Bacillus subtilis and Streptomyces spp. provide tractable experimental systems for studying cellular responses to adverse environmental conditions. During conditions of extreme nutrient limitation, these prokaryotes exhibit a wide range of adaptations, including the production and secretion of antibiotics and enzymes and the formation of aerial mycelium and spores. In response to these conditions, all bacteria, but not eukaryotic microorganisms, exhibit a “stringent response,” during which the unusual guanosine tetraphosphate, ppGpp, accumulates intracellularly. This is accompanied by a marked reduction in the GTP pool, due to ppGpp inhibition of IMP-dehydrogenase, and immediate repression of rRNA synthesis, due to the binding of ppGpp to RNA polymerase. This review summarizes our studies on the bacterial stringent response and its use in applied microbiology. We found that morphological differentiation results from a decrease in the pool of GTP, whereas physiological differentiation (antibiotic production) results from a more direct function of ppGpp. That is, we found that the Streptomyces GTP-binding protein Obg functions by sensing intracellular GTP levels and that certain mutations in the RNA polymerase β-subunit circumvent dependence on ppGpp in antibiotic production. X-ray crystallographic analysis provided a structural basis for the ppGpp regulation of transcription. On the basis of these findings, we have developed the novel concept of “ribosome engineering,” focusing on activation of dormant genes to elicit cellular function fully. Ribosome engineering can be applied to strain improvement, screening of novel metabolites, plant breeding, cell-free translation systems, and the treatment of tuberculosis.
The ecological biochemistry of flavonoids, in which I have been engaged for 25 years, is summarized in this review article. The review covers (1) a survey of rare bio-active flavonoids in higher plants; (2) the fungal metabolism of prenylated flavonoids; (3) flavonoids antidoting against benzimidazole fungicides; (4) dihydroflavonol ampelopsin in Salix sachalinensis as a feeding stimulant towards willow beetles; and (5) flavones as signaling substances in the life-cycle development of the phytopathogenic Peronosporomycete Aphanomyces cochlioides, a cause of spinach root rot and sugar beet damping-off diseases. Finally recent trends in the ecological biochemistry of flavonoids are briefly described.
Arbuscular mycorrhizae formed between more than 80% of land plants and arbuscular mycorrhizal (AM) fungi represent the most widespread symbiosis on the earth. AM fungi facilitate the uptake of soil nutrients, especially phosphate, by plants, and in return obtain carbohydrates from hosts. Apocarotenoids, oxidative cleavage products of carotenoids, have been found to play a critical role in the establishment of AM symbiosis. Strigolactones previously isolated as seed-germination stimulants for root parasitic weeds act as a chemical signal for AM fungi during presymbiotic stages. Stimulation of carotenoid metabolism, leading to massive accumulation of mycorradicin and cyclohexenone derivatives, occurs during root colonization by AM fungi. This review highlights research into the chemical identification of arbuscular mycorrhiza-related apocarotenoids and their role in the regulation and establishment of AM symbiosis conducted in the past 10 years.
Protein glycosylation is essential for eukaryotic cells from yeasts to humans. When compared to N-glycosylation, O-glycosylation is variable in sugar components and the mode of linkages connecting the sugars. In fungi, secretory proteins are commonly mannosylated by protein O-mannosyltransferase (PMT) in the endoplasmic reticulum, and subsequently glycosylated by several glycosyltransferases in the Golgi apparatus to form glycoproteins with diverse O-glycan structures. Protein O-glycosylation has roles in modulating the function of secretory proteins by enhancing the stability and solubility of the proteins, by affording protection from protease degradation, and by acting as a sorting determinant in yeasts. In filamentous fungi, protein O-glycosylation contributes to proper maintenance of fungal morphology, hyphal development, and differentiation. This review describes recent studies of the structure and function of protein O-glycosylation in industrially and medically important fungi.
The novel cyclic peptide, epichlicin, was isolated from Epichloe typhina, an endophytic fungus of the timothy plant (Phleum pretense L.). Its structure was determined by NMR studies and by mass spectrometry. Enantiomers of 3-amino tetradecanoic acid, a constituent amino acid of epichlicin, were synthesized as authentic standards. The stereochemistry of each amino acid was elucidated through a combination of the advanced Marfey method and chemical manipulation. Epichlicin showed inhibitory activity toward the spore germination of Cladosporium phlei, a pathogenic fungus of the timothy plant at an IC50 value of 22 nM.
Sweet pepper (Capsicum annuum) leaves at the mature stage have strong ovipositional deterrence against Liriomyza trifolii (Burgess) (Diptera, Agromyzidae), whereas the cotyledons are fiercely attacked by the fly. Treatment of the cotyledons with 50 μM and 100 μM of a jasmonic acid (JA) solution caused the plant to acquire strong oviposition deterrence against the leafminer. An HPLC analysis of the JA-treated cotyledons revealed the inducible accumulation of a compound. Based on spectroscopic analysis and chemical methods, the induced compound was identified to be caffeoylputrescine (CP). The accumulated amounts of CP in the cotyledons treated with 0, 10, 50 and 100 μM of JA were 6.0, 43.0, 105 and 140 μg/g fr. wt., respectively. Treatment of the cotyledons with CP resulted in a significant decrease in the number of punctures made by L. trifolii, indicating that the JA treatment enhanced the deterrence against the leafminer by inducing CP accumulation.
A concise synthesis of di-O-methyl-β,γ-dehydrocurvularin, the di-O-methylated derivative of the naturally occurring nematicidal macrolide, β,γ-dehydrocurvuralin, was accomplished by starting from a commercially available aromatic carboxylic acid in a three-step sequence consisting of esterification, Friedel-Crafts acylation, and microwave-promoted ring-closing metathesis.
Pseudomonas jessenii EC-S101 produced hyphal branching-inducing and mitosis-accelerating factors active towards Peronosporomycetes, Aphanomyces cochlioides hyphae. In searching for the active substances, EtOAc-solubles extracted from EC-S101-cultured solid medium were fractionated under the guidance of a paper disc assay using an A. cochlioides mycelium. Two active substances were subsequently isolated and the structure was elucidated by spectroscopic analysis to be (+)-4,5-didehydroacaterin (1) and 3-[(1R)-hydroxyhexyl]-5-methylene-2(5H)-furanone (2), both of which accelerated the mitotic process of A. cochlioides hyphae along with excessive branching at 1.0 μg per disc. These compounds are likely to affect the morphophysiological development of certain eukaryotic organisms in the terrestrial ecosystem.
Curcuma xanthorrhiza Roxb., commonly known as Javanese turmeric, has been reported to possess a variety of biological activities, including anti-inflammatory effects, anticarcinogenic effects, wound healing effects, and serum cholesterol-lowering effects. CPE, crude polysaccharide extract isolated from the rhizome of C. xanthorrhiza using 0.1 N NaOH, consisted of arabinose (18.69%), galactose (14.0%), glucose (50.67%), mannose (12.97%), rhamnose (2.73%), and xylose (0.94%), with an average molecular weight of 33,000 Da. In the present study, we investigated the effect of CPE on nitric oxide (NO), hydrogen peroxide (H2O2), tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2) production in RAW 264.7 cells. The uptake of fluorescein-labeled Escherichia coli was measured to determine whether CPE stimulates the phagocytic activity of RAW 264.7 cells. CPE significantly increased the phagocytosis of macrophages and the release of NO, H2O2, TNF-α, and PGE2 in a dose-dependent manner, and showed a similar activity to lipopolysaccharide (LPS). To study the mechanisms of CPE, we examined induction of iNOS and COX-2. NO and PGE2 were produced as a result of stimulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) respectively. Both modulations of iNOS and COX-2 expression by CPE were evaluated by Western immunoblotting and RT-PCR. Since transcription of these enzymes is under the control of nuclear factor-kappa B (NF-κB), we assessed the phosphorylation of inhibitor κBα (IκBα) through Western immunoblotting. CPE clearly induced phosphorylation of IκBα, suggesting a role as an NF-κB activator. Taking all this together, we conclude that CPE isolated from Curcuma xanthorrhiza stimulates the immune functions of macrophages, which is mediated in part by specific activation of NF-κB.
Oriental Beauty, which is made from tea leaves infested by the tea green leafhopper (Jacobiasca formosana) in Taiwan, has a unique aroma like ripe fruits and honey. To determine what occurs in the tea leaves during the oolong tea manufacturing process, the gene expression profiles and the chemical profiles were investigated. Tea samples were prepared from Camellia sinensis var. sinensis cv. Chin-shin Dah-pang while the tea leaves were attacked by the insect. The main volatile compounds, such as linalool-oxides, benzyl alcohol, 2-phenylethanol, and 2,6-dimethylocta-3,7-diene-2,6-diol, increased during manufacture. The gene expression profiles during manufacture were analyzed by differential screening between fresh leaves and tea leaves of the first turn over. Many up-regulated transcripts were found to encode various proteins homologous to stress response proteins. Accordingly, the endogenous contents of abscisic acid and raffinose increased during manufacture. Thus the traditional manufacturing method is a unique process that utilizes plant defense responses to elevate the production of volatile compounds and other metabolites.
Overexpression of a mercuric ion binding protein, MerP, from the mercury resistance operon genes of Gram-positive bacterial strain Bacillus megaterium MB1 and from Gram-negative bacterial strain Pseudomonas aeruginosa K-62 was found to enhance the mercury resistance level of Escherichia coli host cells, even though they share only 27.3% identity. Immunoblot analysis showed that MerP (BMerP) from Bacillus could be expressed on the membrane fraction of E. coli cells. Treated with 10 μM Hg2+, a recombinant strain harboring the BMerP gene significantly improved, showing a 27% increase in mercuric ion adsorption capacity, 16% better than that of a Pseudomonas merP gene (PMerP)-harboring strain. While multiple heavy metals co-existed, the mercuric ion adsorption capacity of the BMerP-harboring E. coli was not affected while that of the PMerP-harboring strain decreased. These results suggest that BMerP can act as a bio-adsorbent compartmentalizing the toxic mercuric ion on the cell membrane and enhancing resistance.
In a previous study of ours, the superoxide scavenging activity of aqueous extracts from dinophycean red tide flagellates was detected by an electron spin resonance (ESR)-spin trapping method, but not by an L-012 (luminol analog)-dependent chemiluminescence (CL) method. To investigate the discrepancy between the two methods, the effect of ferric–protein complexes on superoxide scavenging activity was examined. The reduced signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)–OOH due to superoxide dismutase (SOD) did not change with the addition of horseradish peroxidase (HRP), while the reduced CL response due to SOD was restored by the addition of different concentrations of HRP. Since HRP is a ferric–protein complex, the effects of other ferric–protein complexes, catalase and hemoglobin, on the reduced CL response due to SOD were examined, and similar results were obtained. As is the case with SOD, the reduced CL response activity due to an aqueous extract from a raphidophycean red tide flagellate, Chattonella ovata, was also enhanced by HRP, catalase, and hemoglobin. ESR spectra analyzed at 77 K indicated that aqueous extracts of Gymnodinium impudicum and Alexandrium affine, both of which are dinophycean red tide flagellates, contained a ferric–protein complex, and that an extract of C. ovata did not. These results suggest that the presence of such a ferric–protein complex is a causative factor in the discrepancy between the ESR and luminol CL methods when determining superoxide scavenging activity.
The pine wilt disease caused by Bursaphelenchus xylophilus (BX), also known as pine wood nematode (PWN), is the most devastating disease of pine trees. In this study, we engineered a highly specific antibody (single-chain fragment variable, scFv) against B. xylophilus cellulase antigen (BXCa). The antibody was raised against highly antigenic cellulase purified from PWN that efficiently hydrolyzed carboxymethyl cellulose. Total RNA was extracted from fresh spleens from BALB/c mice immunized with BXCa, and VH and VL were assembled with a linker following reverse transcriptase-polymerase chain reaction. The final phage display antibody library had a repertoire of about 5×104. We obtained specific engineered antibodies against BXCa after five rounds of affinity selection. The positive phage clones were used to infect Escherichia coli HB2151, and enzyme-linked immunosorbent assay and dot blotting showed that the soluble scFv specifically binded to BXCa. The scFv was sequenced and expressed in E. coli BL21 fused to enhanced green fluorescence protein, which had both green fluorescence and anti-BXCa functions. Using the fusion protein, we located cellulase in live PWN using an inverted fluorescence microscope and a laser scanning confocal microscope. The results strongly suggested that the cellulase was synthesized in the esophageal gland cells. This novel method of detecting and localizing proteins in live PWN might further our understanding of the underlying pathology of pine wilt disease.
The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with β-cyclodextrin at 25 °C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry of the thermal unfolding of the proteins showed that the unfolding temperature t1⁄2 of the two mutant proteins decreased by about 10 °C as compared to the wild-type protein at pH 7.0. At t1⁄2 of the wild-type protein (52.7 °C), the mutant proteins destabilized by about 10 kJ mol−1 in terms of the Gibbs energy change. It was found that the mutant proteins were quite stabilized in terms of enthalpy, but that a higher entropy change overwhelmed the enthalpic effect, resulting in destabilization.
Plasminogen and plasminogen activators play important roles in liver regeneration. Previously, we found that plasminogen potentiates hepatocyte proliferation in the primary culture of rat hepatocytes. Here, we examined how exogenous plasminogen affects the downstream events leading to cell proliferation. The addition of plasminogen to hepatocytes increased urokinase-type plasminogen activator (uPA) activity, but did not affect matrix metalloproteinase (MMP)-9 or MMP-2 activities. To increase uPA activity, plasminogen was required to bind the hepatocyte surface through the lysine-binding site of plasminogen molecule, but neither uPA mRNA nor uPA receptor (uPAR) mRNA was affected by the exogenous plasminogen. In addition, treatment of hepatocytes with an uPA inhibitor, p-aminobenzamidine, inhibited the plasminogen-induced and even EGF-induced hepatocyte proliferation. These results suggest that plasminogen-related control of hepatocyte proliferation is exerted topically by producing a hyperfibrinolytic state on the cellular surface involving the activation of uPA.
For higher plants, light is an important external signal, whereas cytokinin acts as an internal hormonal signal, and both are crucial for almost all aspects of development and physiological states. Here we identified and characterized a unique gene, CGA1, encoding a GATA factor, whose expression was rapidly induced by both the light and cytokinin signals in Arabidopsis thaliana.
We cloned the isovaleryl-CoA dehydrogenase (IVD)-encoding gene from Aspergillus oryzae. The promoter of ivdA was subjected to β-glucuronidase (GUS) reporter assays in which certain amino acids were used as a major carbon source. L-leucine most strongly induced GUS-activity, while in the case of L-glutamate, significantly low activity was found, indicating that ivdA transcription was strongly repressed by glutamic acid.
To elucidate the relationship between the substrate size and geometric shape of the catalytic site of Thermus maltogenic amylase, Gly50, Asp109, and Val431, located at the interface of the dimer, were replaced with bulky amino acids. The kcat⁄Km value of the mutant for amylose increased significantly, whereas that for amylopectin decreased as compared to that of the wild-type enzyme. Thus, the substituted bulky amino acid residues modified the shape of the catalytic site, such that the ability of the enzyme to distinguish between small and large molecules like amylose and amylopectin was enhanced.
The gluA gene, encoding an endo-β-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-β-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-β-1,3-glucanases, but GluA was partially similar to two fungal exo-β-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two β-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-β-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.
PhaP is a major poly[(R)-3-hydroxybutyrate] [P(3HB)]-granule-associated protein. Its gene expression is controlled by an autoregulated repressor, PhaR, in Paracoccus denitrificans. The packing force of the P(3HB) granule by PhaP is greatly influenced by the number of PhaP molecules. In this study, the effects of DNA-binding-ability-reduced mutations of PhaR on morphological change in the cellular granule formation of P(3HB) were examined under a transmission electron microscope using an Escherichia coli recombinant system. Microscopic observation indicated that stronger packing of P(3HB) granules took place when the number of PhaP molecules was increased by reduction in the DNA-binding ability of PhaR.
Two lacto-N-biose phosphorylase (LNBP) isozyme genes were cloned from Bifidobacterium bifidum JCM1254. Alignment of the amino acid sequences of LNBP and its homologs identified 24 completely conserved acidic amino acid residues. All single mutants of Bifidobacterium longum LNBP at residues other than D313N retained considerable activity, suggesting that Asp313 is the putative proton donor residue in LNBP.
S-adenosylmethionine (SAM) accumulated in cultured yeast cells and affected growth in two ways. High levels of intracellular SAM in yeast inhibited early growth, but increased growth in medium without sources of nitrogen and sulfur. Accumulated SAM in the yeast cells was recycled as a nutritional source depending on the sulfur and nitrogen contents of the medium.
A maltose phosphorylase (EC 188.8.131.52; MPase) showed novel acceptor specificity and transferred the glucosyl moiety of maltose not only to sugars but also to various acceptors having alcoholic OH groups. Salicyl alcohol acted as acceptor for MPase from Enterococcus hirae, and the product, salicyl-O-α-D-glucopyranoside (α-SalGlc) was identified. The yield based on supplied salicyl alcohol was 86% (mol/mol).
Sialyl oligosaccharides of human milk/colostrum are generally believed to be of biological significance, for example with respect to anti-adhesion of pathogenic organism, providing precursors for biosynthesis of brain, and so on. However, the levels of each of the sialyl oligosaccharides in human colostrum have not so far been determined. The present study was designed to determine the concentrations of nine major sialyl oligosaccharides in human colostrum, collected during the first 3 d (days 1–3) from the start of lactation. We found that the concentration of 3′-sialyllactose was significantly higher on day 1 than on day 2 and 3, but the levels of 6′-sialyllactose and sialyllacto-N-tetraose a were higher on day 3 than on day 1. These results are consistent with the view that during the first 3 d of lactation, the concentration of sialyl oligosaccharides in human colostrum change in accordance with the physiological demands of newborn infants.
We hypothesized that roasted Glycyrrhizae Radix (Glycyrrhizin Radix Praeparata, GRP) might modify anti-diabetic action due to compositional changes. Then we examined the anti-diabetic effect and mechanism of raw Glycyrrhizae Radix (GR) and GRP extracts and their major respective components, glycyrrhizin and glycyrrhetinic acid. In partial pancreatectomized (Px) diabetic mice, both GR and GRP improved glucose tolerance, but only GRP enhanced glucose-stimulated insulin secretion as much as exendin-4. Both GR and GRP extracts enhanced insulin-stimulated glucose uptake through peroxisome proliferation-activated receptor (PPAR)-γ activation in 3T3-L1 adipocytes. Consistently with the results of the mice study, only GRP and glycyrrhetinic acid enhanced glucose-stimulated insulin secretion in isolated islets. In addition, they induced mRNA levels of insulin receptor substrate-2, pancreas duodenum homeobox-1, and glucokinase in the islets, which contributed to improving β-cell viability. In conclusion, GRP extract containing glycyrrhetinic acid improved glucose tolerance better than GR extract by enhancing insulinotropic action. Thus, GRP had better anti-diabetic action than GR.
We have previously constructed a system which enables the search for factors that could modulate the intestinal calcium transporter, CaT1 (TRPV6; Takano et al., Cytotechnology, 43, 113 (2003)). This system evaluates the CaT1-mediated calcium uptake by using CHO cells stably expressing human CaT1 (CHO-hCaT1 cells). We found that a cheese whey protein digest (CWP-D) increased the calcium uptake by the CHO-hCaT1 cells. CWP-D also enhanced the calcium uptake in human intestinal Caco-2 cells. The in vivo effects of CWP-D were then measured by using rats with enteral feeding. Although enteral feeding decreased the portal calcium concentration, CWP-D partially suppressed the decrease, suggesting that CWP-D could be used for food to enhance calcium absorption.
Epidemiological evidence has sugged that vegetables and fruits may have a role in cancer prevention. The aim of the present study was to examine the anti-proliferative activity of ten related pure compounds from common vegetables and fruits. Studies were conducted on a series of carcinoma cells derived from eight human organs. The results show that linalool possessed the strongest activity against nine carcinoma cells, and that baicalein and luteolin also exhibited a broad spectrum of anti-proliferative activities. Among them, linalool showed the strongest activity against carcinoma of the cervix (IC50: 0.37 μg/ml), stomach (IC50: 14.1 μg/ml), skin (IC50: 14.9 μg/ml), lung (IC50: 21.5 μg/ml) and bone (IC50: 21.7 μg/ml). As for the flavonoids, luteolin exhibited the strongest activity against carcinoma of the stomach (IC50: 7.1 μg/ml), cervix (IC50: 7.7 μg/ml), lung (IC50: 11.7 μg/ml) and bladder (IC50: 19.5 μg/ml), whereas baicalein possessed the strongest anti-proliferative activity against carcinoma of the cervix (IC50: 9.8 μg/ml), stomach (IC50: 16.1 μg/ml) and skin (IC50: 19.5 μg/ml). The present study indicates that linalool possessed the strongest activity against a broad spectrum of carcinoma cells, especially cervical carcinoma cells, suggesting that linalool and flavonoids are partially responsible for the cancer prevention of common vegetables and fruits.
The preventive anti-diabetic effect of dangnyosoko (DNSK), a Chinese herbal medicine, was evaluated in STZ-induced diabetic rats. DNSK was orally administered once a day from 3 d after STZ-induction at 100, 200, and 500 mg/kg for 4 weeks, and the results were compared to those for glibenclamide. Dramatic decreases in body weight and plasma insulin levels and increases in blood and urine glucose levels were detected in STZ-induced diabetic animals with disruption and disappearance of pancreatic islets and increases in glucagon- and decreases in insulin-producing cells. However, these diabetic changes were significantly and dose-dependently inhibited by treatment with DNSK, and DNSK at 100 mg/kg showed more favorable effects than glibenclamide at 5 mg/kg. Based on these results, it is thought that DNSK has favorable effects in ameliorating changes in blood and urine glucose levels and body weight, and that histopathological changes in the pancreas in STZ induce diabetes.
The protective effects of Platycodi radix (PR), the root of Platycodon grandiflorum A. DC, on alcohol-induced fatty liver and possible mechanisms involved in this protection were investigated in rats. Administration of PR significantly prevented alcohol-induced elevation of serum and liver lipids. Furthermore, PR treatment normalized hepatic liver fatty acid binding protein (L-FABP) expression and cytochrome P450 2E1 (CYP2E1) activity in alcohol-treated rats. These results suggest that inhibition of CYP2E1 and regulation of L-FABP by PR play an important role in alcohol-induced hepatoprotection.
The antiallergic properties of a hop water extract (HWE) were studied by evaluating the Evans blue leakage from ICR mice caused by compound 48/80 stimulation, and the histamine release from ovalbumin (OVA)-sensitized BALB/c mice. An oral administration of HWE significantly inhibited the vascular permeability and histamine release. HWE itself did not have any influence on the total and antigen-specific immunoglobulin E (IgE) production in OVA-sensitized mice. These results indicate that HWE exerted an antiallergic effect by inhibiting the release of chemical mediators from mast cells and basophiles.
A new enzyme, NAD+-dependent 4-N-trimethylamino-1-butanol dehydrogenase from Pseudomonas sp. 13CM, was purified 526-fold to apparent homogeneity in 5 chromatographic steps. The enzyme had a molecular mass of 45 kDa and appeared to be a monomer enzyme. The isoeletric point was found to be 4.8. The optimum temperature was 50 °C, and the optimum pHs for the oxidation and reduction reactions were 9.5 and 6.0 respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters, and amino acid terminal sequence. The Km values for trimethylamino-1-butanol and NAD+ were 0.54 mM and 0.22 mM respectively. In the reduction reaction, the apparent Km values for trimethylaminobutylaldehyde and NADH were 0.67 mM and 0.04 mM, respectively. The enzyme was inhibited by SH reagents, chelating reagents, and heavy metal ions. The N-terminal 12 amino acid residues were sequenced.
Glucagon-like peptide-1 (GLP-1), an incretin secreted by intestinal L-cells, can effectively lower blood glucose levels in patients with diabetes. A fusion gene, consisting of 10 tandem repeated GLP-1 analog genes, was expressed at a high level in the yeast Pichia pastoris. SDS polyacrylamide gel electrophoresis (SDS–PAGE), and Western Blotting results showed that fusion protein migrated as a single protein band with a molecular weight of 36 kDa. A biological activity test showed that the GLP-1 analog could significantly lower the level of serum glucose when GLP-1 purified analog was injected into diabetic rats. A potential strategy for large-scale production of fusion protein containing the 10 GLP-1 analogs as discovered, and a single GLP-1 analog was obtained from fusion protein digested with trypsin. This should be inspired foreign expression of medicinal short peptides and be valuable in further research on GLP-1 analog drugs in the treatment of diabetes mellitus.
This paper describes the demulsification of olive-oil/SDS emulsion (DOSE) method for selective isolation of environmental mycobacteria. A soil sample was suspended in olive oil and centrifuged. The supernatant was emulsified on plates together with SDS solution. After incubation, the colonies that had developed on the plates were surrounded by clear zones. The isolates were identified as genus Mycobacterium, and as belonging to a fast-growing group, by 16S rRNA gene sequence analysis.
Penicillin V acylase from Streptomyces mobaraensis (Sm-PVA) showed high acyl-transfer activity in reactions using methyl esters of carboxylic acid (acyl donor) and amino compounds (nucleophile), to produce the corresponding amides. Moreover, Sm-PVA had broad substrate specificity, as indicated by the fact that it catalyzed the efficient synthesis of β-lactam antibiotics, capsaicin derivatives, and N-fatty-acyl-amino acid/N-fatty-acyl-peptide derivatives.