Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis

An isolate from kimchi, identified as Lactobacillus brevis, accumulated γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the culture medium. Optimal culture conditions for growth of L. brevis and production of GABA were 6 % (w/v) l-glutamic acid, 4 % (w/v) maltose, 2 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl₂, 2 g Tween 80/l, and 0.02 mM pyridoxal 5′-phosphate at initial pH 5.25 and 37 °C. GABA reached 44.4 g/l after 72 h cultivation with a conversion rate 99.7 %, based on the amount (6 %) of l-glutamic acid added. GABA was purified using ion exchange column chromatography with 70 % recovery and 97 % purity.     

Occurrence of Endogenous Pollen Growth Inhibitors, 4-Hydroxybenzoic Acid and 4-(β-D-Glucopyranosyloxy)benzoic Acid,in the Pollen of Pinus densiflora†

A Pseudomonas strain capable of using pyrazinamide as the sole source of nitrogen was isolated from soil. An aromatic amidase from the bacterium was purified 400-fold to homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 43,000 by gel filtration on Sephadex G-150 and consisted of two identical subunits. The isoelectric point was at 4.45. Among the compounds tested, pyrazinamide (relative activity, 100%), nicotinamide (60%), and 5-methylpyrazinamide (3.4%) were hydrolyzed at considerable rates. Benzamide, picolinamide, and isonicotinamide were not substrates. Apparent Km of the enzyme for pyrazinamide and nicotinamide were 5.6 × 10 ^?5 m and below 5 × 10^?6 m, respectively. The enzyme was not able to hydrolyze aliphatic amides. The enzyme was most active between pH 6.5 and 10 and 75°C, and was stable between pH 5.5 and 8.5 and below 45°C.     

Occurrence of Isóenzymes of Glutamine Synthetase in the Alga Chlorella kessleri

Two forms (GS₁ and GS₂) of glutamine synthetase have been isolated, separated by ion exchange chromatography, and partly characterized from cells of the green alga Chlorella kessleri. Both forms are present in cells grown autotrophically or heterotrophically on various nitrogen sources, but under all nutritional conditions GS₁ was found to be the major isoenzyme present (60-80%). The activity of both isoenzymes was greatest in cells grown under nitrogen-limiting conditions. Both isoenzymes have molecular weights in the range 340 to 350,000 daltons. GS₁ was found to have a greater thermostability than GS₂: GS₁ was stable at 30°C while GS₂ lost 95% of its activity in 30 minutes. GS₁ was much less sensitive to thiol reactive reagents than GS₂.     

Novel Properties of a Mouse γ-Aminobutyric Acid Transporter (GAT4)

We expressed the mouse gamma-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/ human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/ Cl-/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q10) of 4.3. The GAT4 turnover rate (1.5 s(-l); -50 mV, 21 degrees C) and temperature dependence suggest physiological turnover rates of 15-20 s(-1). No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl- leak current (ICl(leak)) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (ICl(leak)) followed the Cl- Nernst potential. (ii) A Li+ leak current (ILi(leak)) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q10 approximately 1). The two leak modes appeared not to coexist, as Li+ inhibited (ICl(leak)). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/Cl-/GABA cotransport, ILi(leak), and ICl(leak), (Ki approximately 30 microM), and the voltage-induced presteady-state charge movements (Ki approximately 440 microM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jicroumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.     

Occurrence of d-α-Amino-n-butyric Acid in Legume Seedlings

Metabolomics analysis of three Saccharopolyspora spinosa strains (wild type strain WT, ultraviolet mutant strain WH124, and metabolic engineering strain LU104) with different spinosad producing levels was performed by liquid chromatograph coupled to mass spectrometry (LC-MS). The metabolite profiles were subjected to hierarchal clustering analysis (HCA) and principal component analysis (PCA). The results of HCA on a heat map revealed that the large numbers of primary metabolism detected were more abundant in WH124 and less abundant in LU104 during the early fermentation stage as compared to the WT strain. PCA separated the three strains clearly and suggested nine metabolites that contributed predominantly to the separation. These biomarkers were associated with central carbon metabolism (succinic acid, α-ketoglutarate, acetyl-CoA, and ATP), amino acid metabolism (glutamate, glutamine, and valine), and secondary metabolism (pseudoaglycone), etc. These findings provide insight into the metabolomic characteristics of the two high-yield strains and for further regulation of spinosad production.     

Occurrence of l-Saccharopine and γ-L-Glutamylglycine in the Mushroom,Agaricus bisporus

Microfolicoumarin (1), a prenylcoumarin from Cedrelopsis microfoliata, was synthesized from 2,4,5-trimethoxybenzaldehyde in five steps. 1 did not show significant antioxidative activity, but the key intermediates, esculetin (3) and 5-prenylesculetin (6), exhibited strong antioxidative activity in both the superoxide-radical and 1,1-diphenyl-2-picrylhydrazyl radical-scavenging models.     

Occurrence and Some Properties of Two Types of δ-Aminolevulinic Acid Synthase in a Facultative Methylotroph,Protaminobacter ruber

In addition to the α-ALA synthase already reported (Fraction I: molecular weight, 100,000; optimal pH, ca. 8.0), an isozyme (Fraction II: molecular weight, 64,000; optimal pH, ca. 6.4) was found in Protaminobacter ruber grown in the dark after an initial light period (20~30hr). The fraction I- and II-enzymes were separable by gel-filtration through Sepharose 6B. While the former was formed constitutively, the latter was formed inducibly under the conditions for bacteriochlo-rophyll formation. Therefore, the fraction II-isozyme seems to be responsible for the biosynthesis of bacteriochlorophyll.     

Occurrence of Multiple Ribonucleotide Reductase Classes in γ-Proteobacteria Species

Ribonucleotide reductase (RNR) is central to de novo synthesis of deoxyribonucleotides and essential for all living cells. Three classes have been described; class I is oxygen dependent and represented by two subclasses, Ia (NrdAB) and Ib (NrdEF); class II (NrdJ) is indifferent to oxygen; and class III (NrdDG) is oxygen sensitive. More than one class can be found in an organism, reflecting the oxygen status of its environment. We have investigated, by using PCR and Southern blot, the occurrence of the different classes among species of the γ-Proteobacteria. Class III are present in all species tested, but the presence of the other classes varies. Some species contain one unique additional enzyme, class Ia, Ib, or II, whereas others contain two additional enzymes, class Ia and Ib, or class Ia and II. Received: 8 May 2000 / Accepted: 13 June 2000     

Occurrence of δ-Aminovaleric Acid in the Culture Medium of Rumen Ciliate Protozoa

An unknown ninhydrin positive spot on the paper chromatogram of the endogenous metabolites of mixed rumen ciliates was isolated by using ‘Dowex’ 50–X8 column and silica gel column and identified as δ-aminovaleric acid by the data of elementary analysis, specific optical rotation, mass spectrum, paper chromatogram and infrared spectrum.     

Agonist-dependent Endocytosis of γ-Aminobutyric Acid Type A (GABAA) Receptors Revealed by a γ2(R43Q) Epilepsy Mutation

GABA-gated chloride channels (GABA_ARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABA_ARs on the cell surface to better understand the trafficking of GABA_ARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABA_AR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABA_ARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABA_AR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABA_ARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist.     

Occurrence of l-(−)-Pipecolic Acid in the Culture Medium of Rumen Ciliate Protozoa

Effect of phenoxazine derivatives (actinomycin D, l,8-dimethyl-3-aminophenoxazone-2, 3-aminophenoxazone-2, sodium resazurate, gallocyanine, fluorescent blue and capri blue) on amino acid transport in rat small intestine was investigated using tissue accumulation method. Capri blue (CI–51015) inhibited the accumulation of l-leucine, l-alanine and l-valine, and scarcely inhibited that of D-glucose. Kinetic analysis showed that the inhibition of amino acid accumulation by this pigment was non-competitive. Actinomycin D had no effect on amino acids and d-glucose accumulation in vitro at the high and low concentrations of these substances. High concentration of actinomycin D and long period of incubation had no effect, too. Accumulation of amino acids into the intestinal tissue was not significantly decreased or increased by the presence of other phenoxazine derivatives.     

Multiple Propofol-binding Sites in a γ-Aminobutyric Acid Type A Receptor (GABAAR) Identified Using a Photoreactive Propofol Analog

Propofol acts as a positive allosteric modulator of γ-aminobutyric acid type A receptors (GABA_ARs), an interaction necessary for its anesthetic potency in vivo as a general anesthetic. Identifying the location of propofol-binding sites is necessary to understand its mechanism of GABA_AR modulation. [³H]2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (azietomidate) and R-[³H]5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), photoreactive analogs of 2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate (etomidate) and mephobarbital, respectively, have identified two homologous but pharmacologically distinct classes of intersubunit-binding sites for general anesthetics in the GABA_AR transmembrane domain. Here, we use a photoreactive analog of propofol (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol ([³H]AziPm)) to identify propofol-binding sites in heterologously expressed human α1β3 GABA_ARs. Propofol, AziPm, etomidate, and R-mTFD-MPAB each inhibited [³H]AziPm photoincorporation into GABA_AR subunits maximally by ∼50%. When the amino acids photolabeled by [³H]AziPm were identified by protein microsequencing, we found propofol-inhibitable photolabeling of amino acids in the β3-α1 subunit interface (β3Met-286 in β3M3 and α1Met-236 in α1M1), previously photolabeled by [³H]azietomidate, and α1Ile-239, located one helical turn below α1Met-236. There was also propofol-inhibitable [³H]AziPm photolabeling of β3Met-227 in βM1, the amino acid in the α1-β3 subunit interface photolabeled by R-[³H]mTFD-MPAB. The propofol-inhibitable [³H]AziPm photolabeling in the GABA_AR β3 subunit in conjunction with the concentration dependence of inhibition of that photolabeling by etomidate or R-mTFD-MPAB also establish that each anesthetic binds to the homologous site at the β3-β3 subunit interface. These results establish that AziPm as well as propofol bind to the homologous intersubunit sites in the GABA_AR transmembrane domain that binds etomidate or R-mTFD-MPAB with high affinity.     

Age-Related Development of γ-Aminobutyric Acid (GABA)BReceptor Functions in Various Brain Regions of Spontaneously Hypertensive Rats

The age-related development of GABA_Breceptors and their coupling to adenylate cyclase were studied in the brains of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Compared with WKY rats, the specific [³H]GABA binding to GABA_Breceptors showed a significant decrease not only in the posterior hypothalamus, midbrain, hippocampus and striatum of eleven-week-old SHR, which maintain a hypertensive state, but also in the posterior hypothalamus of four-week-old normotensive SHR. Similarly, the GABA_Breceptor agonists (baclofen and DN-2327)-induced suppression of adenylate cyclase activity showed a decrease in the posterior hypothalamus of four-week-old SHR as well as in the posterior hypothalamus and striatum of eleven-week-old SHR. These results suggest that the functions of the GABA_Breceptor in the brain of SHR may be decreased independently from the occurrence of blood pressure elevation and that such changes may even be involved in the pathogenesis of SHR.     

Strict Binding Specificity of Small-Sized Lectins from the Red Alga Hypnea japonica for Core (α1-6) Fucosylated N-Glycans

Small-sized isolectins (9 KDa) from Hypnea japonica belong to a new lectin family. Here, we describe the carbohydrate-binding properties of the three isolectins (hypninA1, A2, and A3) and the amino acid sequence of hypninA3 (P85888). In frontal affinity chromatography with about 100 pyridylaminated oligosaccharides, the isolectins, which had no affinity for monosaccharides, commonly bound only core (α1-6) fucosylated N-glycans, and did not the other oligosaccharides examined, including (α1-2), (α1-3), and (α1-4) fucosylated glycans. The specific binding of hypninA3 with the fucosylated N-glycans (K _a; 0.52–7.58×10⁶ M^?1) was confirmed by surface plasmon resonance analyses on an immobilized glycoprotein with and without core (α1-6) fucose. Such specificity of hypninA is clearly distinct from those of other known fucose-binding lectins, making it a valuable tool for cancer diagnosis and quality control of medicinal antibodies. HypninA3 is a polypeptide composed of 90 amino acids containing four half-cystines.     

Synthesis of 4′-(Hydroxymethyl)Guanosine and a Phosphonate Analogue of Guanylic Acid

Abstract

The synthesis of 4′-(hydroxymethyl)guanosine (7) and the phosphonate analogue 8 of guanylic acid proceed from a common intermediate, 2′, 3′-O-isopropylidene-N ²-(monomethoxytrityl)-guanosine-5′-aldehyde (13).     

In Vitro Studies of a New Semisynthetic Penicillin, 6-(D-α-Sulfoaminophenylacetamido)-Penicillanic Acid

The activity of 6-(D-α-sulfoaminophenylacetamido)-penicillanic acid was determined against 357 clinical isolates of gram-negative bacilli by use of the tube-dilution technique. The majority of the isolates of Pseudomonas species were inhibited by 200 μg/ml or less of this antibiotic. Most of the isolates of Escherichia coli had a minimal inhibitory concentration of 50 μg/ml or less. Seventy-three per cent of the isolates of P. mirabilis, 40% of the isolates of P. morganii, and 45% of the isolates of Enterobacter species were inhibited by 12.5 μg/ml or less, whereas most of the isolates of Klebsiella species and Serratia species were resistant. The activity of this semisynthetic penicillin was affected by the size of the inoculum. The drug was bactericidal against all isolates of E. coli and Proteus species that were sensitive to it, but it was bactericidal against only 32% of the sensitive isolates of Pseudomonas species.     

Formation of Phage-Induced γ-Polyglutamic Acid Depolymerase in Lysogenic Strain of Bacillus natto

The influence of tea catechins on the absorption of starch or sucrose was investigated in vivo. Tea catechins were administered orally to rats before soluble starch or sucrose administration. Saccharide-dosed rats were killed and the blood and the contents of the intestine were collected at intervals over two hours. Catechins of certain concentrations suppressed the increase of plasma glucose levels, thus concurrently suppressing insulin activity. Increased activity of intestinal α-amylase by starch dosing was inhibited markedly in the catechin-administered rats. Sucrase on the brush border membrane was also inhibited by prior catechin administration. From these results it was assumed that orally administered catechins will inhibit intestinal α-amylase or sucrase, thereby deterring the digestion of certain amounts of starch or sucrose and eventually reducing the plasma glucose levels.     

Therapeutic effect of CHF5074, a new γ-secretase modulator,in a mouse model of scrapie

In transmissible spongiform encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrP^C is a mediator of a synaptic dysfunction induced by Aβ oligomers. We tested a novel γ-secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 d longer than vehicle-treated mice (p < 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrP^Sc in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared with the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrP^Sc toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.Key words: TSE, prion, murine model, γ-secretase modulator, therapy