the organic solvent coded "B" and a mixture of each as mobile phase "C". Each mobile phase was degassed in vacuo while stirred for ten minutes and sparged with helium gas.
Initial HPLC of quantities of UM05 retentates representing the purified residue from 65 g wet weight of starting pineal glands was performed on a 1.0�25.0 cm, 300 Angstrom semipreparative C-8 column (Rainin Dynamax) at flow rates of 8.0 ml/min. Residues were dissolved in mobile phase, filtered through a 0.45 micron filter and serially injected in volumes of 0.5-0.8 ml. Column eluates were routinely monitored at 210 nm and fractions were collected. Solvent A: 0.01M trifluoroacetic acid (TFA); solvent B: 0.01M TFA in acetonitrile (ACN):H2 (50:50, v/v). Binary gradients were employed in the following configuration:
______________________________________Time (min): 0 20 40 45 46 50______________________________________% Sol. A: 88 75 50 0 0 88% Sol. B: 12 25 50 100 100 12______________________________________
Under these conditions significant amounts of the antigonadotropin of interest were eluted with a retention between 25 and 35 min. A synthetic oxytocin standard may be applied to the column as a marker because it should also be eluted within this same retention time. The fractions collected between 25 and 35 min were pooled from separate chromatographic runs, concentrated by rotary evaporation, lyophilized and stored at -80� C. until further purification.
Secondary HPLC of the residue containing the antigonadotropin from the first HPLC described above was carried out on a 60 Angstrom, semipreparative C-8 column (Rainin Dynamax). Amounts representing 425 g wet weight starting pineal gland equivalents were injected in 0.9 ml volumes. Solvent A: 0.01 TFA in ACN; solvent B: 0.01M TFA: solvent C: 0.01M TFA in H2 O:ACN (1:1, v/v). HPLC was performed with these ternary gradients at a flow rate of 2.5 ml/min and mobile phase components changing as indicated:
______________________________________Time (min): 0 33 38 40 45 50 55 58______________________________________% Sol. A: 0 0 0 0 0 0 15 40% Sol. B: 80 45 45 43 30 10 0 0% Sol. C: 20 55 55 57 70 90 85 60______________________________________
Under these conditions the antigonadotropin was eluted with a retention time between 32 and 35 minutes, again in the volume containing an oxytocin standard. The fractions collected within this retention time were pooled, lyophilized and saved for further purification by HPLC.
Antigonadotropic peptides in the lyophilized residues from the secondary HPLC were further separated on the 60 Angstrom C-8 column by ternary gradient HPLC with mobile phases which included methanol and ammonium acetate as ion-pairing reagent. Solvent A: ACN:methanol, 2:1 (v/v) in 0.01M TFA; solvent B: 15% solvent A and 85% 30 mM NH4 C2 H3 O2 in 0.01M TFA; solvent C: 50 solvent A and 50% 30 mM NH4 C2 H3 O2 in 0.01M TFA. This chromatography was performed at a flow rate of 2.3 ml/min with mobile phase composition programmed to change as follows:
______________________________________Time (min): 0 30 40 45 50 53 60______________________________________% Sol. A: 0 0 0 0 30 30 0% Sol. B: 88 70 50 40 0 0 88% Sol. C: 12 30 50 60 70 70 12______________________________________
Under these conditions the antigonadotropin was eluted between 42 and 52 min; the fractions collected within this range of retention times were pooled, lyophilized and subjected to additional chromatography.
The lyophilized residue from the tertiary chromatography described above were re-chromatographed on the 60 Angstrom C-8 semipreparative column with the same solvents as described for HPLC 3. Ternary gradient mobile composition was programmed to change as follows:
______________________________________Time (min): 0 10 25 28 40 45______________________________________% Sol. A: 0 0 10 30 30 0% Sol. B: 50 50 20 0 0 50% Sol. C: 50 50 70 70 70 50______________________________________
Under these conditions the pineal antigonadotropic peptide was eluted in a large 210 nm absorbance peak with a retention time of 32.29 min. The fractions in the range of this retention time (between 28 and 34 min) were pooled, lyophilized and stored at -80� C. until final chromatography.
Final isolation of the antigonadotropin was accomplished by HPLC on the 60 Angstrom C-8 column with changes in the composition of the mobile phase. Solvent A: 0.04M TFA in methanol; solvent B: 15% 0.04M TFA and 85% 0.04M TFA in methanol/ACN (4:1, v/v); solvent C: 50% 0.04M TFA and 50% 0.04M TFA in methanol/ACN (4:1, v/v). The column was eluted at a flow rate of 2.0 ml/min with the ternary gradient mobile phase changed as follows:
______________________________________Time (min): 0 35 40 45______________________________________% Sol. A: 0 20 20 0% Sol. B: 50 0 0 50% Sol. C: 50 80 80 50______________________________________
Under these conditions the pineal antigonadotropic peptide was eluted in a single 210 nm absorbance peak with a retention time of 36.8 min. By comparison, synthetic or natural pineal gland oxytocin was eluted in this chromatography with retention time of 34.3 min.
Amino acids were quantitated by routine automated methods. A Beckman model 6300 autoanalyzer was utilized. Samples were hydrolyzed at 110� C. in 5.7N HCl for 24 h. The amino acids Cys and Met were determined by performic acid oxidation of the sample prior to hydrolysis with quantitation as cysteic acid and methionine sulfone. Amino guanidino propionic acid was incorporated as an internal standard. The major amino acids detected in hydroiysates of the residue obtained from the final HPLC step described above included: Thr, Phe, Pro, Tyr, Lys and Ser, ranked in order from the largest (Thr) to the smallest (Ser) amounts recovered (Benson, et al., In: Advances in Pineal Research, Vol. 4, Eds. R. J. Reiter & A. Lukaszyk, Libbey & Co. Ltd., pp. 99-111, 1990).
Primary sequence was determined on small quantities (<2 nM) of isolated peptide derived from the final HPLC purification step. Microsequence analysis was performed by automated methods well know to those skilled in the art. A pulsed liquid phase protein/peptide sequencer, Applied Biosystems model 477A was employed. The following primary sequence of the pineal antigonadotropin was revealed after twelve cycles:
NH2 -Ser-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-COOH
Single symbols: S F P T T K T Y F P
After its identification, the straight chain decapeptide of the present invention was readily synthesized by automated solid phase methods well known to those who are skilled in the art, which methods are set forth in the textbooks "Principles of Peptide Synthesis", Springer-Verlag, 1984; "Solid Phase Peptide Synthesis", J. M. Stewart and J. D. Young, Pierce Chemical Company, Rockford, Ill., 1984 (2nd ed.); G. Barany and R. B. Merrifield, "The Peptides", Ch 1 pp 1-285, Academic Press.
In general, these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amid linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final polypeptide.
Similarly, it is within the skill of the art to use the above-described automated solid phase peptide synthesis to synthesize a peptide having a sequences sufficiently homologous to the sequence: NH2 -Ser-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-COOH, to yield a peptide having a correspondingly similar effectiveness to that of the peptide having the above-described seqence.
Now that the peptide sequence NH2 -Ser-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-COOH has been identified, it is within the skill of the art to identify and isolate the gene coding for that peptide, and prepare the peptide by means of recombinant DNA technology as an alternative to isolating it from the pineal gland or preparing it by means of automated solid phase peptide synthesis.
In order to prepare a peptide having the sequence NH2 -Ser-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-COOH by means of recombinant DNA technology, the gene for the peptide must first be isolated from a cell's total DNA by screening a library of that cell's DNA. The DNA library is screened by use of a probe, a synthetic radiolabelled nucleic acid sequence which can be used to detect and isolate complementary base sequences by hybridization. Knowing the amino acid of the peptide, a probe for the gene can be designed based on the fact that each amino acid of the peptide is specified by a specific codon, or sequential grouping of three nucleotides. The possible nucleotides are adenine (A), guanine (G), cytosine (C) , and thymine (T). The codons which are specific for each of the twenty amino acids found in proteins or peptides are listed below, together with the codons which signal termination of protein sythesis; although there is more than one codon for certain amino acids, no codon specifies more than one amino acid.
______________________________________AMINO ACID CODONS______________________________________Alanine GCA, GCC, GCG, GCUArginine AGA, AGG, CGA, CGC, CGG, CGUAsparagine AAC, AAUAspartic Acid GAC, GAUCysteine UGC, UGUGlutamic Acid GAA, GAGGlutamine CAA, CAGGlycine GGA, GGC, GGG, GGUHistidine CAC, CAUIsoleucine AUA, AUC, AUULeucine CUA, CUC, CUG, CUU, UUA, UUGLysine AAA, AAGMethionine AUGPhenylalanine UUC, UUUProline CCA, CCC, CCG, CCUSerine UCA, UCC, UCG, UCU, AGC, AGUThreonine ACA, ACC, ACG, ACUTryptophan UGGTyrosine UAC, UAUValine GUA, GUC, GUG, GUUTermination Codons: UAA, UAG, UGA______________________________________
Because some amino acids have several possible codons and it is not initially known which of the possible codons will actually code for an amino acid, a set of probes can be designed that covers all possible codons for each amino acid comprising the peptide; such a set of probes is known as a "fully degenerate" set of probes.
A library to be screened can be a genomic library (gDNA), which contains a set of all the DNA sequences found in an organism's cells, or a complementary DNA (cDNA) library, which is much smaller and less complex than a gDNA library, as is used frequently when the tissue source for a given gene is known. Because the peptide was isolated from the pineal gland, the tissue source is known, and therefore it would be much more efficient to screen the cDNA library for the pineal gland rather than screening the genomic library.
When the gene for the peptide is identified and isolated from the cDNA library, it is cloned by inserting or splicing it into a plasmid or vector. The recombinant plasmid or vector with the inserted gene--the cloned gene--is then introduced, or "transfected", into a host cell. The host cell at that point is "transformed" or "transvected". The vector or plasmid is the carrier which brings the DNA or gene of interest into the host cell, and allows the DNA to grow and replicate as the host cell grows and replicates, eventually causing the host cell to express or produce the peptide.
Peptide effectiveness, whether isolated from the pineal gland or produced by means of automated solid phase peptide synthesis or recombinant DNA technology, can be evaluated by means of the in vivo and in vitro procedures described as follows.
The neurotropic pineal antigonadotropin of the present invention was assayed in vivo, as is described below. Natural and synthetic analogs were injected into male and female rats and mice in graded doses. Antigonadotropic effects were assessed after a period of chronic daily subcutaneous injection in a variety of solvents/carriers, e.g. physiological saline or corn oil. After a period of two to four weeks the antigonadotropic effect was manifested as reductions of pituitary and blood levels of gonadotropins and prolactin, coupled with significant reductions in pituitary, gonadal and accessory reproductive organ weights, i.e. weights of the testes, prostate gland and seminal vesicles in males and ovaries and uterus in females. Additionally, blood levels of the gonadal steroids (testosterone in males and estrogens in females) were reduced.
In male mice such changes in reproductive hormones and organ weights are generally associated with significant increases in anterior pituitary contents of dopamine (Benson, B. and I. Ebels, In: The Pineal Gland, Vol. II, Ed. by R. J. Reiter, CRC Press Inc., Boca Raton, pp. 165-187, 1981; Benson, B. et al., In: Role of Peptides and Proteins in Reproduction, Ed. by S. M. McCann and D. S. Dhindsa, Elsevier Sci. Pub. Co., Inc., pp. 111-130, 1983).
Acute effects of subcutaneous injections of the pineal antigonadotropin (PAG) were also readily demonstrated. Provided in the table below is an example in which a single injection of 25 nanograms in mice produced a significant increase in anterior pituitary dopamine content:
______________________________________Postinjection Time (min)Treatment 0' 15' 30'______________________________________Saline 0.545 � 0.039 0.551 � 0.043 0.552 � 0.049PAG 0.539 � 0.046 *0.795 � 0.064 *1.801 � 0.126______________________________________ Values are nanograms per mg of pituitary. PAG = pineal antigonadtropin. * = p < 0.05 vs. control; ** = p <0.01
Intravenous or intracerebroventricular injections of the natural or synthetic pineal antigonadotropin readily produced demonstrable inhibitory effects on pituitary secretion of LH and PRL. Intracerebroventricular infusions inhibited release of pituitary LH, reducing both the amplitude and frequency of pulsatile LH release into the blood.
For intravenous peptide injections anesthetized rats were fitted surgically with indwelling intra-arterial cannulae 24 hours before the tests. The next day samples of arterial blood were withdrawn from the unanesthetized rats immediately before and after the injection of graded doses of the pineal antigonadotropin. Prolactin and LH were measured in the blood samples by standard double antibody radioimmunoassay with kits provided by the National Hormone and Pituitary Program. The acute effects of 5.0 micrograms of the antigonadotropin are illustrated in the table below:
______________________________________Postiniection Time (min)Treatment 0' 15' 30' 45' 60'______________________________________Saline 0.26 0.25 0.28 0.25 0.23PAG (1) 0.25 0.21 0.15 0.10 0.14PAG (2) 0.26 0.20 0.15 0.12 0.11______________________________________
______________________________________Postinjection Time (min)Treatment 0' 15' 30' 45' 60'______________________________________PAG (1) 100 � 11 55 � 6 38 � 6 31 � 4 22 � 5PAG (2) 100 � 8 44 � 6 30 � 3 25 � 3 32 � 6______________________________________ PAG (1) = natural peptide; PAG (2) = synthetic peptide;
Values for LH are expressed as the National Institutes of Health reference standard LH-RP-2. Means�S.E.M. are indicated (n=5)
The pineal antigonadotropic decapeptide stimulates the release of dopamine from the rat hypothalamus in vitro. Rat hypothalamic median eminence strips were rapidly dissected and placed in culture plates containing 100 μl of a Kreb's solution saturated with O2 in accord with the procedure described in Racke, K. and E. Muscholl, J. of Neurochemistry 46:745, 1986. The median eminence strips were maintained at 37� C. under 95% O2 /5%CO2 in a Dubnoff shaking incubator. The media were replaced after a ten minute pre-incubation period. 50 μl samples were taken at 30, 60 and 90 min of incubation for catecholamine determination, with replacement of the removed media. 20 μl of 0.1M perchloric acid were added to each sample before storage at -20�. Media contents of dopamine, norepinephrine and various indoleamine and catecholamine metabolites were determined by HPLC coupled with electrochemical detection in accord with the procedure described in Gregory, V. M., B. Larsen and B. Benson, J. of Chromatography 345:140-144, 1985. As shown in the following table the addition of the synthetic decapeptide effectively stimulates the release of dopamine into the incubation medium:
______________________________________Incubation Time (min)Treatment 30' 60' 90'______________________________________Control 15.8 � 1.7 42.9 � 3.6 75.5 � 9.2PAG (10-7 M) 27.2 � 2.1 61.5 � 5.3 109.3 � 10.8PAG (10-5 M) 35.2 � 7.6 100.2 � 13 182.9 � 13.7______________________________________ Means � S.E. are shown for five samples. *Values represent accumulative pg/μl.
The natural and synthetic compounds of this invention produce antigonadotropic effects mediated by increased release of the catecholaminergic hypothalamic neurotransmitter dopamine. A number of uses are therefore embodied in and flow therefrom. These relate to the fields of reproductive endocrinology, gynecology, oncology, contraception, animal husbandry, veterinary science, neurology, and internal medicine.
The decapeptides of this invention, or appropriate nontoxic salts or effective synthetic analogs thereof, can be administered to female humans for treatment of precocious puberty; contraception; ovulation prevention or delay; synchronization of ovulation; treatment of ovarian and mammary cystic disease; treatment of premenstruai syndrome; the regulation of elevated gonadotropins during menopause; treatment of gonadotropin-, prolactin- and/or steroid-dependent breast tumors; treatment of endometriosis; uterine leiomyoma; uterine carcinoma; hyperprolactinemia associated dysmenorrhea or amenorrhea; pituitary lactotroph microadenoma; and during early first trimester as an abortifacient.
In male humans the decapeptides can be used as therapy for benign prostatic hypertrophy; treatment of prostatic carcinoma; contraception; therapy for diseases which result from excessive gonadal hormone production, e.g. precocious puberty; and hyperprolactinemia associated gynecomastia and impotency.
The decapeptide of the present invention has a number of uses in animals, for example, use for ovulation prevention or delay; pregnancy termination in domestic animals or pets; heat or estrus suppression; induction of luteolysis; functional castration in male food producing animals; treatment of endometriosis; and suppression of proestrous bloody discharge in dogs. The decapeptide can also be used as a drug in common laboratory rodents, rabbits, guinea pigs, dogs, cats, pigs, primates and large farm animals in experiments where reduced gonadotropins and prolactin are experimentally desirable.
The decapeptide of this invention can be used as a neurotropic therapeutical agent in the treatment of Parkinson's or Alzheimer diseases in humans in which catecholamine release is deficient.
Other uses in humans include treatment of sleep-wake disorders; treatment of affective and psychotic disorders associated with catecholamine deficiency; and as a therapeutical agent in the treatment of schizophrenia.
The decapeptide of this invention can be used as an antihypertensive agent since it lowers blood pressure. It can be used to reduce renal glomerular filtration rate and to control sodium excretion through intra- or extrarenal regulators, e.g. adrenal release of aldosterone or pituitary release of antidiuretic hormone.
In the practice of methods to use the compounds of the present invention, an effective amount of the peptide, synthetic analogue, pharmaceutically acceptable nontoxic salt thereof, or pharmaceutical composition containing same is administered to the subject in need of, or desiring, such treatment. These compounds or compositions may be administered by any of a variety of routes depending upon the specific end use, including orally, intravenously, parenterally (including subcutaneous, intramuscular and intravenous administration), transdermally, nasally, or by suppository.
The most suitable route in any given case will depend upon the use, particular active ingredient, the subject involved, and the judgment of the medical practitioner. The compound or composition may also be administered by means of controlled-release, depot implant or injectable formulations as described more fully here below.
In general for the uses herein above described, it is expected that the active ingredient will be administered in amounts between about 0.001 and 5 mg/kg body weight. Preferably, for human therapy, it is expected that the active ingredient will be administered in the range of from about 0.01 to about 1 mg/kg/day. This administration may be accomplished by a single administration, by distribution over several applications or by slow release in order to achieve the most effective results.
The exact dose and regimen for administration of these compounds and compositions will necessarily be dependent upon the needs of the individual subject being treated, the type of treatment, the degree of affliction or need and, of course, the judgment of the medical practitioner. In general, parental administration requires lower dosage than other methods of administration which are more dependent upon absorption.
A further aspect of the present invention relates to pharmaceutical compositions containing as active ingredients a compound of the present invention which compositions comprise such compound in admixture with a pharmaceutically acceptable, nontoxic carrier. As mentioned above, such compositions may be prepared for use for parental (subcutaneous, intramuscular and intravenous) administration, particularly in the form of liquid solutions or suspensions, for suppositories, for oral administration particularly in the form of tablets or capsules,or intranasally particularly in the form of powders, nasal drops or aerosols.
Further details of the preparation of pharmaceutical compositions and formulations, preparation of pharmaceutically acceptable nontoxic salts, and the synthesis of peptides and peptide analogues can be found in U.S. Pat. No. 4,801,577, issued Jan. 31, 1989 to Nestor, Jr., et al., which is hereby incorporated by reference herein. It is expected that the compounds of the present invention can be formulated into pharmaceutical compositions, synthesized, and converted into salts in a manner similar to the peptides disclosed in the patent to Nestor, Jr., et al.
Although the invention has been described with regard to its preferred embodiments, it should be understood that changes and modifications as would be obvious to one having the ordinary skill in this art without departing from the scope of the invention which is set forth in the claims which are appended hereto.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 1(2) INFORMATION FOR SEQ ID NO: 1 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acid residues.(B) TYPE: Amino acid(C) STRANDEDNESS: Unknown(D) TOPOLOGY: Unknown(ii) MOLECULE TYPE: Peptide(iii) HYPOTHETICAL: No(v) FRAGMENT TYPE: N-terminal and internal fragments.(vi) ORIGINAL SOURCE:(A) ORGANISM: Bovine(F) TISSUE TYPE: Pineal gland(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :SerPheProThrThrLysThrTyrPhePro1510
|Cited Patent
|Filing date
|Publication date
|Nov 14, 1974
|Jan 20, 1976
|American Home Products Corporation
|(N-Acetyl)-Pro-D-Phe-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt and intermediates
|Mar 28, 1975
|Mar 2, 1976
|American Home Products Corporation
|PGlu-D-Met-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-Gly-NH2 and intermediates
|Dec 17, 1979
|Mar 3, 1981
|American Home Products Corporation
|Anti-ovulatory decapeptides
|May 8, 1980
|Dec 22, 1981
|The Salk Institute For Biological Studies
|LRF Antagonists
|Feb 23, 1984
|Oct 28, 1986
|The Salk Institute For Biological Studies
|GNRH antagonists IIIB
|Aug 15, 1984
|May 19, 1987
|Asahi Kasei Kogyo Kabushiki Kaisha
|Method for producing an active protein
|Jul 17, 1987
|Jan 24, 1989
|Schally Andrew Victor
|LHRH antagonists
|Feb 5, 1987
|Jan 31, 1989
|Syntex (U.S.A.) Inc.
|Nonapeptide and decapeptide analogs of LHRH useful as LHRH antagonists
|Atassi, M. Z. et al., Proc. Natl. Acad. Sci., 86:67 29 6733, Sep. 1989.
|Atassi, M. Z. et al., Proc. Natl. Acad. Sci., 86:67 29-6733, Sep. 1989.
|Beason, et al., Advances in Pineal Research, 4 (Eds. Reiter & Lukaszyk), pp. 99 111, 1990.
|Beason, et al., Advances in Pineal Research, 4 (Eds. Reiter & Lukaszyk), pp. 99-111, 1990.
|Benson, B. et al., Int. J. Peptide Protein Res., 36: 109 121, 1990.
|Benson, B. et al., Int. J. Peptide Protein Res., 36: 109-121, 1990.
|Benson, B., et al., Amer. Zool., 16:17 24, 1976.
|Benson, B., et al., Amer. Zool., 16:17-24, 1976.
|Benson, B., et al., Pineal Gland, 2:165 187, 1981 (Ed Reiter).
|Benson, B., et al., Pineal Gland, 2:165-187, 1981 (Ed Reiter).
|Benson, B., et al., Society for Neuoscience Abstracts, 17:347, 1991, 21st Ann. Mt. of Soc. for Neursci; Nov. 10 15, 1991.
|Benson, B., et al., Society for Neuoscience Abstracts, 17:347, 1991, 21st Ann. Mt. of Soc. for Neursci; Nov. 10-15, 1991.
|Benson, B., Society for Neuroscience Abstracts, 15:1277, 1989, from the 19th Ann. Mtg. of the Soc. for Neurasci, Oct. 29 Nov. 3, 1989.
|Benson, B., Society for Neuroscience Abstracts, 15:1277, 1989, from the 19th Ann. Mtg. of the Soc. for Neurasci, Oct. 29-Nov. 3, 1989.
|Kazim, L. et al., Biochem J., 203; 201 208, 1982.
|Kazim, L. et al., Biochem J., 203; 201-208, 1982.
|Kazim, L. et al., Biochem. J., 191: 261 264, 1980.
|Kazim, L. et al., Biochem. J., 191: 261-264, 1980.
|Remington s Pharmaceutical Sciences, (Ed. Gennaro et al.), pp. 1688 1691, 1990.
|Remington's Pharmaceutical Sciences, (Ed. Gennaro et al.), pp. 1688-1691, 1990.
|Citing Patent
|Filing date
|Publication date
|Jul 21, 2006
|Nov 19, 2013
|Cms Peptides Patent Holding Company Limited
|Biologically active peptides and their new uses
|Mar 7, 2012
|Nov 19, 2013
|Wai Ming Wong
|Biologically active peptides and their new uses
|Feb 21, 2013
|Feb 25, 2014
|Cms Peptides Patent Holding Company Limited
|Biologically active peptides and their new uses
|Feb 21, 2013
|Apr 22, 2014
|Cms Peptides Patent Holding Company Limited
|Biologically active peptides and their new uses
|Jul 21, 2006
|Sep 4, 2008
|Kong Lam
|Novel Biologically Active Peptides and Their New Uses
|Jul 21, 2006
|Apr 16, 2009
|Cms Peptides Patent Holding Co
|Novel biologically active peptides and their new uses
|U.S. Classification
|514/9.9, 530/378, 514/11.5, 514/21.6, 514/10.1
|International Classification
|Cooperative Classification
|European Classification
|Aug 11, 1998
|Maintenance fee reminder mailed
|Jan 17, 1999
|Lapse for failure to pay maintenance fees
|Mar 30, 1999
|Expired due to failure to pay maintenance fee
Effective date: 19990117


text:
I am running browser IE 8 under Vista. The Spiceworks scan tells me that this browser is not supported.
Any solution ?
4 Replies
Feb 2, 2010 at 3:50 UTC
akp982 is an IT service provider.
- Do you have cookies enabled?
Feb 2, 2010 at 3:52 UTC
Keep IT Simple Technology Group is an IT service provider.
I have the same issue on win7 with ie8 but everything is working fine.
Do you experience any problems or just that error message?
Feb 2, 2010 at 4:05 UTC
cookies and check the enhanced browsing security thingy...... I think that was an issue on the box that i installed SW on as well....
have never had any issues with my FireFox / IceWeasel / chrome / opera browsers........ just saying....
Feb 2, 2010 at 11:53 UTC
running same config without any issues. check the new security settings one by one until you find the culprit adn then determine if not having it enables poses a greater security risk than you are willing to be exposed to. If not , leave it disabled.

subset: commoncrawl


text:
You don't recall what made you think smashing a window with your head was a good idea. It really wasn't.
Countless shards of glass now decorate your face and blood streams freely. The screams stop and the child looks as if he's seen a ghost, or Rosie O'Donnell.
You climb out the window and approach the child with arms wide open. You try to ask the child what is wrong but your attempts to vocalize anything at all are futile. Looks like an errant shard of glass has severed your vocal chord. Nice job, Genius!
The child screams like a banshee and quickly runs in the opposite direction.
"Monster! Monster! Help me!!" the boy cries as he runs; fearful for his life.


text:
Crawler Supply Co Inc.
PO BOX 52729 Baton Rouge, LA 70892-2729
(888) 772-9505
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'Islamic takeover plot' at six Birmingham schools
"Concerted efforts" have been made to run six Birmingham schools in line with strict Islamic principles, headteachers have warned.
"Concerted efforts" have been made to run six Birmingham schools in line with strict Islamic principles, headteachers have warned.
A former staff member at Park View Academy has spoken out over an alleged 'Islamic takeover' at the Birmingham school, which they claim was led by senior members of the leadership team.
Among a range of complaints about the treatment of staff and pupils, the anonymous whistleblower told ITV News Midlands Correspondent Rupert Everlyn they had been called "slanderous" and "vindictive" after complaining of a lack of equality at the school.
"There is not equality for the girls and there is not equality for the female staff. That is not just non-Muslim staff, it is all the female staff," they said.
The teacher also claimed that when they raised alleged homophobia in the school that "wasn't being addressed", they were told by a senior member of staff: "It's what we believe".
Park View Academy is one of 21 schools currently under investigation by Ofsted over the alleged Operation Trojan Horse plot to seize control of governing bodies.
Kieran Trippier has received his first call-up to the England squad after impressive form for Spurs.
The prime minister said the public should remain vigilant following a meeting of the Government's emergency committee Cobra.
The Queen said the Manchester Arena suicide bombing was "very wicked" she visited injured victims at the city's children's hospital.


text:
The UK's second-biggest mortgage lender has doubled the deposit for an interest-only loan.
Spanish-owned Santander has increased the deposit an interest-only borrower must find from 25 per cent of the value of a property to 50 per cent.
The move effectively sounds the death-knell for interest-only mortgages which, just five years ago, accounted for 20 per cent of all first-time buyer deals. Last year, the proportion fell to just 3 per cent as the City regulator – the Financial Services Authority – warned of the risks attached and lenders cut back on granting the loans.
David Hollingworth, associate director at London & Country Mortgages, said: "Such a radical shift by a major lender will be seen by many as another nail in the coffin of interest only."
The move will encourage rival lenders to follow, experts said, which could hit hopes of a housing market recovery.
Mark Harris, chief executive of broker SPF Private Clients, said: "Santander's move could set a precedent, with other lenders following suit.
"But if interest-only becomes beyond the reach of all but the wealthiest of borrowers, then it will hamper the housing market recovery, as there will be less choice for borrowers."
Five years ago it was still possible to get 100 per cent interest-only mortgages. However, anyone who took one out then is now likely to be facing the misery of negative equity – owing more than the property is worth.
Doubling the deposit could leave people unable to get a new deal.
David Black, analyst at Defaqto, said: "Only 29 per cent of mortgages will now permit an interest-only loan and most demand a 25 per cent deposit. The move to a 50 per cent deposit could lead to a significant problem for many interest-only borrowers."
Interest-free mortgages
73,000 Number of first-time buyers who took out interest-only mortgages in 2007.
5,300 Number of first-time buyers who took out interest-only mortgages in 2011.Reuse content


text:
Colombia's Paulina Vega was crowned Miss Universe Sunday, beating out contenders from the United States, Ukraine, Jamaica and The Netherlands at the world's top beauty pageant in Florida.
The 22-year-old model and business student triumphed over 87 other women from around the world, and is only the second beauty queen from Colombia to take home the prize.
The last time Colombia won the crown was in 1956 when Luz Marina Zuluaga took home the title.
Vega, wearing a long silver sequined gown, was beaming and tearful as she accepted her sash and crown from reigning Miss Universe, Venezuelan Gabriela Isler.
She edged out first runner-up, Nia Sanchez from the United States, hugging her as her win was announced.
Vega spoke proudly of her country, and said in the question round Colombia "has been a role model for the rest of the world."
"We are persevering people, despite all the obstacles, we keep fighting for what we want to achieve. After years of difficulty, we are leading in several areas on the world stage."
Vega won highest possible score of 9.9 in every presentation at the Miss Universe Colombia contest, according to the pageant's website.
The five-foot-nine (180 centimeter) model from Barranquilla is the granddaughter of legendary tenor Gaston Vega and 1953 Miss Atlantico, Elvira Castillo.
She beat out Ukraine's Diana Harkusha, Yasmin Verheijen from the Netherlands and Miss Jamaica Kaci Fennell, who were also top five finalists.


text:
|Message view
|« Date » · « Thread »
|« Date » · « Thread »
|jhickman <joseph.hick...@nfib.org>
|Re: Administered Objects
|Thu, 15 Jun 2006 12:56:15 GMT
>From the link: "Note that the ActiveMQ Broker will only create server side resources for destinations when messages are actually sent to them.", which seems to bear out the behavior that I've witnessed -- there are messages in the journal/DB table, but when the broker re-starts and I attach via JMX, there are no queues in existence (even though messages do still exist). My problem statement is that I need to read messages, if they exist. The link implies that I must first write at least one message before I can read the others. Is this the case? -- View this message in context: http://www.nabble.com/Administered-Objects-t1788152.html#a4882420 Sent from the ActiveMQ - User forum at Nabble.com.
|View raw message


text:
Saturday, 20 June 2009
True Grimoire Goes Edition
We are delighted to have the Goes Edition of The True Grimoire back from our binders and they are all signed and on their way to their new homes.
The quarter binding in veiny vellum ensures that each book is unique.
It is a delectable talismanic volume which will be treasured and worked with.
The Death's Head edition is still in stock and causing quite a stir.
Jake Stratton-Kent is already deep into the second volume of the Encyclopaedia Goetica which we will be publishing in due course.
Saturday, 6 June 2009
Howlings now available
A second edition of this acclaimed volume of practical working with the grimoires
In stock now for immediate dispatch 6/6/09
HOWLINGS various
edited by Alkistis Dimech
HOWLINGS is an octavo book of two hundred and ten pages, four sumptuous colour plates, handsomely designed and bound in peacock blue and gilt stamped cloth in a limited second edition of 666 copies.
This Edition is numbered sequentially from 334 to 999 inclusive.
The text remains the same as the first edition, though redesigned throughout.
A copy can be yours for the princely sum of thirty-three English pounds plus postage.
HOWLINGS comprises fourteen original and extensive essays which explore the Grimoires. These are passionate, informed and evocative pieces which create a unique testament to the vibrancy of the modern magickal current.
Our writers pursue knowledge, power and wisdom through the pages of:
The Picatrix
The Goetia, or Lesser Key of Solomon
Four Books of Occult Philosophy
The Voudon Gnostic Workbook
Liber 231
And the Grimoire itself
Our esteemed writers are,
David Rankine
Donald Tyson
Peter Grey
David Beth
Stafford Stone
Paul Hughes-Barlow
Krzysztof Azarewicz
Jack Macbeth
Thea Faye
Aleq Grai
Hafiz Batin
and the anonymous author of The Grimoire of Pharaon
Taking the established experts and ranging them alongside raw and hungry voices, this is a quite beautiful monstrosity of staggering scope. With the actual accounts of working magickians from a whole range of traditions, this is a unique collection.
Prepare to be delighted, inspired and intoxicated.
Wednesday, 3 June 2009
We would like to thank those who attended the Occulture Festival in London.
It is vital that the magickal community has the opportunity to meet face to face.
There are few opportunities to find so many magicians in one place who are willing to share the results of their hard won work.
Scarlet Imprint put all of our support behind these real world events.
Jake Stratton-Kent gave a powerful account of his work with the Grimorium Verum.
Dave Evans debunked the Amado Crowley myths.
Stephen Skinner explained a little more of his experience with grimoires, which we found invaluable.
We were able to begin to put forward the practice we are doing with Babalon and the Beast, which will eventually find it's way into print in Seven Heads/Seven Veils.
There is so much more to come.
It is our wish that Occulture, and the other magickal festivals live up to their potential.
To that end we will also be attending the Equinox Festival in London and the Esoteric Book Conference in Seattle.
This is only the beginning.
In Nomine Babalon
Peter Grey
More images and video content should be online soon at the Occulture website.


text:
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Situated within walking distance of the Cape Town Stadium, Wilton Lodge offers centrally located accommodation in Green Point. This B&B boasts an outdoor pool and barbecue facilities. Limited free WiFi is available. Certain rooms at Wilton Lodge are equipped with a flat-screen TV, a fan and a desk. Linen and towels are provided. Some have an en-suite bathroom fitted with a shower while other rooms have access to shared bathrooms. Use of a microwave and BBQ facilities are available. Guests can enjoy a basic Continental breakfast at Wilton Lodge. The B&B is within a short walk of a variety of restaurants, pubs and shops. The property has a front desk that can assist with arranging tours and car hire. Guests can enjoy a game of pool in the lounge area or relax in the entertainment area by the poolside. The V&A Waterfront is within 1.2 miles of Wilton Lodge, while Long Street and the city centre are within 1.9 miles. Cape Town International Airport is 14.3 miles away.


text:
GBP/CHF: buy targets - 1.5200 and 1.5440
By: Dmitriy Chernovolov
- GBP/CHF approaches round resistance level 1.5000
- Next buy targets - 1.5200 and 1.5440
GBP/CHF has been under strong bullish pressure lately – after the price broke above the resistance level 1.4840 (which stopped the earlier minor impulse wave (i) at the start of this month). The breakout of this resistance level accelerated the active impulse wave (iii) – which belongs to the C-wave of the intermediate ABC correction (2) from the start of May.
The pair is currently approaching the strong round resistance level 1.5000. If the price breaks above this resistance level - GBP/CHF can then rise to the next buy targets at 1.5200 (former strong resistance from March) and 1.5440.


text:
There are geological formations in China that have rightfully earned the nickname "the rainbow mountains." These colorful landscapes decorate the Zhangye Danxia Landform Geological Park in Gansu Province with colors not usually found in land formations. The mountains boast vertical stripes of vibrant reds, blues, purples, greens, and yellows.
How did this natural formation, a UNESCO World Heritage Site since 2009, gain its crayonbox colors? The mountain range is made up of densely packed layers of minerals and rocks of different hues, which were formed tens of millions of years ago. When the island that would become present-day India collided with the Eurasian tectonic plate over the course of millions of years, the result was this stunning mountain range. The layers of colors got crumpled up by the collision, exposing vertical lines of varying shades. But this geology is not unique. The Spectrum Range in British Columbia is similar to China's rainbow mountains. Watch the video below to learn about more amazing places on our planet.
Did You Know Everest Isn't The Tallest Mountain?
Speaking of amazing mountains...
Share the knowledge!
Key Facts In This Video
Mt. Everest is still growing at a rate of approximately 4 millimeters per year. 00:39
If measured from its base, Denali (formerly Mt. McKinley) is much taller than Mt. Everest. 02:00
If we measure in distance from the center of the Earth, then Mt. Chimborazo in Ecuador is the tallest mountain on the planet. 03:02


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Education News and Training
News George Spenceley obituary
My husband, George Spenceley, who has died aged 91, was a climber, photographer and lecturer.
In 1955-56, he was a member of a survey expedition to Antarctic South Georgia, where the Spenceley glacier was named after him. In 1957 during an expedition in the Jugal Himal of Nepal he was the sole survivor of an avalanche.
He was born in Harrogate, North Yorkshire. When George was a small boy, his father took him up Ingleborough, one of the highest mountains in the Dales. He pointed out some tents far below that belonged to members of the Yorkshire Ramblers Club and told George that one day he would be a member. George joined the club soon after the outbreak of the second world war, then a recently qualified RAF pilot.
After more than 40 wartime operations, he was shot down in 1942, in one of the early 1,000-plane raids over Germany. He spent several weeks in hospital, then three years as a prisoner of war. Returning home in 1945, he married Marjorie, with whom he had three sons, Julian, Adrian and Nicholas.
He became a geography teacher, but also continued walking, climbing and exploring, and turned his adventures into illustrated talks for schools and societies all over Britain.
After his divorce from Marjorie, we married in 1977 and settled in Oxfordshire. George continued his successful career as a lecturer, mainly during the winter months between September and April. Together we canoed the full length of the river Danube in 1979-80, followed by the Mississippi in 1984. At the age of 84, George became the oldest man to reach the Annapurna Sanctuary in the Himalayas.
He is survived by me and his sons.


text:
Durham Univ. (United Kingdom)
Observatoire de Paris (France)
ONERA (France)
Observatoire Astronomique de Marseille-Provence (France)
UK Astronomy Technology Ctr. (United Kingdom)
Heriot-Watt Univ. (United Kingdom)
EAGLE is a multi-object 3D spectroscopy instrument currently under design for the 42-metre European Extremely Large Telescope (E-ELT). Precise requirements are still being developed, but it is clear that EAGLE will require (~100 x 100 actuator) adaptive optics correction of ~20 - 60 spectroscopic subfields distributed across a ~5 arcminute diameter field of view. It is very likely that LGS will be required to provide wavefront sensing with the necessary sky coverage. Two alternative adaptive optics implementations are being considered, one of which is Multi-Object Adaptive Optics (MOAO). In this scheme, wavefront tomography is performed using a set of LGS and NGS in either a completely open-loop manner, or in a configuration that is only closed-loop with respect to only one DM, probably the adaptive M4 of the E-ELT. The fine wavefront correction required for each subfield is then applied in a completely open-loop fashion by independent DMs within each separate optical relay. The novelty of this scheme is such that on-sky demonstration is required prior to final construction of an E-ELT instrument. The CANARY project will implement a single channel of an MOAO system on the 4.2m William Herschel Telescope. This will be a comprehensive demonstration, which will be phased to include pure NGS, low-order NGS-LGS and high-order woofer-tweeter NGS-LGS configurations. The LGSs used for these demonstrations will be Rayleigh systems, where the variable range-gate height and extension can be used to simulate many of the LGS effects on the E-ELT. We describe the requirements for the various phases of MOAO demonstration, the corresponding CANARY configurations and capabilities and the current conceptual designs of the various subsystems.
Richard M. Myers ; Zoltán Hubert ; Timothy J. Morris ; Eric Gendron ; Nigel A. Dipper, et al."CANARY: the on-sky NGS/LGS MOAO demonstrator for EAGLE", Proc. SPIE 7015, Adaptive Optics Systems, 70150E (July 10, 2008); doi:10.1117/12.789544; http://dx.doi.org/10.1117/12.789544
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About Us
Mentoring changes lives, and Big Brothers Big Sisters is America's leader in one-to-one youth service. BBBS has over a century of proven success in creating positive friendships that benefit children, volunteers, families, neighborhoods, and communities. In the Fargo–Moorhead area, BBBS has been a program of The Village Family Service Center since 1973. School based mentoring was added in 1998.
It Works!
National research reveals that Littles are more likely to show positive behaviors in school and at home than children not matched with a Big. Our matches are effective because professional staff ensure that the relationships are safe and rewarding for everyone involved. Read about a real BBBS success story from right here in Fargo.
More about the impact of our mentoring programs.
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Throughout history, poverty is the normal condition of man.Advances which permit this norm to be exceeded -- here and there, now and then -- are the work of an extremely small minority, frequently despised, often condemned, and almost always opposed by all right-thinking people. Whenever this tiny minority is kept from creating, or (as sometimes happens) is driven out of a society, the people then slip back into abject poverty. This is known as "bad luck." -- Robert A. Heinlein
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Category Archives: Movies
Movies that won't play.
I've decided to follow up on an earlier post (Movies I'd watch) where I lamented on the interesting movies that get made but the majority of intelligent movie fans will not get to see in their home town. A fair … Continue reading
Posted in All the News not fit to print., Blogbits, Movies Leave a comment
This! Impresses me.
Encoding a Classic Film, Computer Operating System in DNA NEW YORK GENOME CENTERYaniv Erlich and colleagues encoded large media files in DNA, copied the DNA multiple times, and still managed to retrieve the files without any errors, they reported in … Continue reading
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Drip, drip, drip…
Inspector General: Clinton emails had intel from most secretive, classified programs EXCLUSIVE: Hillary Clinton's emails on her unsecured, homebrew server contained intelligence from the U.S. government's most secretive and highly classified programs, according to an unclassified letter from a top … Continue reading
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Halloween Science (from the American Council on Science and Health) Spoilers!
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Sin City: The sequel…A personal view.
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Do little boys still have heroes?
Actor James Garner, whose whimsical style in the 1950s TV Western "Maverick" led to a stellar career in TV and films such as "The Rockford Files" and his Oscar-nominated "Murphy's Romance," has died, … He was 86. The first … Continue reading
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Why are the search engines all run by assholes?
Google responds to problems with searches for Dinesh D'Souza's 'America' By Hollie McKay NEW YORK – Conservative filmmaker Dinesh D'Souza wants to know why his latest docudrama "America: Imagine a World Without Her" was buried by mega search engine Google.
****SPOILER**** Game of Thrones
Can I ask you something? See what I did here, or up above, a Spoiler Alert. Very simple. So why the hell can't the print and on-line newspapers, on-line journals, emails from Tor (and it seems) the backs of the … Continue reading
Yea! Go Cap!
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Maleficent is Magnificent Angelina
She has been the best thing in so many movies…even when she didn't take her clothes off. Lately I haven't been going to that many movies. Magnificent Maleficent Angelina, I'm looking forward to this one. Reportedly, they cast her real … Continue reading
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Oba Ma Hamu Una Da Dance Mix - Djz TiGeR.mp3
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Lilly Launches Latest Innovation In Insulin Delivery
Lilly announced the launch of the new HumaPen® SAVVIOTM this week at the Diabetes UK Professional Conference in Manchester.
Engineered to resemble a small personal accessory rather than a medical device, Lilly's latest innovation was designed following feedback from people with diabetes who shared their aspiration to manage diabetes discreetly. The HumaPen SAVVIO is a next-generation insulin delivery device for use with the Humalog® and Humulin® brands of insulin.
Since every experience with diabetes is unique and personal, Lilly launched a landmark market research project in six countries to gain insight into the daily lives of people with diabetes. The research team spent a day-in-the life with patients in their own homes to gather real-life understanding of how they use their insulin devices and the challenges they face. Insulin users' individual lifestyles were candidly discussed and observed and this research was used to design the new HumaPen SAVVIO to meet their needs and help them feel more at ease when using their medication1.
"I want my patients to feel confident about managing their condition and being able to get on with everyday living," says Susan Stockley, Lead Diabetes Specialist Nurse from East Surrey Hospital. "People with diabetes handle their devices every day and to some, the device reminds them that they are on important medication and 'different'. This new insulin pen blends in with their accessories and feels personalised, plus the size means they can be more discreet when administering."
The new insulin delivery device looks like a fashionable pen and features a compact, attractive yet durable design, and is available in several different colours in the UK; blue, graphite, green, silver, pink, and red. While being aesthetically pleasing and highly functional, HumaPen SAVVIO has undergone rigorous testing on a variety of quality, accuracy of dosing and durability measures to ensure people are receiving their medication safely and appropriately1. The HumaPen Savvio is easy to dial and dose and features smooth glide force an easy-to-use cartridge holder.
"The process of developing HumaPen SAVVIO reflects our commitment to 'people led' innovations as it required an understanding of people's emotional responses to their medical treatment", says Dr Arash Tahbaz, Lilly Senior Medical Advisor. "Our research and the time spent with people using insulin therapy on a daily basis gave us meaningful insights, which have been transferred to HumaPen SAVVIO. We're pleased to bring this latest offering in the Lilly Diabetes portfolio to the UK market and we will continue to put patients first."
HumaPen SAVVIO joins Lilly's broad portfolio of insulin pens that provide personalised solutions for people living with diabetes.
HumaPen SAVVIO is available for use with Lilly's 3 mL insulin cartridges, which include the Humalog and Humulin brands of insulin.
[1] Lilly Data on File
Source: Lilly Diabetes
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Disclaimer: This is written for fun, solely for the enjoyment of those who cannot stand thinking inside the box. I make no money of this, and none of the characters are mine.
AN: I know most people do this at the end, but all work is edited by IMFACI
Prologue – Lost
I am alone. Completely and utterly alone. My parents are there, my friends are there, truth be told I see a town full of people around me, and yet I am alone. My mother gives me a hug and I can feel nothing, I know her arms encircle me, and I know she pushes as much as she can, yet I feel not even a glimmer of sensation. My father claps my shoulder, and he is a strong man, has to be to work on the farm, and still nothing.
Can you imagine what I go through? I touch a tree and I can't really do anything. I can't hold it, I can't truly touch it, feel the grain of the bark, how warm or cool it is, the texture of the grain. I would give anything for that because in the end I can't. No wind can truly touch me, no matter how cold, and the warmth of the sun is meaningless to me. I feel its energy cascade over me, I feel something inside whenever I bask in its light, but no warmth. Nothing. It's utterly maddening. I can taste, I can hear, I see, but touch eludes me, because in the end I have to walk as if the world is made up of egg shells.
I can truly take hold of nothing, my strength so great. I would crush it all.
It started when I was a baby, according to my parents. They would discover dirty diapers that I had never made a fuss over. My dad still laughs that they went through the least diapers of any parents they knew, because he simply didn't tell them that he had gone. Mom told me once she had not been paying attention to a bottle, she had fed me and had no complaints. She didn't realize how hot it had been until she tried to dump the portion I did not finish and it scalded her.
As I got older it got worse, I discovered my strength early on, ripping off one of the barn doors when I had gone out to help my dad. One of the hinges snapped violently and it glanced off me and flew into an old tree that we had near the barn, embedding so deep that my dad could not get it out without cutting the tree down. It's there to this day.
I have made friends, Pete Ross and Lana Lang, but I rarely see them. My parents thought it best I learn at home until my powers stopped behaving so erratically. It's why I keep this journal, because without it I am so utterly alone…
Journal of Clark Kent
He was ethereal and yet real, the most powerful of his race, and also the most distant. He had watched creation come and go, and known the true feelings of despair as he was forced to kill his own children. For them it was a slow death, to him it was but a blink of the eye. He had traveled far from his once home to view the life of his last son. There were so many of his kind on this world, he sometimes wondered what they saw so interesting in this race.
"Rao," a soft voice said. He didn't look up to know that it was one of the Greek Pantheon.
"Yes," he said dryly.
"Why have you invaded our domain," Ares growled out.
"The Great Spirits would take exception with that claim, son of Zeus," Rao said calmly, looking away from his son to the God of War. "What do you want?"
"The others are nervous," Ares' hand hovered over his blade.
"They have every right to be, betrayer," Rao said calmly. A wave of his hand and Ares froze in place. "You think a puny god of war can match my power?"
"When I get free…"
"Empty threats, Ares," Rao growled out. He waved his hand again and the frozen god of war flew through the air into the ether. Rao walked forward calmly, his eyes unreadable as the ancient god stood to meet his foe.
"We will find a way to kill you," Ares growled out. "When it's done we will ascend to your throne."
"Your father tried it years ago," Rao smirked at that. "He ripped apart space and time, and summoned the powers of the Titans before him and still it wasn't enough. Do you think you truly have the strength to topple me?"
Ares growled. "What are you doing here old man?"
"Oh, nothing," Rao looked wistfully at the stars at this. He hated setting one of his children up like this, but the time of the awakening was at hand. If he didn't find his connection with this world, their savior would become their destroyer. "Just looking in on my last son."
Ares growled, knowing he was referring to the Kent boy. Zeus had railed for weeks for not being able to bring the boy into his sphere of influence, but Rao had separated the Kryptonians from all other lines. His intention had been for them to be protectors of the creations the others had made; he wanted them under the influence of none.
"You know boy, I have faced the endless void before, brought forth stars that had existed since before you were even thought of," Rao chuckled at the indignant look the God of War gave him. "Remember your place boy. I have removed pantheons for less spoken against me."
Ares knew he referred to the Mayans and their gods and goddesses. The civilization as a whole had grown decadent and for all their achievement Rao refused to let anyone grow too decadent. It was the reason that Zeus had come to preach balances in the world. Too far one way, or the other, and Rao would swoop in and cull everything, whether they be mortal or god.
"Leave this place, my last son will not be your pawn," Rao waved his hand, freeing the god of war.
"We will see," Ares disappeared in a flash of thunder.
Rao sighed heavily; sometimes human form was so strange. He wondered how his son would cope, he could see the tinges of the madness, that drove many of his kind before, creeping in. He turned back to the ether and looked at his son. "Be strong my son, and I hope you live true to the light I see in you. I would hate to have to end everything once again."