Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production.

Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.     

A new method for stoichiometric analysis of proteins in complex mixture--reevaluation of the stoichiometry of E. coli ribosomal proteins

A novel way is presented for determination of the stoichiometry of ribosomal proteins in the ribosome. The 70S E. coli r-proteins, completely separated on a two-dimensional gel system, were used throughout our experiments. The method is based on our previous observation that the amount of Coomassie R bound to a protein molecule is directly proportional to the number of positive charges on that protein. By plotting the amount of bound Coomassie as a function of the number of positive charges of each r-protein, and relating the experimental amount of the dye bound to each r-protein to the value obtained from the linear regression line based on all (a total of some 50 proteins), one can obtain the molar concentration of every protein in the ribosome. A parallel experiment can be carried out, which relates the radioactivity contributed by 3H-labeled amino acid in each r-protein to its amino acid content in that molecule. The two sets of data, which are completely independent of each other, are well correlated. Further verification of the validity of our procedure is provided by the fact that we found the known proportions of four copies of L7/L12 and one copy of S6 per ribosome. The rationale behind the present study was our finding that recalculation of Hardy's data (Hardy, S.J.S. (1975) Mol. Gen. Genet. 140, 253-274), with the accurate molecular weight value of the r-proteins provided by Giri et al. (Adv. Protein Chem. (1984) 36, 1-78), raises some doubt with regard to his experimental results, although we agree with his final conclusion that E. coli ribosome is homogeneous with respect to its proteins.     

In vivo anti inflammatory effects of the M20 IL-1 Inhibitor: I. Effects on acute inflammatory parameters

Previous studies have described an IL-1 Inhibitor produced by a myelomonocytic line developed in our laboratory (Eur J Immunol 1986; 16: 1449). This IL-1 Inhibitor was secreted by the M20 line constitutively in addition to IL-1, from which it could be separated. We have recently shown that the M20 IL-1 Inhibitor is distinct from the IL-1ra.In vitro this factor inhibited IL-1 induced proliferative responses as well as PGE₂ secretion by IL-1 induced fibroblasts. We also showed for the first time (Lymphokine Research 1988; 7(3): 268) that an IL-1 inhibitor can reduce IL-1 induced inflammatory effects. This study describes the specific effect of the M20 IL-1 Inhibitor on IL-1 induced parameters of inflammation: fever, leukocytosis and local foot pad swelling or lymph node enlargement. Purified preparations of the IL-1 Inhibitor, when injected together with IL-1, or before the IL-1, reduced fever, leukocytosis, foot pad swelling and lymph node enlargement caused by IL-1. Similar responses were obtained by injection of IL-6 or TNF, but were unaffected by the IL-1 Inhibitor, when injected together.These results indicate that the M20 IL-1 Inhibitor acts specifically on IL-1 induced responsesin vivo. The potential importance of this factor as an anti-inflammatory and immune regulatory factor, is supported by the findings of this study.Abbreviations IL-1 Interleukin 1 - IL-6 Interleukin 6 - IL-1ra Interleukin 1 receptor antagonist - TNF tumor necrosis factor     

Role of endogenous gibberellins in germination of melon (Cucumis melo) seeds

The effect of the plant growth retardants ancymidol. mefluidide and uniconazole on germination of two melon accessions differing in their ability to germinate at 14°C was examined. The accessions were the cold sensitive Noy Yizre'el and the cold tolerant Persia 202. The three growth retardants were able to delay the germination of intact Noy Yizre'el seeds, but did not affect that of intact Persia 202 seeds. On the other hand germination of decoated seeds of both accessions was unaffected by these inhibitors at normal oxygen concentration, but was inhibited at 5% oxygen. When gibberellin-like activity was measured by a dwarf rice biological assay following HPLC fractionation, it was found that seeds of Persia 202 contained much more gibberellin-like activity than Noy Yizre'el seeds. Among the extracted compounds several endogenous gibberellins were identified by combined gas chromatography-mass spectrometry (GC-MS). They included GA₄, GA₂₀, GA₁ and GA₃ in Noy Yizre'el and GA₃₄, GA₂₀, GA₁ and GA₈ in Persia 202. It is suggested that the better germination of intact Persia 202 seeds, compared to Noy Yizre'el seeds at low temperature and low oxygen concentration, is due to a higher endogenous level of GA and a better seed coat permeability to oxygen.     

Isolation and characterization of an intermediate steroid metabolite in diosgenin biosynthesis in suspension cultures of Dioscorea deltoidea cells.

The aglycon form of the steroidal sapogenin furost -5-ene-3 beta, 22,26-triol, 3 beta- chacotrioside 26 beta-D-glucopyranoside was isolated from cell suspension cultures of Dioscorea deltoidea and its molecular structure was determined by mass spectrometry and 1H and 13C n.m.r. spectroscopy. From kinetic studies and incorporation experiments with [1-14C]acetate it was concluded that the steroidal compound (in the glycoside form) is an intermediate in vivo in diosgenin biosynthesis. It accumulated in growing cells of D. deltoidea and was metabolized to diosgenin (in the glycoside form, i.e. dioscin ) in non-dividing cells.     

Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses.

The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that determines the host range and surface antigenicitiy of virions. We have purified radioactive DNA (cDNAgp) complementary to at least a portion of the env gene for viral subgroups A and C; complementary DNA was synthesized with purified virions of wild-type avian sarcoma virus, and RNA from a mutant with a deletion in env was used to select DNA specific to env by molecular hybridization. The genetic complexity of cDNAgp for subgroup A (ca. 2,000 nucleotides) was sufficient to represent the entire deletion and most or all of the env cistron. The deletions in env in two independently isolated strains of virus (Bryan and rdNY8SR) overlap, and cDNAgp represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletions in the adjacent viral gene src. Laboratory stocks of viral subgroups A, B, C, D and E do not contain detectable amounts of env deletions when tested by molecular hybridization; hence, segregation of deletions in env is a less frequent event that the segregation of deletions in the viral transforming gene src (Vogt, 1971). We found extensive homology among the nucleotide sequences encoding the env genes of virus strains indigenous to chickens (subgroups A, B, C, D, and E) although subgorups B, D and E appear to differ slightly from subgroups A and C at the env locus. By contrast, viruses obtained from pheasant cells (subgroups F and G) have env genes with little or no relationship to env genes of chikcen viruses. According to available data, viruses of subgroup F arose by recombination between an avarian sarcoma virus and viral genes in the genome of ring-necked pheasants, whereas subgroup G viruses may be entirely endogenous to golden pheasants.     

Regulation of sterol biosynthesis in sunflower by 24(R,S),25-epiminolanosterol, a novel C-24 methyl transferase inhibitor

Whereas sitosterol and 24(28)-methylene cycloartanol were competitive inhibitors (with Ki = 26 microM and 14 microM, respectively), 24(R,S)-25-epiminolanosterol was found to be a potent non-competitive inhibitor (Ki = 3.0 nM) of the S-adenosyl-L-methionine-C-24 methyl transferase from sunflower embryos. Because the ground state analog, 24(R,S)-oxidolanosterol, failed to inhibit the catalysis and 25-azalanosterol inhibited the catalysis with a Ki of 30 nM we conclude that the aziridine functions in a manner similar to the azasteriod (Rahier, A., et al., J. Biol. Chem. (1984) 259, 15215) as a transition state analog mimicking the carbonium intermediate found in the normal transmethylation reaction. Additionally, we observed that the aziridine inhibited cycloartenol metabolism (the preferred substrate for transmethylation) in cultured sunflower cells and cell growth.     

Identification and biochemical analysis of novel olfactory-specific cytochrome P-450IIA and UDP-glucuronosyl transferase

Two major transmembranal polypeptides of bovine olfactory epithelium were identified by SDS electrophoretic analysis of Triton X-114 solubilized membranes. Both polypeptides were present in large amounts in membranes of the olfactory epithelium but were barely detectable in membranes of the nasal respiratory epithelium. Both polypeptides are enriched in the deciliated epithelium as compared with isolated cilia. One of them is a glycoprotein with an apparent molecular mass of 56 kDa (gp56); the other is an unglycosylated protein with an apparent molecular mass of 52 kDa (p52). Sequence analysis of peptides obtained by CNBr cleavage of purified gp56 indicates that it is highly homologous to UDP-glucuronosyl transferase (UDPGT). Parallel analysis shows that p52 is highly homologous to cytochrome P-450 sequences of the IIA subfamily. This protein is assigned the name P-450olf2. Polyclonal antibodies were raised against synthetic peptides corresponding to gp56 and p52 peptide sequences. Immunoblots with these antibodies reveal the following properties of gp56 and p52: (1) they are enriched in the microsomal fraction of the bovine olfactory epithelium; (2) they are possibly specific to the olfactory epithelium, as we could not detect reactivity in microsomes derived from respiratory epithelium or lung, and only a very small amount of basal reactivity was seen with liver microsomes; (3) cross-reacting proteins exist in microsomes derived from the rat olfactory epithelium. These results are consistent with a mechanism whereby the microsomal enzymes are involved in odorant modification and clearance from the nasal tissue.