Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient gamma(c)/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance.     

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: Characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism.Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring.All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized.GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed.We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.     

Hybridization and karyotype variability of three endemic Fritillaria L. (Liliaceae) in Argolis Peninsula (Greece)

Abstract

The Argolis Peninsula covers the north-eastern part of Peloponnisos and is surrounded by the Gulf of Argosaronic. The area hosts three species of the genus Fritillaria: F. graeca, F. rhodocanakis and F. spetsiotica. Fritillaria graeca is a Greek endemic taxon and its distribution includes Peloponnisos, C & E Sterea Ellas, C Evia and in proximity to Sterea Ellas, Salamis and Kea islands, while the stenoendemic F. rhodocanakis and F. spetsiotica are mainly found on Idra and Spetses islands respectively. The last two taxa are included in the Red Data Book of Rare and Threatened Plants of Greece, while F. rhodocanakis is also included in the IUCN Red List. Hybridization among them is a common phenomenon in the areas where they coexist, leading to an array of morphologically and karyologically intermediate forms. The current study presents the taxa’s karyomorphometric analysis for the first time and reveals hybrids’ cytological variety, including differences in marker chromosomes, polyploidy and the number of B-chromosomes.     

Phenanthrene degradation by estuarine surface microlayer and bulk water microbial populations

Paired surface microlayer and bulk water samples from five sites in the Great Bay Estuary, New Hampshire, were examined with regard to numbers of bacteria,¹⁴C-phenanthrene biodegradation potentials, and organic and inorganic chemical characteristics. Microlayer samples were generally enriched in nutrients (N and P), dissolved organic matter, and culturable heterotrophic bacteria compared with their corresponding bulk waters. Microlayer samples from marina environments were also enriched in aromatic hydrocarbons, as determined by UV spectrophotometric and fluorometric analyses, and demonstrated substantial phenanthrene biodegradation activity in the assay employed. Biodegradation activity of marina bulk water samples ranged from nil to levels exceeding those exhibited by microlayer samples. No diminution of biodegradation activity was observed after filtration (1.2 m effective retention) of microlayer water, indicating that the responsible organisms were not particle-associated. Phenanthrene-degrading bacteria, enumerated by counting clearing zones in a crystalline phenanthrene overlay after colony development on a phenanthrene/toluene agar (PTA) medium, were superior to epifluorescence direct counts or standard plate counts on PTA or estuarine nutrient agar in predicting¹⁴C-phenanthrene biodegradative activity.     

The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex

The yeast nuclear gene ATP4, encoding the ATP synthase subunit 4, was disrupted by insertion into the middle of it the selective marker URA3. Transformation of the Saccharomyces cerevisiae strain D273-10B/A/U produced a mutant unable to grow on glycerol medium. The ATP4 gene is unique since subunit 4 was not present in mutant mitochondria; the hypothetical truncated subunit 4 was never detected. ATPase was rendered oligomycin-insensitive and the F1 sector of this mutant appeared loosely bound to the membrane. Analysis of mitochondrially translated hydrophobic subunits of F0 revealed that subunits 8 and 9 were present, unlike subunit 6. This indicated a structural relationship between subunits 4 and 6 during biogenesis of F0. It therefore appears that subunit 4 (also called subunit b in beef heart and Escherichia coli ATP synthases) plays at least a structural role in the assembly of the whole complex. Disruption of the ATP4 gene also had a dramatic effect on the assembly of other mitochondrial complexes. Thus, the cytochrome oxidase activity of the mutant strain was about five times lower than that of the wild type. In addition, a high percentage of spontaneous rho- mutants was detected.     

-alkoxyphenol leukotriene B4 receptor antagonists

A series of -alkoxyphenols containing a tetrazole acid sidechain have been prepared as antagonists of leukotriene B₄ receptors. These compounds were tested as receptor antagonists of human neutrophil and guinea pig lung membrane leukotriene B₄ receptors. Compounds in this series were found to be up to 18-fold more potent than LY255283. These results indicate that the acyl group of the 1,2,4,5 substituted hydroxyacetophenone class of LTB₄ antagonists is not critical to antagonist potency.     

Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.     

Expression of class I genes in the major histocompatibility complex: identification of eight distinct mRNAs in DBA/2 mouse liver

The mouse H-2 multigene family includes the genes coding for the major transplantation antigens and for genes located in the Qa-TIa region. We have studied a collection of class I cDNA clones made from liver mRNA of DBA/2 mice (H-2d haplotype) and found that at least six distinct class I genes are transcribed, including three genes of the Qa-TIa region. Two of these six genes each yield two distinct mRNAs, resulting from alternate splicing. Altogether, liver cells may express at least eight distinct class I polypeptides, of which three might be secreted, while one may be a new presumptive nonpolymorphic surface antigen.     

Zooplankton composition of ten reservoirs in southern Brazil

The zooplankton of ten reservoirs of Sao Paulo State was analyzed as part of a larger project, Typology of Reservoirs of São Paulo State.Twenty-four genera of Rotifera, six species of Copepoda and at least nine species of Cladocera were found in samples collected on four occasions in 1979. In general, Rotifera dominated in most reservoirs, although fluctuations occurred during the year.The reservoirs were arranged in four groups, according to zooplankton density, whose range was 10 to 500 i 1^–1.The average composition of Crustacea, in number of species at any one time is comparable to those of other water bodies, being a little higher than that of Colorado lakes.The number of species of limnetic Cladocera in Brazil is between those of Holarctic Region and Tropical Asia. Ceriodaphnia cornuta and Bosminopsis deitersi, and a few species of Daphnia are typical of Brazilian zooplankton. Thermocyclops crassus is common in the southern reservoirs but T. minutus seems to be more widely distributed in Brazil. Calanoida occurred in relatively few reservoirs in São Paulo and usually one species at one time. Brachionus and Keratella were more abundant closer to the Equator then to the Tropics, where other genera seem to be more abundant.The range in size of the planktonic Crustacea is relatively small when compared to temperate lakes, being similar to that of other tropical lakes.