Cloning and analysis of structural genes from Streptomyces pristinaespiralis encoding enzymes involved in the conversion of pristinamycin IIB to pristinamycin IIA (PIIA): PIIA synthase and NADH:riboflavin 5'-phosphate oxidoreductase.

In Streptomyces pristinaespiralis, two enzymes are necessary for conversion of pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA), the major component of pristinamycin (D. Thibaut, N. Ratet, D. Bisch, D. Faucher, L. Debussche, and F. Blanche, J. Bacteriol. 177:5199-5205, 1995); these enzymes are PIIA synthase, a heterodimer composed of the SnaA and SnaB proteins, which catalyzes the oxidation of PIIB to PIIA, and the NADH:riboflavin 5'-phosphate oxidoreductase (hereafter called FMN reductase), the SnaC protein, which provides the reduced form of flavin mononucleotide for the reaction. By using oligonucleotide probes designed from limited peptide sequence information of the purified proteins, the corresponding genes were cloned from a genomic library of S. pristinaespiralis. SnaA and SnaB showed no significant similarity with proteins from databases, but SnaA and SnaB had similar protein domains. Disruption of the snaA gene in S. pristinaespiralis led to accumulation of PIIB. Complementation of a S. pristinaespiralis PIIA-PIIB+ mutant with the snaA and snaB genes, cloned in a low-copy-number plasmid, partially restored production of PIIA. The deduced amino acid sequence of the snaC gene showed no similarity to the sequences of other FMN reductases but was 39% identical with the product of the actVB gene of the actinorhodin cluster of Streptomyces coelicolor A(3)2, likely to be involved in the dimerization step of actinorhodin biosynthesis. Furthermore, an S. coelicolor A(3)2 mutant blocked in this step was successfully complemented by the snaC gene, restoring the production of actinorhodin.     

A STAT factor mediates the sexually dimorphic regulation of hepatic cytochrome P450 3A10/lithocholic acid 6 beta-hydroxylase gene expression by growth hormone.

Adult male rodents have a pulsatile profile of growth hormone (GH) release, whereas female rodents have a relatively steady-state pattern with uniform, albeit lower levels of GH. The expression of a number of sexually differentiated hepatic proteins is primarily determined by these plasma GH profiles and only secondarily regulated by gonadal hormones. An important subset of these sexually dimorphic proteins is cytochrome P450s. CYP3A10/6 beta-hydroxylase is a cytochrome P450 that catalyzes the 6 beta-hydroxylation of lithocholic acid. CYP3A10/6 beta-hydroxylase is expressed only in male hamsters; however, mimicking the male GH secretion pattern in females induces expression of the gene to male levels. Using chimeric CYP3A10/6 beta-hydroxylase promoter/luciferase reporter genes transfected into hamster primary hepatocytes, we have shown a GH-mediated induction of promoter activity. A combination of 5'-deletion constructs, heterologous promoter constructs, and specific mutagenesis was used to localize the DNA element involved in the GH-mediated regulation of CYP3A10/6 beta-hydroxylase promoter activity, which resembles a STAT binding site. Footprint and gel shift analyses confirmed that the expression of the protein binding to this site is regulated by GH and that the DNA-protein complex can be partially supershifted by anti-STAT-5 antibodies. This protein is 50% more abundant in male than in female hamster livers, is absent in hypophysectomized female livers, and is restored when hypophysectomized females are injected with GH in a manner that masculinizes female hamsters in terms of CYP3A10/6 beta-hydroxylase expression. The system characterized and described here is ideally suited for dissecting the molecular details governing the sexually dimorphic expression of liver-specific genes.     

Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana

Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana . Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis . Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age-dependent in planta senescence and dark-induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis . The results further suggest that the three genes function at a common step of age-dependent and dark-induced senescence processes. It is further shown that one of the mutations is allelic to ein2-1 , an ethylene-insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of Arabidopsis .     

The larval development of lateral musculature in gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax

Fibre-type differentiation of the lateral musculature has been studied in Sparus aurata (L.) and Dicentrarchus labrax (L.) during larval development. Histochemical and ultrastructural techniques show two presumptive muscle layers and two germinative zones of presumptive myoblasts. At hatching, myotomal muscle consists of a monolayer of thin undifferentiated cells near the skin (first germinative zone) overlying another mono-layer of small diameter fibres extending hypaxially and epaxially away from the transverse septum. Below this, there is a much thicker, deep layer of fibres, generally large in diameter and polygonal in shape. The presumptive myoblasts are located between these two layers of fibres in the second germinative zone. Initially, the superficial and deep muscle fibres show high and low myosin ATPase activity, respectively. Both layers grow by generating new fibres from the two mentioned germinative zones. At the end of larval life, the superficial layer changes its histochemical profile from high to low myosin ATPase activity and, at the same time, intermediate or pink muscle fibres can be observed by oxidative activity (the NADH-TR reaction). Morphometric analysis shows a significant increase in mean fibre diameter during successive ages, as shown by the Student's t-test (hypertrophic growth). Skewness and kurtosis values of fibre diameters point to the generation of a new fibre population from the germinative zones (hyperplastic growth).     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Immobilization of Lipase from Mucor Miehei Onto Poly (Ethylene) Based Graft Copolymers

Lipase from Mucor miehei was immobilized covalently onto hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate (PE-HEMA). This hydrolysis of the copolymer was achieved using 0.1 M NaOH over different periods of time, under controlled conditions. The graft copolymers and their hydrolyzed equivalents were characterized by scanning electron microscopy (SEM) and by differential scanning calorimetry analysis (DSC). Water sorption studies were undertaken to provide a measure of relative hydrophobicity of the samples.

The lipase immobilization reaction was studied in order to assess the effects of controlling various important parameters. These include the nature of the buffering medium, the time over which the immobilization was allowed to occur, the concentration of the activating and coupling agent used (CMC) and the concentration of enzyme employed during attempts at effective immobilization. The immobilized lipase was used in the hydrolysis of triolein (glycerol trioleate). From this study, the apparent K_M, the optimum pH for hydrolysis and the optimum temperature for hydrolysis were revealed.

The suitability of hydrolyzed poly(ethylene)-g.co-HEMA as a support in the immobilization of lipase was assessed by determination of the amount of lipase coupled to the support and by assessment of the retention of activity of the immobilized lipase after its exposure to the immobilization reagents, procedure and conditions.