Electrophysiological Characterization of the Mechanosensitive Channel MscCG in Corynebacterium glutamicum

Corynebacterium glutamicum MscCG, also referred to as NCgl1221, exports glutamate when biotin is limited in the culture medium. MscCG is a homolog of Escherichia coli MscS, which serves as an osmotic safety valve in E. coli cells. Patch-clamp experiments using heterogeneously expressed MscCG have shown that MscCG is a mechanosensitive channel gated by membrane stretch. Although the association of glutamate secretion with the mechanosensitive gating has been suggested, the electrophysiological characteristics of MscCG have not been well established. In this study, we analyzed the mechanosensitive gating properties of MscCG by expressing it in E. coli spheroplasts. MscCG is permeable to glutamate, but is also permeable to chloride and potassium. The tension at the midpoint of activation is 6.68 ± 0.63 mN/m, which is close to that of MscS. The opening rates at saturating tensions and closing rates at zero tension were at least one order of magnitude slower than those observed for MscS. This slow kinetics produced strong opening-closing hysteresis in response to triangular pressure ramps. Whereas MscS is inactivated under sustained stimulus, MscCG does not undergo inactivation. These results suggest that the mechanosensitive gating properties of MscCG are not suitable for the response to abrupt and harmful changes, such as osmotic downshock, but are tuned to execute slower processes, such as glutamate export.     

Spermatozoa ultrastructure of the pink shrimp Farfantepenaeus paulensis (Decapoda: Dendrobranchiata)

Braga, A.L., Nakayama, C.L., Suita de Castro, L.A. and Wasielesky, W. 2011. Spermatozoa ultrastructure of the pink shrimp Farfantepenaeus paulensis (Decapoda: Dendrobranchiata). —Acta Zoologica (Stockholm) 00 : 1–6. The spermatozoa ultrastructure of the pink shrimp Farfantepenaeus paulensis was investigated in this morphological study. Spermatophores and spermatozoa were analyzed by electron microscopy. The pink shrimp spermatophore is divided into two regions: the appendage and the spermatophore main body, where spermatozoa are grouped in a spermatic mass. Pink shrimp spermatozoa are unistellate and are composed of main body and single spike. The spermatozoa body comprises a perinuclear cytoplasmic band, nucleus, acrosomal cap, and subacrosomal region. The spermatozoa cell mean total length was 10.71 μm, the mean body diameter was 5.56 μm, and the mean spike length and diameter were 5.15 μm and 0.85 μm, respectively.     

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.     

A novel,potent, and orally active VLA-4 antagonist with good aqueous solubility: trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5 mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F = 54%) in monkeys.     

Purification and Properties of L-Methionine γ-Lyase from Aeromonassp.

Four types of β-xylosidases from a concentrated culture filtrate of Pénicillium wortmanni IFO 7237, designated as xylosidase-1, -2, -3, and -4 were purified to homogeneity on SDS polyacrylamide gel electrophoresis by an alcohol precipitation, DEAE-Sephadex A-25 ion exchange chromatography, and isoelectric focusing. The molecular weights of xylosidase-1, -2, -3, and -4 were estimated to be 110,000, 195,000, 210,000, and 180,000 respectively and their isoelectric points to be 3.7, 4.28, 4.6, and 4.8. The pH optima of β-xylosidase activities were from 3 to 4.5. The optimum temperature for enzyme activities was from 55°C to 65°C. On the enzymic hydrolysis of phenyl ß-d- xyloside, the reaction product of each enzyme was found to be β-d-xylose with retention of configuration. All the four ß-xylosidases were free of α-xylosidase and ß-glucosidase activities. All the enzyme activities of four β-xylosidases were strongly inhibited by Hg²⁺ and N- bromosuccinimide. With respect to the hydrolysis patterns and HPLC analysis of hydrolyzates from xylooligosaccharides, xylosidase-2 was totally different from other three as a distinct enzyme. Xylosidase-1 was also in a separate group although xylosidase-3 and -4 showed closely related action patterns as a different group.     

The Piperaceae Amides I: Structure of Pipercide,A New Insecticidal Amide from Piper nigrum L.

It was evidenced that mutagenic principles in tryptophan pyrolysate, 3-amino-1,4-dimethyl-5H pyrido(4,3-b) indole and 3-amino-1-methyl-5H pyrido(4,3-b) indole (abbreviated as Trp-P-1 and Trp-P-2, respectively) bind to DNA without activation by rat liver microsomes. The bindings of Trp-P-1 and Trp-P-2 were not random and did not introduce strand scissions into DNA. Trp-P-1 bound more easily than Trp-P-2. The bindings of these mutagenic principles to DNA were concluded by using negatively superhelical simian virus 40 (SV40) DNA from following experimental data. (1) The intensity of ethidium bromide (EtBr)-DNA fluorescence by illumination with UV light and the electrophoretic mobility of superhelical DNA in agarose gel decreased as a function of the amounts of Trp-P-1 and Trp-P-2. (2) In vitro RNA synthesis catalyzed by Escherichia coli DNA-dependent RNA polymerase and nick-translation catalyzed by Escherichia coli DNA polymerase I (Kornberg enzyme) were inhibited significantly on DNA treated with Trp-P-1 and Trp-P-2. (3) The negative superhelicity of SV40 DNA introduces unpaired regions into DNA. These regions can be cleaved by single-strand-specific S1 endonuclease to generate unit length linear duplex molecules. It was found that this S1-sensitivity of DNA decreased by treatment with Trp-P-1. (4) The cleavage patterns of Trp-P-1 treated DNA with five restriction endonucleases were investigated. The protection of the cleavage site by the drug was observed against HincII, HindIII and EcoRII, whereas not against HaeIII and HinfI. These results show that the binding of Trp-P-1 to DNA is not random. Identical results were also obtained in Trp-P-2.

However, the bindings of Trp-P-1 and Trp-P-2 were not so tight, and phenol extraction of the complex dissociated these drugs from DNA.     

N-Acetylornithine-δ-aminotransferase-deficient and N-Acetylglutamokinase-deficient Arginine Auxotrophs of Corynebacterium glutamicum

An N-acetylglutamokinase-deficient arginine-requiring mutant, KY9390 and an N-acetylornithine aminotransferase-deficient arginine-requiring mutant, AA-7 were derived from a wild-type strain and an l-arginine-producing mutant of Corynebacterium glutamicum, respectively. KY 9390 accumulated 7.5 mg per ml of N-acetylglutamic acid and AA-7 accumulated 2 mg per ml of N-acetylglutamate-γ-semialdehyde, intermediates of arginine biosynthesis, in a culture medium.

The production of N-acetylglutamate-γ-semialdehyde by AA-7 was not affected by the concentration of l-arginine in the medium, whereas that of N-acetylglutamic acid by KY 9390 was inhibited by the addition of l-arginine in the medium.     

A New Basic Triple Salt Containing Magnesium Hydroxide

The quinary system KCl-K₂SO₄-MgCl₂-MgSO₄-Mg(OH)₂-H₂O and associated eight systems K₂SO₄-MgSO₄-Mg(OH)₂-H₂O, MgCl₂-MgSO₄-Mg(OH)₂-H₂O, KCl-MgCl₂-Mg(OH)₂-H₂O, KCl-K₂SO₄-Mg(OH)₂-H₂O, MgSO₄-Mg(OH)₂-H₂O, MgCl₂-Mg(OH)₂-H₂O, K₂SO₄-Mg(OH)₂-H₂O and KCl-Mg(OH)₂-H₂O were investigated at 50° The solid phases of these systems were the new basic triple salt (NS salt B), MgCl₂ · 3Mg(OH)₂ · 8H₂O, MgSO₄ · 5Mg(OH)₂ · 3H₂O, carnallite, leonite, kieserite, hexahydrite, bischofite, potassium chloride, potassium sulfate and magnesium hydroxide and the crystallization fields of these salts in nine systems were determined.     

Studies on l-Threonine Fermentation

Fifteen strains of bacteria were treated with ultraviolet light or N-methyl-N′-nitro-N-nitrosoguanidine to derive auxotrophic mutants, which were screened for their ability to produce l-threonine. A number of auxotrophs were derived from each strain. Among them, those which produced a large amount of l-threonine were found in Aerobacter aerogenes, Serratia marcescens and Escherichia coli, the members of the family Enterobacteriaceae. Nutritional requirements of these threonine producers were proved to be methionine, lysine, or α, ε-diaminopimelic acid (DAP).

In A. aerogenes and E. coli, double and triple auxotrophs were derived with futher mutational treatment. As a, rule, imposition of additional block led to the increase of l-threonine production. In E. coli, many triple auxotrophs (DAP^?, Met^?, He^?) and their isoleucine revertants were screened for their ability to produce l-threonine. Enhancement of l-threonine production was achieved with these mutants.

One of the isoleucine revertants, KY8280, was used to investigate some cultural conditions. As a result, l-threonine accumulation reached to a level of 13.8 mg/ml with the medium containing 7.5% fructose.