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.     

-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.     

Secretory cell activity in the hamster seminal vesicle following castration. A morphometric ultrastructural study

The ultrastructure of hamster seminal vesicle epithelium was studied 7, 14, 21 and 28 days after castration using a stereological approach. The results show that castration promotes epithelial reorganization, mainly characterized by reduced epithelial cell size and number, decreased rough endoplasmic reticulum and Golgi complex, increased lysosomes and lipid droplets, increased apical secretory granule size and number, and increased intracellular secretory products per average epithelial cell. It is concluded that after testosterone withdrawal the secretory activity of hamster seminal vesicle epithelial cells, although reduced, is not abolished, and that exocytosis is relatively more reduced than secretory protein production. We suggest that an extracellular androgen source is responsible for secretory activity not being lost in the epithelial cells of castrated hamster seminal vesicle.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

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.     

2-Methylacetoacetyl-coenzyme A reductase from Ascaris muscle: purification and properties

2-Methylacetoacetyl-CoA and 3-keto-2-methyl pentanoyl-CoA have been proposed to be intermediates in the synthesis of 2-methylbutyrate and 2-methylvalerate, respectively, by Ascaris lumbricoides muscle. These volatile acids are major fermentation products of Ascaris metabolism. 2-Methylacetoacetyl-CoA reductase has been purified 532-fold from Ascaris muscle to yield a homogeneous preparation which contained a single protein species as observed on discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purification procedure utilized subcellular fractionation, affinity chromatography on NAD+ agarose, and ion-exchange chromatography on DEAE-cellulose. A constant activity ratio for ethyl 2-methylacetoacetate and acetoacetyl-CoA was observed during purification, indicating that the same enzyme catalyzed both reactions. In addition, the purified protein catalyzed the NADH-dependent reduction of ethyl-3-keto-2-methyl pentanoate at essentially the same rate as it did ethyl 2-methylacetoacetate. The purified enzyme is a basic protein with an isoelectric point of 8.45 at 4 degrees C. The molecular weight of the native protein (Mr = 64,000 by exclusion chromatography) and the size of the subunit (Mr = 30,000 by dodecyl sulfate-polyacrylamide electrophoresis) indicate that the enzyme is composed of two subunits of the same molecular weight. Substrate-specificity studies, undertaken with the purified protein, demonstrated that the ethyl esters can substitute for the coenzyme A derivatives but this substitution results in an active substrate only when a branched 2-methyl group is present. The straight-chain ethyl ester is inactive. Kinetic constants for the substrates and nucleotides were determined. The role of the CoA esters as the physiological substrates for the Ascaris enzyme is substantiated. When assayed in the reductive direction with ethyl 2-methylacetoacetate as substrate, the activity of the purified enzyme was inhibited not only by coenzyme A as previously reported, but also by acetyl-CoA. The physiological implications of these inhibitions are discussed.