Partial purification and properties of γ-glutamyltranspeptidase from mycelia of Morchella esculenta

Three -glutamyltranspeptidase (enzymes I, II and III) were partially purified from the cell free extracts of the cultured mycelia of Morchella esculenta Fr. The molecular masses of enzymes were 155,000 (I), 219,000 (II) and 102,000 (III). All of them catalyzed both hydrolysis and transpeptidation of various -glutamyl compounds. -l-Glutamyl-cis-3-amino-l-proline occurring in the cultured mycelia of this fungus was a good substrate for both reactions. K _m values for hydrolysis were in the order of 10^-4 to 10^-5 M, and those for transpeptidation were in the order of 10^-2 to 10^-4 M. The enzymes were inhibited by a -glutamyltranspeptidase inhibitor, l-serine plus borate.Abbreviations -GTP -glutamyltranspeptidase - HPLC High-performance liquid chromatography     

The proline biosynthesis in living organisms

Summary In this article we review recent work on the physiology of proline and ¹-pyrroline-5-carboxylate (P5C) in living organisms and consider recent progress in our understanding of the role of P5C synthetase in collagen metabolism and the regulation of urea cycle in vertebrates. Much of this recent progress has been made possible by advances in our knowledge of the enzymes and genes involved in proline biosynthesis in man. The availability of well characterized P5C synthetase deficiency in man has been an impetus for the cloning of the cDNA encoding for this enzyme from man and facilitated the establishment of the phenotype-genotype relationships in P5C synthetase deficiency in higher vertebrates.Abbreviations GK -glutamyl kinase - GPR -glutamyl phosphate reductase - P5CR ¹-pyrroline-5-carboxylate reductase - GSA glutamic--semialdehyde - P5C ¹-pyrroline-5-carboxylate - P₁ Inorganic phosphate - AMP, ADP, ATP Adenosine 5-mono-, di-, triphosphate - NAD⁺, NADH nicotinamide adenine dinucleotide, and its reduced form - NADP⁺, NADPH nicotinamide adenine dinucleotide phosphate, and its reduced form; DEAF: diethylaminoethyle - OAT ornithine amino transferase; CHO: Chinese hamster ovary - IGF-1 insulin-like growth factor-1 - P5CDH pyrroline 5carboxylate dehydrogenase - IMP inosine 5-monophosphate     

Synergistic effects of TGIF and interferonγ (IFN-γ) on tumor cell growthTGIF and IFN-γ

Interferon- (IFN-) and tumor growth inhibitory factor (TGIF) were inducedin vitro in the supernatant from mixed culture of human peripheral blood mononuclear cells (PBMC) and OK-432. TGIF activity was determined by growth inhibition of a human gastric adenocarcinoma cell line, MK-1 cells, and IFN- activity was measured by radioimmunoassay. The production of TGIF and IFN- was time-dependent, reaching its maximum around 48 hrs. Although there was no significant correlation between TGIF production and IFN- production, combination of a subthreshold concentration of recombinant IFN- (rIFN-) and TGIF induced significant growth inhibition of MK-1 cells. This fact indicates that the effects of rIFN- and TGIF are synergistic. The antiproliferative effect of these cytokines are highly species-specific, and their synergistic effects were also species-specific. rIFN--sensitive and -resistant clones were successfully established from the original MK-1 cell line; those clones are both sensitive to TGIF. Synergistic antiproliferative effects were found when the rIFN--sensitive clone, but not the resistant clone, was used as a target, suggesting that the synergistic effects require the target cells' sensitivity to IFN-. These results indicate that the synergistic effects of TGIF and IFN- may produce a clinical antitumor action in cancer patients receiving OK-432 administration.     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

γ-Glutamyl conjugation of 5-hydroxytryptamine (serotonin) in the earthworm (Lumbricus terrestris)

The conversion of 5-hydroxytryptamine to several potential metabolites was examined in the annelid earthworm (Lumbricus terrestris). 5-hydroxytryptamine and some related amines were found to be present in several tissues of the earthworm. Injection of 5-hydroxytryptamine into the body cavity of the earthworm resulted in the production of a -glutamyl conjugate of 5-hydroxytryptamine. Incubations of the anterior nerve cord of the earthworm resulted in the accumlation of considerable amounts of 5-hydroxytryptamine and -glutamyl 5-hydroxytryptamine in the incubation medium. The earthworm did not produce any N-acetyl 5-hydroxytryptamine and only very little 5-hydroxyindoleacetic acid. Experiments involving the injection of radiolabeled 5-hydroxytryptamine or coninjection of radiolabeled glutamic acid with unlabeled 5-hydroxytryptamine into the earthworm resulted in the production of radiolabeled -glutamyl 5-hydroxytryptamine. This work demonstrates that the enzymatic conversion of 5-HT in the earthworm is markedly different from that of vertebrates and insects.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

Evolutionary relationship of the members of the sulphur-rich hordein family revealed by common antigenic determinants

Summary Five monoclonal antibodies raised against an enriched C hordein fraction have been characterized in detail and were found to be specific for the members of the sulphur-rich hordein family. Two antibodies specific for B hordein polypeptides were identified, one of which reacted predominantly with CNBr cleavage class III polypeptides. 1 hordein was recognized by two antibodies, of which one also reacted with 2 hordein and several members of the CNBr cleavage class II B hordein polypeptides. One antibody recognized 3 hordein but cross-reacted at higher antibody concentration with almost all of the B and C hordein polypeptides. The specificity of the monoclonal antibodies was confirmed by Western blotting of one- or two-dimensionally separated hordein from the B hordein-deficient mutant hor2ca and its wild-type Carlsberg II and the 3 hordein-deficient genotype Nevsky. The identification of the hordein-specific monoclonal antibodies was further supported by immune precipitation of in-vitro transcribed and translated 2 hordein, and hor2ca and Carlsberg II mRNA translation products. The monoclonal antibodies were used to screen for mutants in hordein synthesis. Two mutants, one deficient in 1 hordein synthesis and a second in 2 or closely related B hordein polypeptides were identified. A model is proposed for the evolution of the sulphur-rich hordein loci Hor5 and Hor2.     

Isolation of a Δ6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp.strain PCC 7120

The enzyme ⁶-desaturase is responsible for the conversion of linoleic acid (18:2) to -linolenic acid (18:3). A cyanobacterial gene encoding ⁶-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking ⁶-desaturase. Expression of the Synechocystis ⁶-desaturase gene in Anabaena resulted in the accumulation of -linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis ⁶-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a ¹²-desaturase gene linked to the ⁶-desaturase gene. Expression of Synechocystis ⁶-and ¹²-desaturase in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and -linolenic acid.     

γ-Tubulin is a component of the Spitzenkörper and centrosomes in hyphal-tip cells ofAllomyces macrogynus

Summary A monoclonal antibody was used to localize -tubulin in hyphal tip cells of the chytridiomycete fungusAllomyces macrogynus, and its distribution determined with standard epifluorescence and laser scanning confocal microscopy. The results demonstrate that -tubulin is a component of the Spitzenkörper and centrosomes. Immunoblot analysis of total soluble protein extracts separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified a single 56 kDa -tubulin-related polypeptide. Localization of -tubulin to the Spitzenkörper ofA. macrogynus provides evidence that the Spitzenkörper in this fungus functions as a microtubule-organizing center.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - LSCM laser scanning confocal microscopy - MTOCs microtubule-organizing centers - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SPB spindle pole body - YpSs yeast extract-inorganic phosphate-soluble starch     

Inhibition of interferon- γ and interleukin-2 production from lymphocytes stimulated with food antigens by an anti-allergic drug,Tranilast, in patients with food-sensitive atopic dermatitis

N(3, 4-dimethoxycinnamoyl) anthranilic acid (Tranilast) inhibits antibody-mediated hypersensitivity reactions, and is an effective drug for patients with bronchial asthma or allergic rhinitis. Interferon- (IFN-) production of ovalbumin (OA)-stimulated peripheral blood mononuclear cells (PBMCs) from hen's egg-sensitive patients with atopic dermatitis (AD) was significantly higher than those of healthy controls. Tranilast inhibited this IFN- production. Moreover, interleukin-2 (IL-2) production of OA-stimulated PBMCs from hen's egg-sensitive patients with AD was also inhibited by Tranilast. Our results suggest that Tranilast can be used to the patients with food sensitive AD.Abbreviations PBMCs peripheral blood mononuclear cells - OA ovalbumin - BSA bovine serum albumin - AD atopic dermatitis - IL-2 interleukin-2 - IFN- interferon- - Tranilast N(3, 4-dimethoxycinnamoyl) anthranilic acid - IL-4 interleukin 4 - IL-5 interleukin 5     

Sequential changes in MHC antigen expression induced by the v-Ki-ras oncogene

A series of early-passage cell lines were transformed with the v-Ki-ras oncogene with the aim of examining the effect of an activatedras gene on the ability of these cells to express major histocompatibility complex (MHC) antigens. These cell lines were found to undergo multiple phenotypic changes upon transformation and subsequent proliferation. At early passage, the predominant effect ofras was an increased ability to express class II antigens when induced with interferon (IFN). For class I antigens, maximum levels of expression induced with IFN were largely unaffected, however, decreased sensitivity to induction with this lymphokine was noted. With subsequent in vitro or in vivo passage, both class I and class II antigen inducibility was attenuated. The latter phenotypic change was found to be transferable by coculture, implicating a soluble IFN antagonist. Conditioned media fromras-transformed cells treated to activate their latent transforming growth factor (TGF) content mediated similar changes in MHC antigen inducibility, suggesting that TGF\ may be involved in modulating MHC antigen expression inras-transformed cells.     

The Effect of γ-Radiation and Desiccation on the Viability of the Soil Bacteria Isolated from the Alienated Zone around the Chernobyl Nuclear Power Plant

Methylobacterium extorquens, M. mesophilicum, and Bacillus subtilis strains were found to be resistant to -radiation, irrespective of whether they were isolated from the alienated zone around the Chernobyl Nuclear Power Plant or outside this zone. The LD₉₀ of Methylobacterium and B. subtilis strains with respect to -radiation was 2.0–3.4 and 3.7–4.4 kGy, respectively, whereas their LD_99.99 values were 4.5–6.9 and more than 10 kGy, respectively. The high threshold levels of -radiation for Methylobacterium and B. subtilis imply the efficient functioning of DNA repair systems in these bacteria. Unlike Bacillus polymyxa cells, the cells of M. extorquens, M. mesophilicum, and B. subtilis were also resistant to desiccation. Pseudomonas sp., Nocardiasp., and nocardioform actinomycetes were sensitive to both -radiation and desiccation. Similar results were obtained when the bacteria studied were exposed to hydrogen peroxide and ultraviolet radiation. The results obtained indicate that the bacteria that are resistant to -radiation are also resistant to desiccation, UV radiation, and hydrogen peroxide. The possibility of using common laboratory tests (such as the determination of bacterial resistance to UV light and desiccation) for the evaluation of bacterial resistance to -radiation is discussed.     

Antifreeze activities of poly(γ-glutamic acid) produced by Bacillus licheniformis

Various enantiomeric isomers, metals salts and molecular sizes of poly(-glutamic acid), -PGA, produced by Bacillus licheniformis CCRC 12826, were prepared and their antifreeze activities were studied by differential scanning calorimetry. The antifreeze activity of -PGA increased as its molecular weight decreased but was indifferent to its d/l-glutamate composition. The antifreeze activity was cation dependent decreasing in the order Mg²⁺>>Ca ²⁺Na ⁺>>K ⁺ which follows that of inorganic chlorides in that high ionic charge leads to high antifreeze activity. The mechanism by which the cryoprotective effects of -PGA can be explained is still yet to be determined.     

Conformational study of N(epsilon)-(carboxymethyl)lysine adducts of recombinant gammaC-crystallin

N -(carboxymethyl)lysine, an advanced glycation end product, is present in the human lens. The effects of CML formation on protein conformation and stability were studied using the recombinant C-crystallin as a model. Conformational change was studied by spectroscopic measurements such as fluorescence and circular dichroism. Conformational stability was determined by unfolding with heat. The results indicated that no conformational change was observed due to CML formation, but conformational stability decreased. These observations can be explained in terms of the relatively stable structure of -crystallin, especially when compared with other crystallins. The lens nucleus is rich in -crystallin and its stable conformation can assist -crystallin sustained insults and remain soluble.     

Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Summary The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against ₃-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against -MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against -MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against -endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against -endorphin. These results correlate with biochemical data in favour of mainly — if not exclusively — intragranular processing of POMC. Except for ₃-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of -endorphin appears to be a later step in POMC processing, occurring in some mature SV.     

rol genes of Agrobacterium rhizogenes cucumopine strain: sequence,effects and pattern of expression

By sequencing the central region of the cucumopine-type T-DNA of Agrobacterium rhizogenes strain 2659, we identified three open reading frames homologous, to different extents, to ORFs 10, 11 and 12 (rolA, B and C) of the agropine-type (1855) T-DNA. Recombinant Agrobacterium strains encompassing the ORFs of 2659 T-DNA-which we refer to as rol, and -were utilized to infect carrot discs and to obtain transgenic tobacco plants, in order to compare the morphogenetic capabilities to those of the 1855 rol genes. Moreover, a long segment of the 5 non-coding region of rol and rol was fused to the GUS reporter gene and the pattern of expression and the responsiveness to auxin of the constructs was analysed in transgenic tobacco. Differences in the auxin requirement for root induction between the 2659 rol genes and their respective 1855 counterparts were pinpointed. These differences are not due to gene regulation and presumably reflect functional differences in the proteins encoded. Differences were also observed in the pattern of expression of rol in roots of transgenic plants, as compared to rolB. In addition, the pattern of expression of rol-GUS construct in roots was found to be analogous to that observed for a construct driven by two of the five regulatory domains of the rolB promoter.     

HLA class II alleles and measles virus-specific cytokine immune response following two doses of measles vaccine

Measles virus-specific T cells and the production of cytokines play a critical role in the immune response following measles immunization. To understand the genetic factors that influence variation in IFN- and IL-4 responses following measles immunization and to provide insight into the factors influencing both cellular and humoral immunity to measles, we assessed associations between human leukocyte antigen (HLA) class II genes and measles-specific Th1 and Th2-type cytokine responses in peripheral blood lymphocytes from 339 children previously vaccinated with two doses of measles-mumps-rubella vaccine (MMR-II). Median values for measles-specific IFN- and IL-4 secretion levels were 40.73 and 9.71 pg/ml, respectively. The global tests suggested associations between measles-specific IFN- response and alleles of the DRB1 and DQB1 loci (P=0.07 and P=0.02, respectively). Specifically, DRB1*0301, *0901, and *1501 alleles were significantly associated with IFN- secretion. The alleles that suggested evidence of an HLA association with IL-4 secretion were DRB1*0103, *0701, and *1101. Th1 cytokine responses and DQB1 allele associations revealed that the alleles with the strongest association with IFN- secretion were DQB1*0201, *0303, *0402, and *0602. Specific alleles with a suggestive association with low measles-specific Th2 cytokine responses were DQB1*0202 and *0503. In addition, DPB1*0101, *0201, and *0601 alleles provided suggestive evidence of an HLA association with measles-induced IFN- response, while DPB1*0501 was associated with an IL-4 response. These data suggest that IFN- and IL-4 cytokine responses to measles may be genetically restricted in part by HLA class II genes, which in turn can restrict the cellular immune response to measles vaccine.