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US 6406698 B1 Abstract Pharmaceutical compositions comprising a protein serum amyloid P component (SAP) capable of binding viruses, especially myxovirus, e.g. influenza viruses and thereby preventing the transmission and infection with such viruses. Preferred is mucosal administration, e.g. SAP incorporated in a nose spray or nose drops. Also provided is a method for measuring the binding of SAP to the hemagglutinin of the prevailing influenza virus subtype and strain.
Claims(15) 1. A pharmaceutical composition for mucosal administration comprising:
a. a native, synthetic or recombinant serum amyloid P component (SAP) selected from the group consisting of serum amyloid P protein, a subunit of said SAP protein comprising at least one complete SAP polypeptide chain, and a subunit of the SAP polypeptide chain comprising a Ca2+-dependent carbohydrate binding site;
b. Ca 2+;
c. a physiologically acceptable carrier; and
d. a viscosity enhancing factor.
2. The pharmaceutical composition according to
3. The pharmaceutical composition according to
4. The pharmaceutical composition according to
5. The pharmaceutical composition according to
6. The pharmaceutical composition according to
7. The pharmaceutical composition of
8. A kit for the detection and quantitation of SAP binding to influenza virus comprising a container holding SAP in solution, a container holding indicator cells, and a buffer system including Ca2+ ions in a concentration of about 1.2 mM to about 2.0 mM.
9. The kit of
10. A pharmaceutical composition for treating an orthomyxovirus or a paramyxovirus infection, said composition comprising:
a. a native, synthetic or recombinant serum amyloid P component (SAP) selected from the group consisting of serum amyloid P protein, a subunit of said SAP protein comprising at least one complete SAP polypeptide chain, and a subunit of the SAP polypeptide chain comprising a Ca2+-dependent carbohydrate binding site;
b. Ca 2+;
c. a physiologically acceptable carrier; and
d. a viscosity enhancing factor.
11. The pharmaceutical composition of
12. The pharmaceutical composition of
13. A pharmaceutical composition for inhibiting an oxrthomyxovirus or a paramyxovirus infection, said composition comprising:
a. a native, synthetic or recombinant serum amyloid P component (SAP) selected from the group consisting of serum amyloid P protein, a subunit of said SAP protein comprising at least one complete SAP polypeptide chain, and a subunit of the SAP polypeptide chain comprising a Ca2+-dependent carbohydrate binding site;
b. Ca2+;
c. a physiologically acceptable carrier; and
d. a viscosity enhancing factor.
14. The pharmaceutical composition of
15. The pharmaceutical composition of
Description This application is a continuation of International Application No. PCT/DK97/00035 filed Jan. 24, 1997, incorporated hereby by reference in its entirety. The present invention provides pharmaceutical compositions comprising: a) serum amyloid P component in a form capable of binding a virus component, said binding resulting in a reduction of the pathogenic properties of said virus component; and b) a physiologically acceptable carrier. The pharmaceutical composition comprises serum amyloid P component (SAP) (a naturally occurring protein) or a binder-functional subunit thereof. The reduction of pathogenic properties results in prophylaxis, alleviation or cure of infectious diseases caused by said virus component. Especially preferred are compositions formulated for mucosal administration, e.g. nasal administration. In particular, the virus component is a virus carbohydrate, e.g. exposed on a viral hemagglutinin, and the binding results in at least a partial blocking of said hemagglutinin. The virus component may be derived from an orthomyxovirus, e.g. an influenzavirus type A, B or C from humans or animals, a virus from the paramyxoviridae family, e.g. a paramyxovirus, from a herpes virus or from a corona virus. The invention also provides the use of serum amyloid P component (SAP) for the production of a medicament for the prophylaxis or treatment of virus infectious diseases in human beings or animals. The prophylaxis or treatment is obtained by means of SAP in a form capable of binding at least a part of a virus component causing said infectious disease, said binding resulting in a reduction of the pathogenic properties of said virus component. According to the present invention, the serum amyloid P component (SAP) is the native protein, the recombinant protein or a binder-functional subunit thereof, the subunit being one or more complete SAP polypeptide chain(s) or a part or parts of the chain comprising the Ca2+-dependent carbohydrate binding site(s) or synthetic peptides constructed to contain amino acid sequences contributing to said binding sites. The infectious disease may be caused by the above-mentioned viruses. A preferred aspect of the invention relates to a method of preventing infection with influenza virus or a parainfluenza virus and the transmission of said virus by blocking virus infectivity with serum amyloid P component (SAP). In particular, this invention relates to the prophylactic/therapeutic use of SAP in epidemics/pandemics of influenza virus infection in man. Another aspect of the invention relates to the prophylactic/therapeutic use of SAP in epidemics/pandemics of influenza virus infection in horses, swine, and birds. For instance for prophylactic treatment of horses the pharmaceutical composition may be contained in a vehicle for nose spray or a nebulizer, which is applied separately to the nostrils, nose or upper respiratory tract. Viral infections are a major cause of serious diseases for which effective prevention or treatment in many cases are still not available. The present invention is concerned with viral infections caused by the families orthomyxoviridae, paramyxoviridae, herpes viridae and corona viridae. The orthomyxoviridae family include i.a. the orthomyxoviruses influenza viruses types A, B and influenza virus type C. Especially influenza virus type A is a major cause of morbidity and mortality due to respiratory disease, outbreaks of which may occur in world-wide epidemics. The paramyxovirus genus comprises the four serotypes of human parainfluenza viruses, mumps virus, shipping fever virus of cattle and sheep and the avian parainfluenza virus, Newcastle disease virus. Other viruses from the paramyxoviridae family include the Morbillivirus genus (e.g. measles virus, canine distemper virus and rinderpest virus) and the pneumovirus genus (e.g. human and bovine respiratory syncytial virus). The parainfluenza viruses and respiratory syncytial virus are major pathogens causing severe respiratory tract disease in infants and young children. The herpesviridae family comprises several important human pathogens. Important members of this family are herpes simplex types 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus and human herpes virus type 6. The human corona viridae cause common colds and have been implicated in gastroenteritis in infants. The main aspect of the present invention concerns human and animal influenzavirus. A more elaborate discussion of these viruses is therefore deemed proper. Influenzavirus is a genus of viruses (family orthomyxoviridae) which includes influenza virus type A and influenza virus type B. Both A and B types cause sporadic or epidemic influenza in man; type A strains also cause epizootics in pigs, horses or birds (e.g. fowl plague and swine influenza). The viruses are transmitted via aerosols or water or by direct contact. The enveloped virions are pleomorphic, ca. 80-120 nm in diameter. The envelope encloses the genome of 8 molecules of linear negative-sense ssRNA (total Mwt ca. 5�106), the RNA forming helical ribonucleoprotein complexes with protein NP. Proteins PB1, PB2 and PB3, which together constitute a transcriptase, are associated with the NP-RNA complex. The envelope is associated with viral matrix protein M1 (which occurs internally, surrounding the nucleoprotein core), M2 protein (an integral membrane protein, product of a spliced transcript of the same genome segment (1) as M1), and the glycoproteins HA (hemagglutinin) and NA (neuraminidase); HA trimers and NA tetramers form spikes and mushroom-shaped projections, respectively, which radiate from the outer surface of the viral envelope (24). Influenza virus types are divided into subtypes (subgroups) on the basis of antigenic differences in the HA antigens (13 subtypes) and NA antigens (9 subtypes). Each individual virus strain is designated by a formula which indicates the following : type (A, B or C); the animal from which the strain was first isolated (omitted if the host was human); the place of initial isolation; the strain number; the year of isolation; and the particular HA (=H) and NA (=N) antigens. Example: A/duck/Ukraine/1/63(H3N8). In humans, strain A/Singapore/1/57(H2N2)�formerly known as �strain A2��was responsible for the influenza pandemic of 1957 (�Asian flu�), while Strain A/Hong Kong/1/68(H3N2) was responsible for the 1968 pandemic (�Hong Kong flu�). However, these strains are not immutable; influenza viruses undergo ANTIGENIC VARIATION in their glycoprotein antigenic determinants, allowing the viruses to overcome their hosts' immune responses and resulting in new epidemics of influenza. Two kinds of antigenic variation occur: ANTIGENIC DRIFT AND ANTIGENIC SHIFT. Antigenic shift occurs infrequently and has been recorded only in type A strains. It appears to depend on the fact that type A strains can infect animals as well as man, and that, in mixed infections involving strains from different species, genome segments may undergo reassortment, allowing the generation of new virus subtypes (25). Since 1946, three distinct human subtypes �H1N1, H2N2 and H3N2�have arisen, each causing a new pandemic of influenza and each largely replacing its predecessor. Each prevailing subtype has also undergone regular antigenic drift (as a result of small mutational changes in HA and NA antigens), each new variant causing outbreaks and epidemics of influenza. Antigenic drift also occurs, though more slowly, in B- and C-type influenza viruses. Influenza virus C is also a virus of the ORTHOMYXOVIRIDAE. It is physicochemically and morphologically similar to members of the genus Influenzavirus, but differs in containing only 7 RNA segments (total Mwt ca. 4-5�106) and in having only one type of surface glycoprotein (HA) which has both haemagglutinating and receptor-destroying activities, but apparently no neuraminidase activity. It has recently been shown that, contrary to previous assumptions, sialic acid may nevertheless be an essential component of the host cell-surface receptor (26). Influenza virus C primarily infects man, although it has been isolated from pigs in China; it generally causes only mild or subclinical disease of the upper respiratory tract. Antigenic drift occurs slowly, but antigenic shift has not been observed. Paramyxovirus is a genus of viruses of the PARAMYXOVIRIDAE family in which the virions also have both haemagglutinin and neuraminidase activities. The paramyxovirus virion consists of a helical nucleocapsid, composed of the genomic ssRNA and proteins NP, P and L, surrounded by an envelope containing a non-glycosylated protein (M) in the inner layer, and two glycoproteins which extend across the width of the envelope and beyond the outer surface to form spikes. Proteins P and/or L may have transcriptase activity. Protein M plays an important role in virus assembly. The larger of the envelope glycoproteins (designated HN) has cell-binding, haemagglutinating and neuraminidase activities, while the smaller (F) has haemolytic activity and promotes fusion between the virus envelope and the host plasma membrane. Prior Art Related to Vaccination The phenomenon of antigenic variation has made it difficult to produce effective vaccines for influenza. Attempts have been made to prepare attenuated �live� vaccines using reassortant viruses containing e.g. surface glycoprotein antigens from a currently epidemic strain. Temperature-sensitive (cold-adapted) strains have been used as the attenuated �parent�, but the resulting virus tends to be genetically unstable; attempts have therefore been made to use a strain from another species with low virulence for man, e.g. avian strains. An alternative approach has been to prepare synthetic oligopeptide vaccines corresponding to the influenzavirus glycoprotein epitopes (27). With regard to influenza A virus epidemics it is well known that even the use of a new vaccine based on the appropriate virus subtype conveys protective immunity to only 60-70% of the vaccinated population. Furthermore, only a minor part of the susceptible population is usually receiving the vaccine even in verified epidemics. In addition, due to the antigenic drift of the virus and the short duration of the specific secretory IgA antibody response, the immunity is short lasting after vaccination (14). Finally, annual vaccinations may predominantly boost antibody response directed against irrelevant strains to which the individual has once been exposed (original antigenic sin). Prior Art Related to Specific Medicaments Prophylaxis has been attempted with amantadine (Virofral) and the related rimantadine which are drugs for systemic use in the prevention of influenza A virus infections (15). The drugs block uncoating of influenza A virus in the host cell. However, the drugs appear ineffective in protecting household contacts from influenza as drug-resistant mutants of virus develop. In addition, the drugs may affect the central nervous system causing dizziness and insomnia. Consequently, they have found very limited application. In conclusion, prophylactic measures against influenza A virus infections are rather inefficient as documented by the high number of individuals coming down with the disease and the over-mortality primarily due to pneumonia each time an epidemic/pandemic strikes. This in spite of continuous scientific efforts and economic investments in order to improve the vaccines and find new effective chemoprophylactic drugs. Recently, it has been proposed that sialidase-based inhibitors might prevent influenza virus replication and therefore be used in therapeutic or prophylactic treatment (23). However, the effect has not yet been confirmed in humans. Thus, there is a long felt need for novel and effective prophylactic measures against influenzavirus. This has been confirmed in two recent articles (21,22). To date, attempts to develop effective vaccines against the paramyxoviruses and respiratory syncytical virus have not been successful. The drug ribavirin administrated in aerosols has been reported to be beneficial to hospitalized infants with respiratory syncytial virus infections. Effective vaccines against mumps and measles are available. No safe and effective vaccines are available for prevention of diseases caused by the corona and herpes virus families. Antiviral compounds such as vidarabine and in particular acyclovir can alleviate the symptoms in patients with herpes virus infections. In U.S. Pat. No. 5,221,628 A (46), a test kit for detecting or quantitating immune complexes is described. The test kit comprises a container holding SAP, said SAP being characterized in that it preferentially binds immune complexes in the presence of monomeric immunoglobulin. SAP may be bound to a solid support such as a dipstick, latex beads or a microtiter plate. In this document, it has not been suggested to add Ca2+ in order to obtain a inhibitory effect of SAP. A number of infectious diseases in animals, such as infections with influenza A virus and the parainfluenza viruses, shipping fever virus and Newcastle disease virus involve considerable problems. Influenza has been known as the cause of epizootics of respiratory disease in equines for several centuries. It is from an economical viewpoint the most important respiratory disease in horses. Equine influenza has low mortality but high morbidity and it is spreading extremely fast. During the last decades this rapid spread of the disease has caused great economical losses to the horse owners and the equine industry (16). Similarly, outbreaks of Newcastle disease virus can inflict great economic losses on the poultry industry. SAP is a naturally-occurring plasma protein composed of five identical subunits which are non-covalently associated in a disc-like molecule (29). SAP is also a natural constituent of glomerular basement membranes and elastic fibres of the skin and blood vessels (30, 31), the glycosylated SAP monomers have a β-pleated sheet structure and two calcium binding sites (32). The SAP molecule has been found to be resistant to proteolysis in the presence of calcium (33). Recently, the three-dimensional structure of the pentameric molecule has been solved by X-ray analysis of SAP crystals (32). Binding of SAP to ligands is reported to include agarose, zymosan, heparin, fibronectin, the complement components C1q adn C4bp, Fab fragments of IgG and IgG aggregates (1, 10, 34-38). Furthermore, a calcium-dependent binding of sap to glycosaminoglycans, in particular heparan and dermatan sulphates, and to chromatin and DNA has been demonstrated (8, 39, 40). Both calcium-dependent binding and calcium-independent binding of SAP to amyloid AA protein and the amyloid precursor protein β2M globulin were described recently (28). The best defined binding site on SAP is the one for 4,6-cyclicpyruvate acetal on β-D-galactose (41). In addition, SAP has been found to bind to mannose-rich oligosaccharide sequences and mannose-terminated sequences (2, 7). Binding interactions with cells have also been reported. Thus, SAP was reported to bind to receptors on elicited tissue macrophages and to granulocytes (42, 43). In humans, unlike in mice, SAP is not an acute-phase reactant (44). The physiological role of SAP is not known but it may have a function in clearance of DNA in cell death (39). An antagonistic effect of SAP on heparin's anticoagulant activities was recently reported (45). FIG. 1 shows an electron microscopy of negatively stained purified human serum amyloid P component. Mag. 200.000. FIG. 2 shows the dose-response inhibition of virus hemagglutination (HA) when two different influenza virus A strains were preincubated with increasing concentrations of human SAP. FIG. 3 shows that the SAP-mediated inhibition of influenza A virus hemagglutination (HA) is Ca++-dependent. FIG. 4 shows that polyclonal rabbit antibodies against human SAP abolished the SAP-mediated inhibition of influenza A virus hemagglutination. Polyclonal antibodies against C-reactive protein had no effect on the inhibition exerted by SAP. FIG. 5 shows that SAP protected MDCK cells from infection with influenza A Shangdong 9/93 (H3N2) virus. a) MDCK cells plus virus (2 HA units) and SAP (15 μg/100 μl), b) MDCK cells and virus and c) MDCK cells only. The photos were taken on the third day after infection of the cultures. Magn. 250�. FIG. 6a is an electron micrograph showing the binding of gold-conjugated SAP to the spikes of the viral envelope of influenza A/Shangdong/9/93 (H3N2) in the presence of 1.2 M CaCl2. FIG. 6b shows that there was no binding when virus was reacted with gold-conjugated bovine serum albumin. FIG. 7 shows Western blotting analysis of SAP's interaction with influenza A/Shangdong/9/93. a) gold-stained virus preparation, b) strip incubated with SAP in TBS containing 2 mM CaCl2 and c) with SAP in TBS containing 10 mM EDTA. Lane b) shows that SAP exhibits Ca++-dependent binding to a viral peptide of 50-55 kDa. Molecular weight standards are shown in margin. The present invention involves use of the naturally occurring protein, serum amyloid P component (SAP). Even though SAP has been known for many years (3), its physiological function is not known. SAP has been isolated from man and several animal species. Very similar conserved proteins are present in all vertebrates in which they have been sought. As an example, it can be mentioned that there is a 50% homology of amino acid sequence in the N-terminal region between SAP of plaice and man. The concentration of SAP in human plasma is 30-60 μg/ml. It is synthesized in the liver and the biological half-life of human SAP is 24 hours (4). Its molecular weight is about 125 kDa and it consists of 5 subunits forming a ring (pentamer). Each subunit consists of about 205 amino acids with one intrapolypeptide disulphide bond (5). It contains 11% carbohydrate, 4% of which is galactose and mannose, 3% hexosamin and 4% sialic acid (6). SAP is highly resistant to degradation by proteolytic enzymes in the presence of calcium. SAP has been the topic for a number of articles co-authored by the present inventors (9, 18, 20, 28), primarily concerned with its native and purified forms. The overall conclusion is that most native SAP circulates as a single pentamer, a part of which forms complexes with C4b binding protein (20). SAP shows a certain degree of heterogeneity with several isoelectric forms (18). It has an aggregation tendency, which is enhanced by Ca2+, and during purification formation of decamers is observed (20). A pH-dependency of SAP's binding to glycosaminoglycans and amyloid proteins was demonstrated recently (28). The present invention provides pharmaceutical compositions comprising serum amyloid P component (SAP) capable of binding myxovirus and paramyxovirus, especially influenza and parainfluenza viruses, and thereby preventing the transmission and infection with such viruses. It is believed that SAP is also effective in preventing the transmission and infection with the other viruses mentioned above. Preferred is mucosal administration, e.g. SAP incorporated in a nose spray or nose drops. Also provided is a method for measuring the binding of SAP to the hemagglutinin of the prevailing influenza virus subtype and strain. The present invention comprises a method of binding serum amyloid P component (SAP) to myxovirus especially influenza virus. The invention is based on so far unreported recognition that SAP binds selectively to oligosaccharides on the hemagglutinin molecules of different influenza virus subtypes and strains. It can be assumed that SAP also binds to oligosaccharides on cell-binding fusion glycoproteins of parainfluenza viruses. The ability of SAP to bind influenza viruses can be utilized to inhibit or abolish the infectivity of the virus and reduce the spread of virus. To achieve this, SAP is incorporated in a nose spray or nose drops to reduce the transmission of virus produced by epithelial cells in the respiratory tract. The invention also comprises a method of determining SAP's binding to the hemagglutinin of a prevailing influenza virus subtype and strain. Thus, the ability of SAP to bind influenza virus may be utilized in the prophylactic/therapeutic treatment of influenza virus infections. Without wishing to be bound by a particular molecular theory, one of the mechanisms underlying the invention may be the following: The use of SAP according to the invention may interact with the virus components in a manner described below, taking influenza virions as an example. Influenza A virions have 500-1000 spikes on the surface of their envelope, which mediate the attachment to the host cells. About 80-90% of these are hemagglutinins (HA) build from three monomeric subunits, the remainder being neuraminidase (NA). Each HA monomer consists of two disulphide-linked glycosylated polypeptides, HA1 with a MW of about 50 kDa and HA2 with a MW of about 28 kDa. During infection, the virions binds via the HA to sialic acid-containing glycolipid/glycoprotein surface receptors on the host cell. The sialic-binding area on the HA molecule is formed by a grove of 5-6 conserved amino acid residues. Both polypeptide chains in the HA-monomer are glycosylated with 4-6 N-glycosylation sites on the HA1 chain (up to 25% of the weight) and 1-2 on the HA2 chain (app. 6%). All carbohydrates are localized on the surface of the HA-trimer (13). HA subtypes demonstrate varying number and localization of carbohydrate chains. These differences are due to variation in the amino acid sequence, i.e. the existence of Asn-X Ser/Thr sequences. Influenza A/Hongkong/(H3) hemagglutinins have 7 N-glycosylation sites, 6 on the HA1 chain and 1 on the HA2 chain. Five of these are oligosaccharides of complex type (GlcNAc, Man, Gal or Fuc), two are of the simple oligomannoside type (GlcNAc and Man). In comparison, influenza A/Asian (H2) hemagglutinins have 5 N-linked oligosaccharides, all of complex type, 4 on HA1 and 1 on the HA2 chain (25). The compositions and the uses according to the present invention may be based on the following mechanisms: Influenza virus infection is acquired by transfer of virus-containing respiratory secretions from an infected to a susceptible individual. There are large amounts of virus present in such secretions at the time of illness and virus is dispersed in small-particle aerosols produced by sneezing and coughing. A single infected individual may transmit virus to a large number of susceptible individuals. When virus reaches the respiratory tract it attaches to columnar epithelial cells and initiates a replication cycle if not blocked by specific secretory antibodies or prevented by the mucus film in the respiratory tract. The virus attaches to sialic acid on epithelial cell surface receptors via a site on the virus hemagglutinin molecule (13). Without wishing to be bound by any particular hypothesis it can be assumed that when SAP is administered prophylactically a considerable part of the SAP molecules are bound or adsorbed on the epithelial cells so as to exhibit a protective effect against incoming virus. In therapeutic administration the SAP molecules are brought into contact with the virions which are liberated in considerable amounts from the epithelial cells, whereby the spreading of the infection is prevented or limited. Logically, the SAP dosage used for therapeutical purposes will therefore advantageously be higher than for prophylactic administration. Generally, infections of the respiratory tract may be caused by orthomyxoviruses or paramyxoviruses. The infection may spread to involve trachea, bronchi and the lungs. Secondary bacterial infections may occur. The incubation period is 1 to 4 days for influenza virus infections and 1 to 7 days for parainfluenza virus infections. To prevent transmission of virus by virus-carrying small-particle aerosols the pharmaceutical composition according to the invention is administered as early as possible during an epidemic or local outbreak of the respiratory virus disease. According to an aspect of the invention compositions comprising SAP and a specific amount of Ca2+ may be used. A preferred amount of Ca2+ is a final concentration corresponding to the physiological amount, viz. 1.2-1.5 mM. SAP exhibits Ca2+-dependent binding to certain carbohydrates. SAP has been shown to bind to zymosan, agarose and other polysaccharides (17) and to mannose-terminated sequences of polysaccharides and glycoproteins (2). However, its binding to influenza virus components has not been described earlier. SAP has the capacity of multispecific Ca2+-dependent binding to oligosaccharides with terminal N-acetylgalactosamin, glucuronic acid, 6-phosphate-mannose and to certain glycosaminoglycans (7, 8, 9, 10). It has also been reported to bind Ca2+-dependently to heparin, fibronectin, C1q,C4b-binding protein (C4bp) and chromatin (1, 11, 12). Some authors (11) propose that nearly all SAP in the blood is bound to C4bp. In the presence of Ca2+, human IgG and Fab, but not Fc were found to bind to SAP (1). Besides of the active SAP the pharmaceutical compositions according to the invention also comprise a pharmaceutically acceptable carrier. Obviously, said carrier will depend on the chosen mode of administration and may comprise usual preservation agents, such as p-hydroxy benzoate, and/or stabilizers. The presently preferred formulation is a nasal spray which may be formulated in aqueous solution and packed into spray containers either with an aerosol or provided with means for manual compression. A preferred embodiment of a nasal spray comprises SAP in an amount of at least 50 μg/ml in isotonic NaCl, CaCl2 at about the physiological level (1.2-1.5 mM) and further comprising a viscosity enhancing factor, so as to prolong the contact with the mucous membrane. It is assumed that with a spray dosis of ≧50 μg, 1-2 sprays, 2-4 times per day in each nostril is sufficient for prophylactic purposes. The composition may also be administered in the form of nose drops having the same composition as the spray except for the propellant, and presumably having a higher concentration of SAP. It is assumed that 2-4 drops, 2-3 times per day in each nostril is sufficient for prophylactic purpose. In both cases the dosage for therapeutic purposes will depend on the severity of the infection and will be determined in consultation with the responsible physician. The composition may also be formulated as a nasal ointment. Native SAP may be isolated by a combination of different purification methods (17, 18, 19). We have preferred affinity chromatography on Sepharose or DNA-agarose combined with anion-exchange chromatography, and for some preparations, a final affinity chromatography on Protein G-Sepharose. The method of preparing SAP is described in further detail below in Example 1. Modifications of this procedure for isolating native SAP may also be used. The purity of the isolated SAP preparation is analyzed by SDS-polyacrylamide gel electrophoresis, Western blotting and electron microscopy. This characterization of isolated SAP may be supplemented by other methods for analyzing the purity of protein preparations. According to the present invention, the so isolated SAP or SAP produced by recombinant techniques may be utilized to bind influenza virus and prevent transmission of and infection with the virus. Subunits/Peptides of SAP can be prepared which may have the necessary biological functionality and the invention also comprises the use of such subunits/peptides that bind influenza virus and inhibit virus infectivity. In particular subunits of SAP comprising one or more complete SAP polypeptide chain(s) or a part or parts of the chain comprising the Ca++ dependent carbohydrate binding site(s) can be prepared or synthetic peptides constructed to contain amino acid sequences contributing to said binding sites may be used. With respect to bringing SAP in contact with the virus in infected individuals, SAP in liquid phase containing Ca++ is incorporated into a vehicle for nose drops, nose spray or a nebulizer and the drops or spray are applied to the nose or upper respiratory tract. In this way influenza virus released from infected columnar epithelial cells becomes coated with SAP molecules that bind preferentially to the virus hemagglutinin. This inhibits the local spread of virus to other epithelial cells as well as the transmission of virus via small-particle aerosols to other susceptible individuals. With respect to preventing the transmission of influenza virus to susceptible but not infected individuals SAP is brought in contact with the virus by application of SAP-containing drops or spray to these individuals as described above. The virus-carrying small-+particle aerosols produced by influenza virus infected individuals will in this way come in contact with fluid phase SAP that inhibits the infectivity of the virus before it reaches the epithelial cells in the upper respiratory tract. A test kit for determining the binding of SAP to the prevailing influenza virus strain is also part of the present invention. The kit comprises a container holding a solution of SAP and a container with indicator cells. The invention further comprises a method of detecting or measuring the binding of SAP to the prevailing influenza virus strain in an epidemic. Purified native SAP or recombinant SAP and buffer only are incubated with the virus isolate, dilution series are prepared and indicator cells are added. The ability of SAP to inhibit the virus hemagglutination reaction can now be determined by comparing the hemagglutination titer in the two dilution series. Alternatively, the binding of SAP to influenza virus can be detected by conventional immunoassays, including radioimmunoassay, enzyme immunoassays and fluorescence assays. The performance of these assays is known to those skilled in the art. The invention is further illustrated by the following examples. Hemagglutination inhibition (HI) tests were performed to compare the binding of different SAP preparations to several different influenza A virus strains. SAP was isolated from normal human blood plasma by a standard procedure previously described (see Ref. 18). The citrated plasma was stored at −80� C., thawed at 4� C. and recalcified to 20 mM CaCl2. The serum (200 ml) was centrifuged at 3.000 g and 4� C. for 10 min, diluted 1:1 in 100 mM Tris, 150 mM NaCl (TBS) containing 2 mM CaCl2, pH 7.4, and applied to a Sepharose-4B-Cl column, at 4� C., with a bed volume of 162 ml and a flow rate of 0.5 ml/min. The column was washed with the loading buffer until the absorbance at 280 nm was less than 0.001. Bound proteins were eluted with TBS containing 2 mM EDTA, pH 7.4. In some purifications 0.5 mM cyclokapron, 0.5 mM iodoacetamide and 0.5 mM PMSF, final concentrations were added to the Sepharose eluate. The protein-containing fractions were pooled and dialyzed against 10 mM Tris, pH 7.4, before further purification and characterization by anion-exchange chromatography on a MA7Q column (Bio-Rad, Richmond, Calif., USA) (bed volume 3.5 ml) using the Pharmacia FPLC system. Elution was performed with a 0-1 M NaCl gradient for 60 min in 10 mM Tris, pH 7.4. The flow rate was 1 ml/min and 0.5 ml fractions were collected. The protein concentration of SAP preparations was measured by UV absorption using E280=1.84. The purity of SAP in the peak fractions from the anion exchange column was estimated by SDS polyacrylamide gel (4-20%) electrophoresis developed with silver nitrate, by Western blotting, performed as described previously and by electron microscopy (FIG. 1). The SAP may be further purified by passage through a Protein G-column which removes traces of immunoglobulins. For hemagglutination inhibition assay (HI) microtiter plates with round-bottomed wells (Nunc) were used. SAP is diluted in TBS containing 1 mM Ca++ to a final concentration of 3.2 μg/ml and 50 μl is transferred to the first well. From this well a two-fold dilution series in TBS+1 mM Ca++ is prepared. To all wells is added 50 μl virus (whole or HA subunits) containing 4 HA units of virus. The 4 HA units have been determined in a HA test. The microtiter plate is incubated for 30 min at 20� C. with slow agitation. Washed chicken erythrocytes or human type 0,Rh- erythrocytes (50 μl, 1.5% suspension) are added to all wells and the plate is incubated for 30 min at 33� C. The lowest concentration of SAP which inhibits the HA reaction is determined by visual inspection of the plates. The results obtained with two different influenza A virus strains are shown in FIG. 2. Total inhibition of virus HA was seen when virus was incubated with 0.8-1.6 μg SAP/ml. For studying the influence of Ca++ on the binding of SAP to influenza A virus, SAP was diluted in TBS without Ca++ or with the addition of Ca++ at different concentrations or with addition of EDTA (10 mM). The HI test was then performed as described above. The inhibitory effect of SAP on virus HA is Ca++-dependent (FIG. 3) and no inhibition is seen in the absence of Ca++. Other data have shown that physiological levels of Ca++ (1.2-1.5 mM) are sufficient for optimal inhibition of virus HA by SAP. To determine whether specific polyclonal antibodies to SAP were able to interfere with or abolish the HI activity of SAP, SAP preparations were preincubated for 30 min at 20� C. with increasing antibody concentrations. Polyclonal antibodies against C-reactive protein were used as control. The HI test was then performed as described above. The results (FIG. 4) show that polyclonal antibodies against SAP abolished the SAP-mediated inhibition of virus HA. Antibodies against other serum proteins exerted no influence on SAP's binding to the influenza virus. HI tests were also performed to determine whether monosaccharides or glycosaminoglycans would influence the HI activity of SAP. SAP was diluted in TBS with 2 mM Ca++ and preincubated for 30 min at 20� C. with 10 mM monosaccharides or 50 μg/ml glycosaminoglycans. The HI test was then performed as above. Both heparin, heparan sulphate and D-mannose reduced, but did not abolish the antiviral activity of SAP. Heparin is known to bind with high affinity to SAP and the effect of this glycan was most pronounced. To investigate whether denaturated and renaturated SAP would show HI activity two treatments were compared. One treatment comprised the reduction of SAP (1.0 mg/ml) with 1,4-Dithio-L-threitol (0.15 M) for 2 h at 20� C. followed by dialysis for 2 h at 20� C. against TBS. A control SAP sample was treated in the same way with the exception that Dithio-L-threitol was replaced by TBS. The HI test, performed as described above, showed that 1 to 2 μg/ml of the reduced and dialyzed SAP inhibited the HA reaction. The control SAP preparation showed the same inhibitory activity at 0.5-1 μg SAP/ml. The second type of treatment was exposure of SAP (1.0 mg/ml) to pH 4.0 (0.1 M HCl) for 30 min at 20� C. followed by gradual titration with 0.1 M NaOH for 30 min back to pH 7.4. A control SAP preparation was incubated at 20� C. for 1 h. The HI test, performed as described above, showed that SAP's inhibitory activity was not reduced by the treatment as 0.6 μg SAP/ml of both preparations was sufficient to abolish the HA reaction. To illustrate that SAP could inhibit different strains and subtypes of influenza A virus 10 different strains were tested. It is shown in Table 1 that human SAP (0.2-3.2 μg/ml) inhibited the HA reaction of nine of the strains. It is not known whether also the 10th strain (A/Mississippi/1/85/H3N2) would have been inhibited with a SAP concentration exceeding 3.2 μg/ml.
The procedure when the effect of SAP on virus replication and HA production in cell cultures is investigated is to preincubate the virus suspension with from 0 to 40 μg/ml of sterile filtered SAP for 30 min at room temperature before inoculation of the cultures. After 24, 48 and 72 hrs., 50 μl of the culture medium from infected cultures and control cultures is transferred to the first well (containing 50 μl physiological NaCl) of a microtiter plate and a two-fold dilution series is made in NaCl. Chicken erythrocytes (50 μl, 1.5% suspension) are added to all wells, the plate is incubated for 30 min at 33� C. and the HA titer is read by visual inspection of the plate. To demonstrate the effect of SAP on influenza A virus infection of cell cultures MDCK-cells (American Type Culture Collection) were used. Purified, sterile filtered SAP, 0-150 μg/ml, and influenza A virus/Shangdong/9/93 (H3N2) and Taiwan/1/86 (H1N1) are preincubated for 30 min at room temperature. MDCK-cells are grown at 37� C. to near confluency in chamber slides (Nunc) in BME-medium with 4% fetal calf serum with 1% Glutamax and 1% Gentamycin. The cells are washed with serum-free medium whereafter 2 ng/ml trypsin are added to the SAP-virus mixture and 100 μl of this are transferred to the cells. After incubation for 2 h at 33� C. in 5% CO2, serum-free medium with trypsin-CaCl2 is added to a total volume of 1 ml. The cells are incubated for 3 days at 33� C. with 5% CO2. Every day HA tests are performed on the culture medium, the cytopathogenic effect is read in the microscope and the cell cultures are photographed. On the third day the slides are immunostained with FITC-conjugated monoclonal antibodies to influenza A virus antigens (DAKO). Controls cultures without SAP and without virus and SAP are included. To illustrate the effect of polyclonal anti-SAP antibodies on the virus inhibiting activity of SAP, rabbit antibodies against human P-component (DAKO) were incubated with SAP for 24 hours at 4� C. before performing experiments as described above. The effect of SAP on production of the two influenza A virus subtypes in MDCK-cells is shown in Table 2. Using SAP concentrations exceeding 10-20 μg/100 μl no detectable virus hemagglutinin was produced whereas the HA titer of medium from control virus cultures was 64-128.
Furthermore, no cytopathogenic effect was seen after infecting the cultures with virus incubated with these SAP concentrations (FIG. 5). Single, scattered cells in the SAP-treated cell cultures showed positive immunofluorescence probably due to adsorption of viral antigen not resulting in virus replication. In the presence of anti-SAP antibodies, there was no effect of SAP, and the results were the same as obtained with the virus-infected control. In the cell culture system preincubation of SAP with heparin (10 mM) did not abolish SAP's antiviral effect. For electron microscopic investigations of the binding of SAP to influenza A virus SAP molecules were coated with colloidal gold (5 nm) and incubated with virus subtypes for 1 h at 20� C. in the presence of 2 mM Ca++. A drop (7 μl) of the mixture was placed on glow-discharged grid with a holey Triafol film (Merck) covered with a thin carbon film. The drop was left on the grid for 15 sec and the grid was flushed with 2% aqueous uranyl acetate, pH 4.2. The specimens were examined in a Zeiss EM 902 electron microscope. In Ca++-containing medium the gold particles were seen in contact with spikes on the viral envelope (FIG. 6), the degree of coating with the particles varying with the virus strains used. Incubation of virus with gold particles only or with bovine serum albumin coated with colloidal gold showed a random distribution of the gold particles and no selective reactivity with influenza A virions. To illustrate the binding of SAP to influenza A virus subunits SDS-PAGE and Western blotting analyses were performed. SDS-PAGE of A/Shangdong/9/93 and A/Texas/36/91 was performed in 4-20% polyacrylamide gel with discontinuous buffer under reducing and alkylating conditions. The separated viral subunits were electroblotted onto a PVDF-membrane (Immobilon-P, Millipore). The membrane was quenched with TBS containing 0.1% Tween 20 and 0.1% human serum albumin (HSA) and cut into strips. The strips were incubated with 30 to 60 μg SAP in TBS containing 2 mM Ca++, washed and blocked with 0.1% HSA and incubated for 1 h at 20� C. with biotinylated rabbit antibodies (1 μg/ml) to SAP. The strips were washed 3 times and alkaline phosphatase-conjugated avidin (1:3000) was added and incubated for 1 h at 20� C. After 3 washes the protein bands were developed with p-nitrophenyl-phosphate (1 mg/ml) in 9.7% diethanolamine, pH 9.6. Controls were performed in parallel with SAP diluted in TBS containing 10 mM EDTA and without addition of SAP. SAP exhibited a Ca++-dependent binding to a viral peptide of 50 to 55 kDa (FIG. 7), compatible with the molecular weight of the HA1 polypeptide of the viral hemagglutinin. 1. Bristow, C. L. and R. J. Boackle. Mol. Immunol. 23, 1045-1052, 1986. 2. Kubak, B. M. et al. Mol. Immunol. 25, 851-858, 1988. 3. Cathcart, E. S. et al., New Engl. J. Med. 273, 143-146, 1965. 4. Hawkins, P. N. et al. J. Clin. Invest. 86, 1862-1869, 1990. 5. Anderson, J. K. and J. E. Mole. Ann. N.Y. Acad. Sci. 389, 216-234, 1982. 6. Hess et al. Clin. Chim. Acta 173, 331-336, 1988. 7. Loveless, R. W. et al. EMBO J. 11, 813-819, 1992. 8. Hamazaki, H. J. Biol. Chem. 262, 1456-1460, 1987. 9. Nielsen, E. H. et al. APMIS 102, 420-426, 1994. 10. DeBeer, F. C. et al. J. Exp. Med. 154, 1134-1149, 1981. 11. Schwalbe, R. A. et al. J. Biol. Chem. 266, 12896-12901, 1991. 12. Pepys, M. B. et al. Clin. Exp. Immunol. 97, 152-157, 1994. 13. Wilson, I. A. et al. Nature 289, 366, 1981. 14. Couch, R. B. et al. In: Dayton, D. H. et al. (eds). The Secretory Immunologic System. Washington, D.C.: US GPO; 93, 1969. 15. Atkinson, W. L. et al. Arch. Intern. Med. 146, 1751, 1986. 16. Wood, J. and J. Mumford. Vet. Rec. February 8, 126, 1992. 17. Potempa, L. A. et al. J. Biol. Chem. 260, 12142-12147, 1985. 18. Juul S�rensen, I. et al. Int. Arch. Allergy Immunol. 106, 25-31, 1995. 19. DeBeer, F. C. and M. B. Pepys. J. Immunol. Meth. 50, 17-31, 1982. 20. S�rensen, I. J. et al., Scand. J. Immunology 41, 263-267, 21. Gordon, A, Int. Arch. Allergy Immunology 108, 304-308, 1995. 22. Brede, H. D., Int. Arch. Allergy Immunol. 108, 318-320, 1995. 23. von Itzstein et al., Nature 365, 418-423, 1993. 24. Betts, R. F. and Douglas, R. G. In: Mandell, Douglas, Bennett (eds). Principles and practice of infections diseases. 3rd. ed., Churchill Livingstone, 1306-1325, 1990. 25. Webster, R. G., Karger Gazette, no. 57, 1-3, 1993. 26. Herrler, G. et al. Virology 141, 144-147, 1985. 27. Niesters, H. G. M. and van der Zeijst, B. A. M., Microbiol. Sciences 1, 55-58, 1984. 28. Danielsen, B. et al., Biochim. Biophys. Acta, in press, 1997. 29. Painter, R. H., Ann. N.Y. Acad. Sci. 389, 199-215, 1982 30. Dyck, R. F. et al., J. Exp. Med. 152, 1162-1174, 1980 31. Breathnach, S. M. et al., Nature 293, 652-654, 1981 32. Emsley, J. et al., Nature 367, 338-345, 1994 33. Kinoshita, C. M. et al, Protein Sci. 1, 700-709, 1992 34. Pepys, M. B. et al., Lancet 1, 1029-1031, 1977 35. Kubak, B. M. et al., Fed. Proc. 44, 1189, 1985 36. Hamazaki, H., Biochim. Biophys. Acta 998, 231-235, 1989 37. Schwalbe, R. A. et al., J. Biol. Chem. 265, 21749-21762, 1990 38. Brown, M. R. and B. E. Anderson, J. Immunol. 151, 2087-2095, 1993 39. Butler, P. J. G. et al., J. Exp. Med. 172, 13-18, 1990 40. Pepys, M. B. and Butler, P. J. G., Biochem. Biophys. Res. Comm. 148, 308-313, 1987 41. Hind, C. R. K. et al., J. Exp. Med. 159, 1058-1069, 1994 42. Sarlo, K. T., Cell. Immunol. 93, 398-405, 1985 43. Landsmann, P. et al., Eur. J. Biochem. 223, 805-811, 1984 44. Kushner, I., Ann. N.Y. Acad. Sci. 389, 39-45, 1982 45. Williams, E. C. et al., J. Lab. Clin. Med. 120, 159-167, 1992 46. U.S. Pat. No. 5,221,628 A (Northwestern University) Patent Citations
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Food-Related Songs?
- 96
- Follow
So my fiance and I are looking for background music for when we cut the wedding cake. Typical suggestions include Def Leppard's "Pour some sugar on me" (the lyrics seem a bit inappropriate for a family function...) "Sugar Sugar", etc. I'm looking for something unique and not overly sentimental (we want a fun rather than overly sentimental atmosphere). I tossed out the idea "C is for Cookie" since we have odd senses of humor, but I think it might be too odd. So, what are your favorite food-related songs?
Kcchan, Check out the soundtrack to "The Big Night". (The Big Night is based upon an Italian dinner that is going to make or break the restaurant.) The theme song is frivelous, melodic and people cannot resist smiling when they hear it. And, after all, it IS the Big Night, right?
0 -
Hmm...the ones that immediately come to mind, which may or may not be appropriate for wedding cake cutting:
Food, Glorious Food
Let's Call the Whole Thing Off (you say potayto, I say potahto) (just how odd is your sense of humor? ;-)
Short'nin' Bread (mama's little baby...)
Weird Al's Eat It
If I Knew You were Coming I'd Have Baked a Cake
Can she bake a cherry pie, Billy Boy
Fish heads, fish heads
Alice's Restaurant (not sure there's actually any food in there)
Scenes from an Italian Restaurant0 -
Ice Cream by Sarah McClachlan
Sweetest Thing by U2
Cherry Pie by Warrant (not family friendly!)
Parsley Sage Rosemary & Thyme (not the song's real name, I'm blanking . . . )by Simon & Garfunkel
Candy Man by Christina Aguilera
Hungry Eyes by Eric Carmen
Two Pina Coladas by Garth Brooks
Banana Pancakes by Jack Johnson0 -
So we've ruled out a cheesy wedding band playing "The bride cuts the cake" to the tune of "The Farmer in the Dell"...?
Maybe "Mairzy Doats"?
0 -
re: personalcheffie
The OP said she (he?) would like a fun, not overly sentimental atmosphere at the wedding. I thought Savoy Truffle was a good choice for a song.

subset: commoncrawl


text:
Meijer Interview Questions
Interview Experience
Interview Experience
Getting an Interview
Getting an Interview
Interview Difficulty
Interviews for Top Jobs at Meijer
- Cashier (32)
- GM Clerk (8)
- Gas Station Team Member (5)
- Merchandise Associate (4)
- Intern (4)
- Utility Worker (4)
- Stocker (3)
- Bakery Clerk (3)
- General Merchandiser (3)
- Service Clerk (3)
- Merchandising Analyst (2)
- Grocery Stocker (2)
- Department Manager (2)
- Pharmacy Technician (2)
- Produce Clerk (2)
- Food Clerk (2)
- Clerk (2)
- Area Team Leader (2)
- Inventory Control Analyst (2)
- Retail Clerk (2)
- Manager (2)
- Team Member (2)
- Third Shift Stocker (2)
- Third Shift Stocking Team Member (2)
- Systems Monitor (2)
- Store Director In Training (2)
- Customer Database Marketing Specialist (1)
- Food Services Clerk (1)
- Prepared Foods Team Leader (1)
- Cashier/Service Desk (1)
- Application Details
I applied through college or university. The process took 4+ weeks – interviewed at Meijer in February 2015.
Interview DetailsAfter an on campus career fair, they held next day interviews. It was a 30 minutes interview. About a week later, I received a phone call from HR requesting a second round interview. They provided transportation to this interview in Grand Rapids, MI. The on site interview consisted of three 30-minute interviews and a personality/IQ assessment.
Interview Questions- Tell me about a time when using email was not a sufficient form of communication. Answer Question
Accepted OfferPositive ExperienceAverage Interview - Application Details
I applied online. The process took 2 weeks – interviewed at Meijer.
Interview DetailsIt takes three interviews and hours of mindless tests that teach you nothing and is very redundant. You have to talk to the store director, your department manager, and another manager and they ask you tons of questions that don't really mean anything. As long as you can answer, you are all right. The computer training process is dumb and you will pass. They never give you any real training for the job.
Accepted OfferNegative ExperienceEasy Interview - Application Details
I applied online. The process took a week – interviewed at Meijer.
Interview DetailsToured the store and talked with managers in three different departments, including the store manager. Questions were behavior-based for the most part. My conversation with the store manager centered around my school experience. I later heard that they were most impressed that I dressed up and was conscientious and articulate. All in all it was a painless and positive experience.
Interview Questions- This was my first job, so questions about previous customer service experience were a challenge. They understood and didn't really care that I couldn't answer. Answer Question
Accepted OfferPositive ExperienceEasy Interview - Application Details
I applied online. The process took 5 weeks – interviewed at Meijer.
Interview DetailsDidn't hear back for two weeks after applying. Then had a panel interview at a hiring event. Then 2 weeks later I met with the store director and we discussed the position. I started working a week later.
Interview Questions- Tell me about yourself. Answer Question
Negotiation DetailsAble to negotiate pay and vacation time.Accepted OfferNeutral ExperienceAverage Interview - Interview Details
I was interviewed by three different people from three different departments of Meijer. The first interview was the head pharmacist, the next two were team leaders from grocery and store management. I was impressed by the professionalism and thoroughness of the questions. Once I was hired there was a little bit of rushing through paper work and being asked to sign things that you didn't have the opportunity to read. They said, "don't worry, you'll have access to a copy that you can read later". Other than rushing through the new hire process I felt the standard/quality of the team leaders in all the departments was high and that most employees who had been at the store for a long time appreciated the company enough to remain lifers.
Interview Questions- Describe an unethical situation that you were forced into and and how you responded. View Answer
Negotiation DetailsThey said, "Your starting pay is 9/hr." I said, "Ok."Accepted Offer - Application Details
I applied online. The process took 2 weeks – interviewed at Meijer.
Interview DetailsThe process has changed since I got hired several years ago. Got a phone call to set up a time for an interview. Went in and the interview lasted 20 minutes at the most. Only had one interview with my current manager and started within the next couple weeks. Now all candidates go through at least 2 or 3 interviews and start within a couple weeks.
Interview Questions- Tell them a time at work when I had to make a difficult ethical choice. View Answer
Negotiation DetailsSince my job is in retail there really isn't any negotiation.Accepted OfferPositive ExperienceEasy Interview - Application Details
I applied online. The process took 2 weeks – interviewed at Meijer in November 2014.
Interview DetailsApplied online for the Overnight stocker. I received a call about a week later to come in for an interview. When I arrived I went to the service desk and told them that I was there for an interview with the store manager. They told me to go back and wait in the employee break room. I was kinda weirded out by this because I felt like I wasnt suppose to be there but an employee who was at the door could tell that I was lost an assured me that It was ok for me to wait back there. I waited about 20 minutes and finally a lady came out of the office and told me that I would be interviewing with her instead of the store director. She was extremely nice and it was more like a conversation than an interview. I felt like I had the job before I walked in. After our "Conversation" She told me that they would do my background check and they would call me in for orientation when they received it. About a week later they called me in for orientation in which we filled out paperwork. I started working 2 days later.
Accepted OfferPositive ExperienceEasy Interview - Application Details
I applied in-person – interviewed at Meijer.
Interview DetailsEveryone was friendly and welcoming, which was what prompted me to want to work there. There is a lot of negativity and gossip which the management does not control. The interview process was not representative of the actual working conditions. I feel mislead that the majority of employees are bitter and sullen, not the work atmosphere I expected from my interview.
Negotiation DetailsN/AAccepted Offer - Application Details
I applied online. The process took 2 weeks – interviewed at Meijer.
Interview DetailsA week after applying online I was called and had a phone interview that went extremely well. She then scheduled my second interview in person the same day with two managers. This also went over extremely well. The third interview was about a week later with the store director. Not a very friendly lady but it was a standard interview. As soon as she finished with the questions she simply told me I would not be hired because I was not available on weekends. I would have been understanding of this if it had not been on my application that I was not able to work weekends as well as being asked at the beginning of all three interviews! It was a complete waste of my time.
Interview Questions- Had to explain why my previous managers were unaccountable for other employees slacking off on the job. Answer Question
No OfferNegative ExperienceEasy Interview - Interview Details
It took about a week and a half. I applied online at the company website and got a phone call. I had 2 to 3 interviews. One with the manager and the other with the store director. Then both at the same time. A call came in 2 days later from HR offering the position.
Interview Questions- Describe a challenge that you tackled. Answer Question
Accepted OfferAverage Interview
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Advanced Search
Consulting and publishing firm Network Frontiers has released the Q1 2008 Unified Compliance Framework (UCF), a database used in compliance management systems. UCF maps hundreds of regulations, including privacy information, HIPPA, PCI-DDS and Medicaid/Medicare mandates, into a master hierarchal framework. This latest version of the UCF consolidates cross platform configuration management controls into a single set of controls.
"The UCF harmonizes IT controls from over 400 international regulatory requirements, standards, and guidelines from both technical and legal perspectives," said Dorian Cougias, CTO of the company. "Rather than testing and asserting compliance for each individual regulation, IT organizations use the UCF to save...time and money by distilling compliance requirements to their essence and asserting compliance across multiple authority documents simultaneously."
Created by law firm Latham and Watkins, UCF was first introduced to the market in 2006 and has been integrated into products from Computer Associates and NetIQ. The framework has been used by multiple education institutions, including the University of Delaware, the University of Fairfax, Carnegie Mellon University, the University of Utah, and the University System of Georgia.
"CA GRC Manager integrates the Unified Compliance Framework as an essential resource for the mapping of current controls and regulations as well as the rapid support of future compliance requirements," said Marc Camm, VP of governance, risk and compliance products at CA. "Since the UCF harmonizes thousands of controls, our customers are able to simplify compliance and proactively address new and updated regulations."
"We selected the Unified Compliance Framework because it is the standard for integrated compliance," said Matt Ulery, director of product management at NetIQ. "The UCF allows us to deliver the leading controls content and focus on our core competency of delivering IT and security management solutions."
The UCF organizes real-world IT processes into 12 "IT Impact Zones," each dealing with one area of policies, standards, and procedures, such as audits and risk management. Within each zone, a company can map the overlap between multiple authority documents, create a control list for each zone and clarify any conflicts created by overlapping authority documents.
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text:
Question from EtherealJester
Why wont these monsters let me sleep?
No matter what I do these freaking monsters keep spawning by my bed when I sleep. I'm pretty sure I've already satisfied the requirements for a safe bed but this still keeps happening.
1. My bed is in an enclosed house.
2. None of the walls in my house are made of dirt.
3. My house is lit by torches in every conceivable spot.
4. My bed is not touching any walls.
5. To my knowledge my house isn't near any dungeons.
I've got 2 block thick glass windows in my house but I don't see how that could cause any problems.
4 years ago
My house is not all that big though. It couldn't have a width of anything more then 10 blocks from wall to wall on either side.
4 years ago
How high is your ceiling?
4 years ago
Top Voted Answer
Maybe the monsters act like Freddy Krueger now and haunt your dreams. So, don't sleep. Shoot some adrenaline in your eye.
4 years ago
Mobs (Monsters) should only be able to spawn 24 blocks from your location. The torches aren't helping you; the Mobs do spawn in light. So try making a smaller enclosure and reduce your lighting and see if that helps. Also you can't enter bed from the foot, only the sides.
4 years ago
This is a known bug in the current version of minecraft, current workaround is placing your bed five or more spaces away from the walls.
4 years ago
From what I've heard, the 1.5_01 patch made it so that monsters can pass through doors when you are sleeping -- this was an unintended side-effect of letting wolves move through doors. I haven't run into this problem myself, but you could try blocking off your door with dirt before you go to sleep and see if that fixes it for you.
4 years ago
This is a widely known bug, while looking at the bugs section earlier today in the wiki it seems that it has to do with bed placement and facing, and there is a rumor of mobs being able to trespass walls that are 1 block thick during your sleep. You can check the wiki.
4 years ago
Make an entire room for a bedroom.
4 years ago
This question has been successfully answered and closed
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text:
Pronunciation: /ˌin(t)ərˈlinēāt/
Definition of interlineate in English:
Another term for interline1.
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- Three, I had no other feasible way to respond to the scope and breadth of David's remarks except by interlineating.
Late 17th century: from medieval Latin interlineat- 'interlined', from the verb interlineare.
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TDK Life On Record Wireless Charging Cube (Photo: TDK Life On Record)
TDK Life on Record (NYSE: IMN) today announced the addition of four new wireless speaker products to round out its premium audio line. These new products mark the next evolution of the TDK Life on Record audio portfolio, creating an ecosystem of products that give consumers high-fidelity audio with universal input flexibility and portability in all areas of their lives. Building on the success of its initial audio line launch in 2010 and highlighted by its proprietary "Signature Sound" and sleek designs, the new TDK Life on Record products now integrate Bluetooth® technology to deliver a truly wireless audio experience. Now, audiophiles are free to utilize their mobile devices in any way they choose and still easily play music -- no docking required -- all without sacrificing audio quality.
TDK Life on Record Wireless Boombox (Photo: TDK Life on Record)
Debuting at the prestigious IFA trade show in Berlin in September and available in the U.S. in October, the new line features a Wireless Weatherproof Speaker, Wireless Boombox, Wireless Sound Cube and Wireless Charging Speaker. All four of the new wireless audio products were created to offer an easy connection with Bluetooth A2DP enabled mobile devices in a wide variety of locations and settings. The new line builds upon the high quality of the initial TDK Life on Record audio products with clean, striking designs carefully crafted to make bold visual statements, while delivering high-performance audio. These new additions have already won a 2012 silver "IDEA" design excellence award from the Industrial Designers Society of America.
When used with a Bluetooth AAC enabled device, the products utilize AAC codec technology to deliver superior quality and near-lossless audio. The AAC technology in the TDK Life on Record line removes a layer of compression typically found in Bluetooth-enabled audio devices, giving the listener the truest sound possible. The universal input flexibility also allows users to be more social with their music, sharing music no matter what mobile device they use.
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MandyMG's Canterbury, UK
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Howletts Wild Animal Park
Bekesbourne Lane
"Excellent day out"
A must see if you're in the Canterbury area. A wide variety of animal in fabulous surroundings. The ...
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The Phoenix
67 Old Dover Road
"Lovely, friendly pub"
This lovely little pub has a great landlord and landlady. Nothing is too much trouble and you're alw...
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Old Gate Inn
162-164 New Dover Road
"Good selection on menu"
This is a comfortable restaurant with casual and more formal seating. The layout is quirky, with noo...
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Howletts Wild Animal Park
Bekesbourne Lane
"Great, whole day out"
Well maintained zoo with animals in decent amount of space. Gorillas are a must see! Remember to be ...
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Cafe des Amis
95 St Dunstans Street
"Yummy and relaxed"
This restaurant has a quirky, slightly bohemian feel. The food is of a great standard, with great po...
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Deeson's Restaurant
25/26 Sun Street
"Posh nosh"
We were a largish party and were pleased with our meals on the whole. Each dish was well proportione...
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The Goods Shed, Station Road West
"Good food"
Very good food, service a little 'moody' but not so bad that I wouldn't return. The menu is small bu...
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Marlowe Theatre
The Friars
"Lovely new revamp"
Great programme of up coming events. Comfortable seats and a decent menu in the little restaurant. A...
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Gulbenkian Theatre
Giles Lane
"Small and friendly"
Excellent little theatre, much better value than the equally exciting Marlowe. Some great, less well...
read more


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Definition of no longer in English:
Not now as formerly: they no longer live here
More example sentences
- After all, they are the ones who have forgotten that we no longer live by the law of the jungle.
- Sorry to have to let you know that Steve is no longer living with us and I am now a single mum.
- First, the working class and the oppressed can no longer go on living in the old way.
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Worried about the data retention scheme? Protect your privacy with this simple to use software. It's the service I use at home and at work. - Chief -
Thank God it's footy season!
The latest from the Hunt/Suns Situation, preview the NAB Challenge matches from round 1, look at upcoming rule changes, umpire trials, GWS and Gold Coast financials and womens footy.
With The_Wookie, Messenger and Mike__
Labor MP accused of rorting union credit Part II
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White Paper: Intel® Atom™ processor Z670/Z650 -based reference board single and dual stream video decode results, recommendations. (v.001, June 2012)
The embedded Intel® Atom™ processor Z6xx series with Intel® SM35 Express Chipset is optimized for low power, battery-capable, embedded tablet designs.
Bill of Materials: Provides a list of required component parts for the Alpine Bay customer reference board—Oak Trail Platform.
Updates to the Intel® SM35 Express Chipset Datasheet, including device and documentation errata, specification clarifications and changes.
Maxim MAX8958* Power Management IC for Intel® Atom™ processor-based platforms key specifications, overview, technical documents, and ordering info.
Information about the Nuvoton NPCE791A0Dx* family of embedded controllers, which has an embedded RISC core and is targeted for portable applications.


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"This is a perfect way to personalize the Baby Wish Cards with your future baby's name, etc. I was also able to include a pic of the sonogram on back and also include a pic of the parents. It's so sweet!"
"Loved the design of this invitation. Easy to incorporate the baby carriage theme for the shower as well. Used the modified rectangular envelopes and ordered the solid color back."
"I could not be happier with my baby shower invitations, they were even better than I imagined they would be. The cards are great quality and with a gorgeous shimmer!! And the print looks fantastic! I can't wait to send these out!! Also very impressed that they arrived in 1 week all the way to Australia!! I will be ordering my Thank You cards and Birth Announcements with Tiny Prints as well."
"This card was better than I expected. The quality, the printing and the customer service was out of this world. I will always use tiny prints for future needs and I will be recommending this product to everyone,"
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The Degas Wears Prada
August 19, 2013
At the Metropolitan Museum of Art in New York, this summer's exhibition "Punk: Chaos to Couture" endured one of the angriest and most universal critical drubbings in recent memory.
Eleven Astonishing Works of Art Made from Books
August 13, 2013
The 25 contemporary artists featured in Art Made From Books (forthcoming from Chronicle Books) may use similar materials, but their work exhibits extraordinary range—themes that range from nostalgia and the passage of time, to history and nature, to
Why the Impressionists Still Matter
August 08, 2013
Tired of the Impressionists? "Impressionists on the Water" at the Legion of Honor Museum in San Francisco will re-awaken your love.
The Departure of LA MOCA Director Jeffrey Deitch: How the Box Office Took Over the Modern Art Museum
July 26, 2013
Jeffrey Deitch embodied the fashion-forward approach to art. Now he's fashion-forwarded himself out of a job.
"Steel Skyscrapers Reflecting Down"
July 22, 2013
The Instagram photography of Alex Ogle.
Spectacle for Spectacle's Sake
July 10, 2013
Turrell is a minor poet with the ambitions of a megalomaniac.
Playboy in Marfa
June 26, 2013
There is nowhere immune from the market and from the marketers, not even the middle of the desert.
The Neutralization of Chinese Art
June 24, 2013
Just how much artistic freedom does China allow?
Must-See Summer Art Shows
June 19, 2013
Three idiosyncratic exhibits at the Met, MoMA, and Neue Galerie
About that Rand Paul Cover Photo...
June 17, 2013
An interview with Platon, who photographed this issue's cover with Rand Paul, discussing the experience, his art, plus various portraits throughout.


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Firewall Router VPN with biult in Switch, Wan, Console port
© Robert Szczepanek - See portfolioPricing
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Country | United Kingdom of Great Britain and Northern Ireland |
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Conceptual | Technology Computers Connecting/Dialing |
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UCSB has taken over the #1 position on the U.S. News and World Report's
Zakiya Luna's research is in the areas of social change, sociology...
We are turning FIFTY! Sociology's PhD program was founded during the
Help us contribute to educate the researchers of the future.
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Apple Case Design in 2013, Part 2: On Apple Design Specifics
According to internal Apple research that was publicized last year, roughly 80% of iPhone owners use cases to protect their devices, and obviously iPod and iPad cases are popular, too. iLounge recently reached out to some of the biggest case designers in the industry to gather their thoughts on various aspects of case design. Here's part two of our three-part series on the state of Apple case design in 2013, focusing on how developers accommodate Apple's published case design recommendations; part one can be found here, and discusses developers' priorities and general standards for protection.
iLounge: Why do some cases have such huge holes around the rear camera?
Michael Pizzimenti, Director of Sales & Marketing, X-Doria: When the iPhone 4 was first launched with the flash, a lot of cases had small openings that caused photo issues due to reflection and discoloring. A larger camera hole opening will insure that the photo you take remains clean and crisp. Additionally, a lot of cases use a black ring around the opening to make extra sure that flash reflections do not pose an issue.
Jay Jun, Marketing Associate, Spigen: We choose to keep it as close to the camera shape as possible. We know of Apple's specifications regarding the recommended camera cutout, but after R&D testing we've concluded that the camera quality is unobstructed as long as we used a black ring inside the cut-out. We take a lot of pride in our ability to complement the original design of the device and would never cut a bigger hole than necessary.
Brian Thomas, President and CEO, Otterbox: With the quality of smartphone cameras rivaling those of pocketable point-and-shoot cameras, we do our absolute best to ensure that we don't hinder the phone's ability to take a great picture; there is a delicate balance between protection and function that needs to be achieved. We take into account the manufacturer's recommendations regarding the camera and the flash, but we also do our own internal testing and work with external testing resources to ensure quality photos in all lighting conditions.
Bryan Hynecek, VP of Design, Speck: Design of the camera hole on Speck cases is all about enabling the best photos possible. The size and our black interior band reduces glare and prevents any capture of the color of the case.
Steve Bau, Managing Director, SwitchEasy: There is nothing worse than taking a picture of an unforgettable moment, and there is a flare from the flash or a rim of the case is in that picture. That is the reason why most of the me-too companies follow the safe route and over-dimension the camera hole. For aesthetic reasons, SwitchEasy likes to minimize the size of the camera holes. Understanding the problems described above, our R&D department spends a lot of time and effort to test different shapes and finishes to optimize the functionality.
iLounge: Apple specifies an open "keepout area" for Lightning connector in its iPod, iPhone, and iPad dimensional drawings, but the area is way smaller than is needed for speakers with Lightning docks. How much of a challenge is accessory compatibility when you're creating cases?
X-Doria: Maintaining the phone's full functional use is one of the most important things we do with case design. Anything that hinders that experience is a reflection on the case, not the phone. Therefore, we start all design by understanding how the device is going to be used on a daily basis. While it is challenging to design, understanding this before beginning the work definitely makes things simpler.
Barry Sween, Design Director, Belkin: We try whenever possible to accommodate for dock connection. With hard shell cases this is usually not a problem because they are strong enough to stay attached firmly, even with a large opening. Sometimes, to gain the advantages of softer, more hand friendly and tactile case materials, making a large dock connector opening would mean limiting the structural integrity of the case. That means we have to make a compromise, and we usually feel that everyday comfort and protection is the most important consideration.
Otterbox: At OtterBox, we always engineer the cases for compatibility with OEM-included accessories. While we can't always ensure compatibility with third-party accessories, we do always open up connector and headphone ports as large as the design will allow without hindering protection in an attempt to accommodate the wide variety of accessories out there. Likewise, new accessories are always becoming available. We do our best to listen to customer comments and try to incorporate their feedback into designs.
Doug Broadhurst, Marketing Manager, Scosche: We recognize that most consumers will need to connect their device when the case is installed, so we do our best to keep these areas as open as possible without compromising the case's structural integrity.
SwitchEasy: At SwitchEasy, we like to make our case designs use case specific. And in any case, we need to keep in account the mobile device we make the case for and its dedicated charging connector such as the iPhone 5 and the Lightning connector. But for a value-driven brand like ours, it depends on the use case whether we decide to keep in account the hundreds of external products that are available on the market; a minimal and functional product like our Nude will have a lot more compatibility than a Monsters or a Bonds case, as their primary design objective is not compatibility, but protection and identity.
Coming tomorrow, part three discusses innovation and future trends in case design. Part one on design priorities and protection is here.
Related Stories
- Apple Case Design in 2013, Part 3: On Changes, Innovation, and the Future
- Apple Case Design in 2013, Part 1: On Protection + Priorities
- Interview: Soundfreaq On Apple + The Accessory Market
- Interview: How Soundfreaq Designs + Prices Speakers
- Tapbots: The iDesign Interview
- SwitchEasy: The iDesign Interview
If you have a comment, news tip, advertising inquiry, or coverage request, a question about iPods/iPhones/iPad or accessories, or if you sell or market iPod/iPhone/iPad products or services, read iLounge's Comments + Questions policies before posting, and fully identify yourself if you do. We will delete comments containing advertising, astroturfing, trolling, personal attacks, offensive language, or other objectionable content, then ban and/or publicly identify violators.
Recent News
- Revamped Apple music service not coming until WWDC; new Apple TV later this year?
- Larger iPad production delayed until later this year, USB 3.0 ports to be added?
- Apple, others likely to reach $415M settlement in poaching lawsuit [Update: settlement approved]
- Apple and HBO in talks for 'HBO Now' service launch
- Report: Next-gen 'iPhone 6s' to feature 2GB RAM, Apple SIM
- Apple looking to settle with electric car battery company
- Apple pushing out fix next week for 'FREAK' security flaw
- Report: Apple Watch retail strategy aims to push iPhone sales
- Apple back on top in phone sales for first time since 2011
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- Mobile Home Siri remote
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- ExoLens Professional Photography System for iPhone 6
- Incipio offGRID Shine for iPhone 6
- Seidio Obex for iPhone 6
- RooCase GlacialTough for iPhone 6 Plus, VersaTough for iPhone 6 + 6 Plus
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FTCE (REA) - Professional Education Test The Best Teachers'
REA … Real review, Real practice, Real results. Take your teaching career to the head of the class. FTCE Professional Education Test - 4th Edition For Florida Licensure Are you prepared to excel on the FTCE? * Get to know the test, how it is scored, and much more. * Set up a study schedule by following our flexible, results-driven timeline. * Take one of the practice tests to discover what youJob's Belief and Faithfulness Rewarded by God
MY STORY I was born in Waynesboro, Georgia to the proud parents, Annie Miller and Smith Biggens. My mother had 10 children, having been married twice. My brother, sister and I were the three children born to her second husband. My paternal grandmother was named Sylvia, therefore, my father thought it was befitting to name me after one so dear to his heart. At age 16 my parents passed away. In 1946Praising and Worshipping Jesus from the Heart Through Psalms and Songs
Praising and Worshipping Jesus from the Heart Through Psalms and SongsThis book is for believers who want a closer relationship with God. It is for those who want to accept Jesus as their Lord and Savior. Praise and worship needs to occur on a daily basis. True worship consists of a feeling from the heart; humility, and love. True worship frees us from bondage. The psalms and songs throughout thisBalanced Assessment: From Formative to Summative
Learn how to integrate formative and summative assessments seamlessly into instruction. The research, rationale, strategies, and examples provided in this book will help teachers develop their own repertoire of formative and summative assessments to monitor, grade, and make inferences about a student's ability to meet standards and curriculum goals. Exercises at the end of each chapter provideThe Nature and Nurture of Learners
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Prospective and in-service teachers are the intended readers of this book. Teaching involves much more then dispensing knowledge. Teaching is a process of arranging activities that will enable individuals to learn and behave appropriately. The appropriateness of the activities depends on the degree they interact with the status of the targeted individuals. Just as physicians need to know about


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iPhone Video proof of concept posted
iPhone developer Drunknbass has been working amazingly hard for some time now getting video recording working on the iPhone. I've been following his effort and seeing him struggle with some major obstacles. It's not a simple thing. There are issues about capturing the data and producing output in real time. And here, sometimes events outpace us. I was hoping to post a real video with this write-up but since the software has already been leaked, I decided to go ahead and write about what currently exists.
Drunknbass's program is groundbreaking. It records 5 seconds of video at 10 fps and then immediately plays it back and proves that usable video capture is possible.
Read more about his work and visit his website at monsterandfriends.com.
Software Updates
more updates- Apple Remote Desktop updated with Yosemite support
- OS X Yosemite 10.10.2, iOS 8.1.3 updates now available
- Sports Illustrated 120 SPORTS channel comes to Apple TV
- Logic Pro X update brings AirDrop support, new effects, tools, and more
- Parallels Access 2.5 released, adds file manager, computer-to-computer remote access
- The Google Translate iOS app is about to get a lot smarter


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'Calvary': The Wages of Sin
Threat of death is the dramatic engine that drives 'Calvary'
Don't be beguiled by the delicious wit or the flow of rich language in John Michael McDonagh's "Calvary." Or be beguiled as long as you like—the film, set in Ireland, is easy to enjoy from the start almost to the finish—but keep in mind that the title expresses the filmmaker's deep seriousness.
More Reviews
Brendan Gleeson is Father James, a sharp-tongued priest in a small rural parish on Ireland's spectacular west coast. In a more innocent era he might have been played, with sly ebullience, by Barry Fitzgerald, but this priest, in this cynical time, is a perfect fit with Mr. Gleeson's gift for taut irony. A troubled man with a troubled daughter—he was married before entering the priesthood—James is also an exemplary man who may pay the ultimate price for his probity. A parishioner has threatened to kill him—not because he's guilty of any transgression but because the death of an innocent man will call more attention, or so the would-be killer contends, to his suffering as a child, when he was repeatedly molested by a priest. (In an fine supporting cast, the standouts are Dylan Moran, Chris O'Dowd, Aidan Gillen, M. Emmet Walsh and Kelly Reilly, who is heartbreakingly lovely as James's daughter, Fiona.)
Related Reading
Threat of death is the dramatic engine that drives "Calvary," Mr. McDonagh's strong second feature; his first, a broad buddy comedy called "The Guard," starred Mr. Gleeson as a corrupt Irish cop and Don Cheadle as a tightly-wound FBI agent. For much of the time the engine hovers near idle while Father James makes his rounds through a town that abounds in picturesque characters, several of them plausible candidates for the assassin, whose identity becomes known to the priest but not to us. In the end, "Calvary" hurtles to a climax that's followed by a quick recap of the town's griefs and tribulations. It's a film of modest means and great ambition, a darkly comic drama concerned with nothing less than the place of faith, and an embattled Church, in modern life.
DVD //Streaming //Download
'In Bruges' (2008)
Brendan Gleeson and Colin Farrell are hit men, and Ralph Fiennes is their scarily explosive boss, who sends them to Bruges, Belgium, with instructions to wait —not for Godot, though there's a bit of that feeling, but for further instructions about who gets killed next. The violence can be shocking, but Martin McDonagh's film is also very funny, and a morality play that's deeply affecting. Jordan Prentice is a racist dwarf and Clémence Poésy plays Chloe, a woman who is not, but also is, the person she seems to be.
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'Wild Tales' Review: A Collection From the Edge
'McFarland, USA' Review: Running Against Expectations


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Prime Minister Stephen Harper vowed Friday to review Ottawa's role in the regulation of exotic animals as questions mounted over why Environment Canada delivered the python that killed two boys to a pet store in New Brunswick.
Environment Canada said one of its wildlife officers brought the African rock python to Reptile Ocean in Campbellton 11 years ago, even though the species was banned in the province without a special permit.
Mark Johnson, a spokesman for the federal department, said in an email that Environment Canada was asked to help take the snake to Reptile Ocean after it was abandoned at the SPCA in Moncton, N.B. His email did not address why the department would transport a banned animal, but he said department records indicate Reptile Ocean was operating as a zoo when the snake arrived at the facility in August 2002.
New Brunswick's Department of Natural Resources said only accredited zoos can apply for a permit to own a banned species, including the African rock python in question.
But Canada's Accredited Zoos and Aquariums said it is the only recognized national body to accredit zoos in the country and it has no record of Reptile Ocean ever applying for accreditation.
The Natural Resources Department declined to say whether Reptile Ocean was issued a permit for the python. But it has said it was unaware of the snake's existence until the deaths of four-year-old Noah Barthe and his six-year-old brother Connor this week.
No one from the department was made available for an interview. Spokeswoman Anne Bull declined to say in an email whether Reptile Ocean was ever considered a zoo, citing the ongoing police investigation and privacy concerns. She was unable to say whether unaccredited zoos can legally operate in the province.
Animal welfare advocates said the case in New Brunswick highlights major gaps in laws governing exotic animals.
"In this instance, there were laws prohibiting it, but clearly enforcement was either non-existent or inadequate," said Nick Wright, a lawyer and executive director of Animal Justice Canada.
"There's no uncertainty that the African rock python is a species that should not be permitted to be kept in captivity in the way that it was."
Melissa Matlow, a Toronto-based animal advocate, said she was appalled that the python would be taken to Reptile Ocean without the appropriate measures in place.
"If (that) is confirmed to be true, then it's shocking that Environment Canada would just drop a snake off at a place that sounds like it didn't have proper permits," said Matlow, a spokeswoman for the World Society for the Protection of Animals.
"I think our regulations are grossly inadequate across the country."
Harper said the federal government will determine whether it should play a role in the regulation of exotic pet shops.
"We're going to look at all that has happened here to get all of the facts," Harper said in Miramichi, N.B., after an unrelated funding announcement.
"My understanding is that these types of establishments are regulated mainly by provincial governments, but at our level as well, we will try to ascertain exactly what has occurred and if there is a federal role, what needs to be done about that."
Matlow said most provinces fail to properly inspect pet stores, zoos and other similar facilities, adding that in many cases, inspections are done only when a complaint has been made.
New Brunswick's Department of Natural Resources should have known there was an African rock python at the store in Campbellton, she said.
"There should have been an inspection and there should be an inventory of all the animals that he has there to ensure all the animals are legal and permitted."
The store is owned by Jean-Claude Savoie, a family friend of the boys who took them shopping and to a farm before hosting a sleepover Sunday along with his son. Savoie has not returned repeated messages for comment.
Zoo staff seized 23 reptiles and euthanized four alligators Friday that belonged to Reptile Ocean.
— With files from Michael Tutton in Miramichi, N.B., and Geordon Omand in Halifax
Copyright 2013 Canadian Press.
All rights reserved. This material may not be published, broadcast, rewritten, or redistributed.