Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.

Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K _m values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and K _i values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K _i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.Abbreviations PEG polyethylene glycol - PMT putrescine-N-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We are grateful to C.R. Waspe, M.G. Hilton and P.D.G. Wilson for assistance with the provision of roots from fermenters. We thank W. Martin and S.D. Barr, Chemistry Department, University of Glasgow, and T.A. Smith, Long Ashton Research Station, Bristol, for the supply of compounds not commercially available, as indicated in the text. For helpful discussion and comment, we are grateful to A.J. Parr, W.R. McLauchlan and P. Bachmann. H.D.B, thanks the Science and Engineering Research Council for a research studentship and the Agricultural and Food Research Council Institute of Food Research for additional support.     

Polymorphism of the Thymidylate Synthase Gene of Pneumocystis carinii from Different Host Species

Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400-bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat-derived P. carinii was hybridized with the amplified products from rat- and mouse-derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplifed from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host-species specific. A specific probe which recognized all human P. carinii isolates was defined.     

Catabolism of Tryptophan by Trypanosoma evansi

ABSTRACT. Trypanosoma brucei gambiense , which causes human African trypanosomiasis, catabolizes the aromatic amino acid tryptophan via an initial aminotransferase catalyzed reaction to form several indole end products, which have been suggested to contribute to the pathogenesis of trypanosomiasis. To determine if this same pathway exists in T. evansi , the closely related trypanosome pathogen of domestic animals, tryptophan catabolism was examined in vitro and in vivo. As is the case with human African trypanosomes, T. evansi catabolized tryptophan to form indole-3-pyruvic acid and smaller amounts of indole-3-acetic acid and indole-3-lactic acid. Large concentrations of indole-3-pyruvic acid are excreted in urine of trypanosome-infected mice. However, indole-3-ethanol could not be detected in incubates of T. evansi or T. b. gambiense , even though the latter species had previously been reported to form this neutral metabolite. A new, previously unreported tryptophan metabolite was isolated and partially characterized from incubates of T. evansi and T. b. gambiense. Although the functional significance of tryptophan catabolism to trypanosomatids remains obscure, the pathway is quantitatively significant in all species examined thus far.     

<Emphasis Type="Italic">Dunaliella salina</Emphasis> exopolysaccharides: a promising biostimulant for salt stress tolerance in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

Microalgal exopolysaccharides represent a potential sustainable alternative for the enhancement and protection of agricultural crops including management of both biotic and abiotic stress. In the present study, we investigated the potential of Dunaliella salina exopolysaccharides (PS) to attenuate the effect of salt stress on growth of Solanum lycopersicum, which was grown under different salinity levels (3 and 6 g L^?1 NaCl). The effects of PS treatment on plant growth, osmoprotectant molecules, protein content, and antioxidant enzymes activities of tomato plants under salt stress were analyzed. A metabolomics study showed that the exopolysaccharides released by D. salina contained sulfated moiety along with carbohydrates and uronic acids. The application of sulfated exopolysaccharides on tomato plants alleviated the salt stress and mitigated the decrease in length and dry weight of the plant’s shoot and root systems, as well as that of potassium (K⁺), and K⁺/Na⁺ ratio. Furthermore, the increase in proline, phenolic compounds, Na⁺, and antioxidant enzymes (CAT, POD, SOD) activities caused by salt stress were attenuated after the exopolysaccharide treatment. GC-MS metabolomics analysis showed that PS treatment allowed the activation and/or inhibition of various metabolic pathways involved in the plant’s tolerance to stress such as jasmonic acid-dependent pathways. This study shows the potential of microalgal exopolysaccharides for enhancing tomato tolerance to salt stress and highlights the possibility of their use as plant growth biostimulants under harsh environmental conditions.     

Central administration of alpha-melanocyte-stimulating hormone changes lipid metabolism in chicks

Alpha-melanocyte-stimulating hormone (MSH) is well known as an anorexigenic peptide in the brain of mammals. In addition to this, brain alpha-MSH enhances heat production (HP), indicating that the peptide acts as a catabolic factor in the regulation of energy metabolism. The anorexigenic effect of alpha-MSH is also observed in chicks (Gallus gallus), but no information has been available for its effect on HP. The present study was performed to examine whether intracerebroventricular (ICV) injection of alpha-MSH increases HP in chicks. The injection of alpha-MSH (10 and 100 pmol) did not affect oxygen consumption, carbon dioxide production and HP during the 1 h post-injection period. This result was supported by another result that ICV injection of alpha-MSH did not affect locomotion activity in chicks. In contrast, the respiratory quotient was significantly lowered by the ICV injection of MSH. We also found that alpha-MSH significantly increased plasma non-esterified fatty acid concentrations. In summary, brain alpha-MSH appears to exert generally catabolic effects on lipid metabolism in the chick, but does not appear to be involved in the regulation of HP.     

The Phlesirtes complex (Orthoptera,Tettigoniidae, Conocephalinae,Conocephalini) reviewed: integrating morphological,molecular, chromosomal and bioacoustic data

The tettigoniid genus Phlesirtes Bolivar and its allies are reviewed. Morphological, ecological and molecular data prompt the erection of the new genus Chortoscirtes gen.n. with type species Xiphidion meruense Sjöstedt. The genera Phlesirtes, Chortoscirtes, Karniella and Naskreckiella are characterized by morphological characters supported by molecular, acoustic, ecological and chromosomal data. Four species, Chortoscirtes pseudomeruensis sp.n. , C. masaicus sp.n. , C. puguensis sp.n. and C. serengeti sp.n. , are described from localities in northern and coastal Tanzania and one Karniella, K. crassicerca sp.n. , is described from Uganda. The following comb n. are proposed: Phlesirtes kibonotensis (Sjöstedt) and Phlesirtes kilimandjaricus (Sjöstedt). Subtribal status is proposed for the four investigated African genera. A key to the Chortoscirtes species is provided.     

Molecular cloning of biologically active Rauscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat.

Rauscher and Friend spleen focus-forming viruses (R- and F-SFFVs) cause similar progressive erythroleukemias dependent upon a virus-encoded membrane glycoprotein. Moreover, these SFFV glycoproteins are immunologically related to each other and to the recombinant-type glycoproteins encoded by the env genes of dual tropic murine leukemia viruses. To better understand these diseases and the viral origins, we isolated a pathogenically active molecular clone of R-SFFV proviral DNA, sequenced its 3'-terminal 2,163-base-pair (bp) region, and compared these sequences with previously determined sequences of F-SFFV. The 516-bp R-SFFV long terminal repeat is highly homologous to those of F-SFFV and Friend murine leukemia virus, although only the latter contains a 65-bp direct repeat in its U3 region. The env gene of R-SFFV encodes a glycoprotein with 408 amino acids that is identical in its basic domain organization to the glycoprotein of F-SFFV. Thus, the junctions between the dual tropic-related and ecotropic sequences occur at the same nucleotide, and both SFFV env genes contain identical 585-bp deletions in their ecotropic domains and single-bp insertions which cause premature terminations at the same amino acid in their ecotropic p15E domains. Consistent with their independent origins, however, the env sequences of R- and F-SFFV are distinctive in both their 5' dual tropic-related and 3' ecotropic-related domains. Furthermore, there are several consistent amino acid differences between the polycythemic F-SFFV sequences and the anemia-inducing R-SFFV sequence. The striking similarities of the independently formed F- and R-SFFV env genes imply that all of the glycoprotein domains arranged in a precise organization may be required for its leukemogenic activity     

tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster

Developmental analysis of a newly isolated maternal effect grandchildless mutant, tudor (tud), in Drosophila melanogaster indicates that tud+ activity is required during oogenesis for the determination and/or formation of primordial germ cells (pole cells) and for normal embryonic abdominal segmentation. Regardless of their genotype, progeny of females homozygous for strong alleles (tud1 and tud3) never form pole cells, apparently lack polar granules in the germ plasm, and approximately 40% of them die during late embryogenesis exhibiting severe abdominal segmentation pattern defects. Females carrying weak allele, tud4, produce progeny with some functional pole cells and form polar granules approximately one-third the size of those observed in wild-type oocytes and embryos. No segmentation abnormalities are observed in the inviable embryos derived from tud4/tud4 females.     

Circulating proalbumin associated with a variant proteinase inhibitor

The unique finding of normal proalbumin in human plasma provides an insight into the mechanism of propeptide cleavage. Proalbumin, present as 1–5% of the total albumin, was found in a boy whose prime problem was the presence of a mutant proteinase inhibitor, α₁-antitrypsin Pittsburgh (358Met→Arg) [2]. The infeerred structure of human proalbumin was confirmed as ArgGlyValPheArgArgAlb. On incubation with various enzymes (trypsin, tryptase, thrombin, chymotrypsin, chymase and cathepsin B), only trypsin was capable of converting proalbumin to albumin. There was no conversion when proalbumin was incubated with whole blood, plasma or serum. However, intravenous injection of proalbumin into a rat resulted in complete conversion to albumin, the half-life of this process being 6 h. We conclude that propeptide cleavage is dependent on a serine proteinase which is inhibited intracellularly, by the mutant inhibitor, and that all the albumin in the boy was secreted as proalbumin, but was subjected to a separate cleavage process after export from the hepatocyte.     

The outline structure of the T-cell alpha beta receptor.

From an analysis of the immunoglobulins of known structure we derive a list of 40 sites crucial for the conserved structure of the variable domains. We show that, with marginal exceptions, the sequences of the T-cell alpha beta receptors contain, at sites homologous to these 40, the same or very similar residues. Thus the V alpha-V beta dimer has a framework structure very close to that of the immunoglobulins. Further comparisons show that parts of the surface of the V alpha-V beta framework are hypervariable. They also show that the loops that form the antigen-binding site are similar in size to those commonly found in the immunoglobulins but have different conformations. Only limited sequence variations occur in the first loop of the antigen-binding site in both V alpha and V beta. This, and their geometrical arrangement, suggest that they mainly interact with the MHC proteins.