Trypsin catalyzed hydrolysis of new chromogenic arginine substrates

Trypsin catalyzed hydrolysis of seven new chromogenic arginine substrates, N alpha-benzyloxycarbonyl-L-arginine-3-nitro-5X-anilide (X = H, CF3, SO2CH3, F, Cl, Br and I) were studied. These substrates are suitable for studying electronic effects on trypsin activity. The Km and kcat values for the hydrolysis of each substrate were determined and found to differ significantly for the various substrates. The Hammett plot of the catalytic rate constants gave a straight line with a negative rho value (-0.82) thus indicating that electron withdrawing substituents retard the trypsin catalyzed hydrolysis of the new anilide substrates.     

Purification of acetylcholinesterase from horse erythrocytes using affinity chromatography

A commercial preparation of water-soluble acetylcholinesterase from horse red cells has been purified to a specific activity of 2380 U/mg of protein (a 1660-fold purification) by a twofold affinity chromatography on the known sorbent of Sepharose-p-[NH-(CH2)5-C(O)NH(CH2)5C(O)NH-]-C6H4-N+(CH3)3 X Br- at pH 7.5. A selective elution of the enzyme was carried out from 10 mM of the phosphate buffer solution which contains 0.2% of triton X-100. Subsequent desorption of the enzyme proceeded with 5 mM of phenyltrimethylammonium bromide introduced into the buffer. Such effective preparations of acetylcholinesterase have not been previously produced. Effectiveness of the affinity sorbents considerably depends on the nature of the ligand which is covalent-linked with a Sepharose matrix and on the length of the attachment spacer arm ("insert") between them. A reversible inhibitory effect of certain ligands (tetramethylammonium, phenyltrimethylammonium) and their derivatives on acetylcholinesterase is estimated in comparison.     

Transforming potential of a myc-containing variant of feline leukemia virus in vitro in early-passage feline cells.

We studied a naturally occurring variant of feline leukemia virus (FeLV) in which the oncogene myc has substituted for a portion of the viral structural genes (myc-FeLV). myc-FeLV was rescued by replication in the presence of FeLV as helper, and its biological activity was examined in early-passage feline cells in vitro. Infection of leukocytes from peripheral blood, spleen, or thymus, or of kitten fibroblasts did not immortalize these cells or alter them morphologically. Northern blot (RNA blot) analysis of virion RNA prepared from the supernatant of infected cells demonstrated the 8.2-kilobase genome of FeLV, but did not demonstrate the 5.0-kilobase genome of myc-FeLV. Apparently, the myc-FeLV genome was lost in the absence of the selective pressure of transformation. In contrast, infection of embryonic fibroblasts with myc-FeLV(FeLV) rendered these cells capable of greatly increased, if not infinite, proliferative potential. The cells were morphologically altered compared with controls and were only loosely adherent to the substrate. The cells failed to proliferate in semisolid medium and did not form tumors when inoculated subcutaneously into athymic mice. Blot analyses demonstrated the presence and expression of integrated proviral DNAs of both FeLV and myc-FeLV in these cells. They appear, then, to represent cells partially transformed by infection with myc-FeLV(FeLV). The action of feline v-myc in early-passage cells in vitro was compared to that of avian v-myc.     

Simian virus 40-transformed human cells that express large T antigens defective for viral DNA replication.

Many types of human cells cultured in vitro are generally semipermissive for simian virus 40 (SV40) replication. Consequently, subpopulations of stably transformed human cells often carry free viral DNA, which is presumed to arise via spontaneous excision from an integrated DNA template. Stably transformed human cell lines that do not have detectable free DNA are therefore likely to harbor harbor mutant viral genomes incapable of excision and replication, or these cells may synthesize variant cellular proteins necessary for viral replication. We examined four such cell lines and conclude that for the three lines SV80, GM638, and GM639, the cells did indeed harbor spontaneous T-antigen mutants. For the SV80 line, marker rescue (determined by a plaque assay) and DNA sequence analysis of cloned DNA showed that a single point mutation converting serine 147 to asparagine was the cause of the mutation. Similarly, a point mutation converting leucine 457 to methionine for the GM638 mutant T allele was found. Moreover, the SV80 line maintained its permissivity for SV40 DNA replication but did not complement the SV40 tsA209 mutant at its nonpermissive temperature. The cloned SV80 T-antigen allele, though replication incompetent, maintained its ability to transform rodent cells at wild-type efficiencies. A compilation of spontaneously occurring SV40 mutations which cannot replicate but can transform shows that these mutations tend to cluster in two regions of the T-antigen gene, one ascribed to the site-specific DNA-binding ability of the protein, and the other to the ATPase activity which is linked to its helicase activity.     

Proteins present in bovine papillomavirus particles.

Analysis by two-dimensional gel electrophoresis and silver staining of heavy full, light full, and empty bovine papillomavirus particles has shown that the major capsid protein L1 is highly modified. Besides exhibiting at least 13 isoelectric point variants of approximately the same molecular mass (54 kilodaltons), it is suggested that an additional heavier protein chain (69 kilodaltons) is also derived from L1 by glycosylation. These modifications may stabilize the particle structure. Treatment with neuraminidase reduces the number of modification products detectable, with a concomitant increase in the more basic forms of L1. Although it was not possible to detect histones in any of the preparations, proteins of similar molecular mass were detected. Therefore, it is suggested that the basic tails of L1 bind to the DNA in a manner similar to that of histone. Calculation of the theoretical mobilities of the papillomavirus proteins shows good agreement with the actual position of L1 and its isoelectric point variants and suggests that two of the proteins with molecular masses similar to those of the histones may actually be coded by the bovine papillomavirus E7 and E5 open reading frames.     

Genetic and biological analyses of a herpes simplex virus intertypic recombinant reduced specifically for neurovirulence.

RS6 is a herpes simplex virus intertypic recombinant derived from type 1 strain 17 syn+ and type 2 strain HG52. With a 50% lethal dose of about 10(5) PFU after intracerebral inoculation of mice, RS6 was approximately 100,000 times less neurovirulent than either of its wild-type parental viruses were. When compared with strains 17 syn+ and HG52, RS6 replicated intermediately in primary mouse embryo fibroblasts in vitro at 38.5 degrees C (mouse temperature) and to wild-type peak titers in mouse feet in vivo. In contrast, following intracranial inoculation of mice, RS6 replicated significantly less well than did either of its parental viruses in brains. The genetic defect(s) responsible for the reduced neurovirulence of RS6 was stable after in vitro and in vivo serial passage, was not manifested as temperature-sensitive plaquing in vitro, and did not affect thymidine kinase expression. These data indicate that RS6 has a genetic defect(s) specifically affecting its ability to replicate in the mouse brain. Using marker rescue technologies, we increased the neurovirulence of RS6 and localized one genetic determinant(s) involved with the reduced neurovirulence of this agent to 0.72 to 0.87 map units (and, tentatively, to 0.79 to 0.83 map units) of the herpes simplex virus genome. When coupled with the work suggesting that thymidine kinase expression is essential for efficient replication in nerve tissues and earlier reports from this laboratory and others, the results presented in this study indicate that more than one herpes simplex virus gene is involved with neurovirulence.     

Lack of competition results in efficient packaging of heterologous murine retroviral RNAs and reticuloendotheliosis virus encapsidation-minus RNAs by the reticuloendotheliosis virus helper cell line.

We constructed recombinant reticuloendotheliosis virus (Rev)-derived and murine leukemia virus-derived vectors to characterize the specificity of packaging retroviral RNAs in Rev proteins. Using this approach, we further localized the Rev encapsidation sequence (E) to a 144-nucleotide region and determined that there are sequences in both the 5' and 3' halves of this region which are necessary in cis for viral replication. We found that the Rev E, like the murine leukemia virus E (psi), is position independent (R. Mann and D. Baltimore, J. Virol. 54:401-407, 1986). Also, a 156-nucleotide region of the Rev intron enhanced replication in a cis-acting fashion in the presence, but not in the absence, of helper virus. Finally, we showed that packaging of E- and heterologous retroviral genomes occurred efficiently in the Rev helper cell in the absence of competing E-containing (E+) viral RNAs.     

Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites.