Identification of acetic acid bacteria isolated from Indonesian sources,especially of isolates classified in the genus Gluconobacter

Sixty-four strains of acetic acid bacteria were isolated from Indonesian sources such as fruits, flowers, and fermented foods by the enrichment culture at pH 3.5. Forty-five strains were routinely identified as Acetobacter strains because of their oxidation of acetate and lactate to carbon dioxide and water and their Q-9 isoprenolog, corresponding to 70% of all the 64 acetic acid bacteria isolated. Eight isolates were identified as Gluconacetobacter strains because of their oxidation of acetate and lactate and their Q-10 isoprenolog, occupying 13% of all the isolates. The remaining 11 isolates, accommodated in the genus Gluconobacter because of no oxidation of acetate and lactate and because of their Q-10 isoprenolog, accounted for 17% of all the isolates. They were divided into two groups based on DNA base compositions. One comprised the seven isolates, which had high G1C contents of DNA ranging from 60.3 to 63.5 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter oxydans at values of 64-94% of DNA relatedness. The other comprised the remaining four isolates, which had low G+C contents of DNA ranging from 57.5 to 57.7 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter frateurii at values of 63-77% of DNA relatedness. The high values of DNA relatedness, 84 to 96%, were obtained between the type strains of Gluconobacter cerinus and Gluconobacter asaii.     

Colocalization of GLUT3 and choline acetyltransferase immunoreactivity in the rat retina

Toward elucidating the functional aspects ofGLUT3, a primary neuronal glucose transporter isoform in the vertebrate central nervous system, this study examined its expression in cholinergic amacrine cells made identifiable by the presence of acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT), in the rat retina. Double-immunofluorescence staining of adult rat retinal tissue with anti-GLUT3 and anti-ChAT antibodies revealed characteristic stratified GLUT3 immunoreactivity (GLUT3-IR) in the inner plexiform layer (IPL) that was identical to the arborization pattern of ChAT-positive neuronal processes there. In addition, approximately 30-50% of intensely GLUT3-immunoreactive cell bodies in the inner nuclear layer and ganglion cell layer showed ChAT-IR, while the majority of ChAT-positive cell bodies were also intensely GLUT3 immunoreactive. Analysis at the cellular level using retinal cells in culture revealed similar findings. These results collectively indicate that cholinergic amacrine cells constitute the major component of GLUT3-expressing cells in the rat retina. It is expected that the link demonstrated here between GLUT3 expression and cholinergic amacrine cell population will provide clues for further analyzing GLUT3 function in the retina.     

Signal detection theory applied to three visual search tasks--identification, yes/no detection and localization

Adding distracters to a display impairs performance on visual tasks (i.e. the set-size effect). While keeping the display characteristics constant, we investigated this effect in three tasks: 2 target identification, yes-no detection with 2 targets, and 8-alternative localization. A Signal Detection Theory (SDT) model, tailored for each task, accounts for the set-size effects observed in identification and localization tasks, and slightly under-predicts the set-size effect in a detection task. Given that sensitivity varies as a function of spatial frequency (SF), we measured performance in each of these three tasks in neutral and peripheral precue conditions for each of six spatial frequencies (0.5-12 cpd). For all spatial frequencies tested, performance on the three tasks decreased as set size increased in the neutral precue condition, and the peripheral precue reduced the effect. Larger set-size effects were observed at low SFs in the identification and localization tasks. This effect can be described using the SDT model, but was not predicted by it. For each of these tasks we also established the extent to which covert attention modulates performance across a range of set sizes. A peripheral precue substantially diminished the set-size effect and improved performance, even at set size 1. These results provide support for distracter exclusion, and suggest that signal enhancement may also be a mechanism by which covert attention can impose its effect.     

Isolation and characterization of a cellulolytic <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> T4 strain from sugar refinery wastewater

A novel, cellulolytic, bacterial thermophilic strain, T4, was isolated from sugar refinery wastewater in southern Taiwan. This isolate, a Gram-negative, motile, aerobically growing sporulating rod, can secrete thermostable endocellulase (endo-1,4--D-glucanase, EC 3.2.1.4) and hydrolyze carboxymethylcellulose (CMC), phosphoric acid-swollen cellulose, Avicel, filter paper, and salicin. When strain T4 was grown in CMC medium, the cellulolytic enzyme activity in culture supernatants was stable up to 70°C. More than 10% of the original activity was still detectable after heating to 100°C with a pH 7.0 for 1 h. Based on 16S rDNA sequence analysis, DNA base composition, phenotypic and physiological characteristics, as well as DNA–DNA hybridization, strain T4 was classified as Geobacillus thermoleovorans T4 (DSM 14791 = CCRC 17200). We also demonstrated that the type species G. stearothermophilus (DSM 22 = ATCC 12980) could hydrolyze amorphous and crystalline (filter paper) celluloses at a rate of 13 and 14%, respectively, in comparison with strain T4.     

Thioredoxin liquefies and decreases the viscoelasticity of cystic fibrosis sputum

The persistent and viscous nature of airway secretions in cystic fibrosis (CF) disease leads to airway obstruction, opportunistic infection, and deterioration of lung function. Thioredoxin (Trx) is a protein disulfide reductase that catalyzes numerous thiol-dependent cellular reductive processes. To determine whether Trx can alter the rheological properties of mucus, sputum obtained from CF patients was treated with TRX and its reducing system (0.1 microM thioredoxin reductase + 2 mM NADPH), and liquid phase-gel phase ratio (percent liquid phase) was assessed by compaction assay. Exposure to low Trx concentrations (1 microM) caused significant increases in the percentage of liquid phase of sputum. Maximal increases in percent liquid phase occurred with 30 microM Trx. Additional measurements revealed that sputum liquefaction by the Trx reducing system is dependent on NADPH concentration. The relative potency of the Trx reducing system also was compared with other disulfide-reducing agents. In contrast with Trx, glutathione and N-acetylcysteine were ineffective in liquefying sputum when used at concentrations <1 mM. Sputum viscoelasticity, measured by magnetic microrheometry, also was diminished significantly following 20-min treatment with 3, 10, or 30 microM Trx. Similarly, this reduction in viscoelasticty also was dependent on NADPH concentration. Further investigation has indicated that Trx treatment increases the solubility of high-molecular-weight glycoproteins and causes redistribution of extracellular DNA into the liquid phase of sputum. Recognizing that mucins are the major gel-forming glycoproteins in mucus, we suggest that Trx alters sputum rheology by enzymatic reduction of glycoprotein polymers present in sputum.     

Signaling via histamine receptors in cat duodenal smooth muscle cells

Histamine produced concentration-dependent contractions in cat duodenal smooth muscle cells that were obtained by enzymatic digestion of smooth muscle with collagenase F. Pyrilamine, an H1 receptor antagonist, inhibited the contractile response while famotidine, an H2 receptor antagonist, augmented it. In cells with selectively preserved H1 receptors, produced by pretreatment with pyrilamine followed by inactivation of all unprotected receptors with N-ethylmaleimide, histamine-induced contraction was significantly augmented as compared with control cells. Pertussis toxin (PTX) had no effect on contraction, suggesting that the H1 receptor is coupled to a PTX-insensitive G protein. Gi2, Gi3, Go, Gs, and Gq subunits were present in cat duodenum, and histamine-induced contraction was inhibited by Gq antibody after cell permeabilization. Neomycin, a PLC inhibitor, inhibited the histamine-induced cell contraction, but not rhoCMB, a PLD inhibitor, or DEDA, a PLA2 inhibitor. Heparin, an IP3 receptor inhibitor, inhibited contraction whereas chelerythrine, a PKC inhibitor, had no effect. We conclude that histamine-induced contraction in cat duodenal smooth muscle cells is mediated by H1 receptors coupled to a PTX-insensitive Gq protein and results in activation of phosphatidylinositol-specific phospholipase C (PI-PLC).     

Characterization of Lactobacillus sakei by the type of stereoisomers of lactic acid produced

Lactobacillus sakei strains were characterized by the shift of the type of stereoisomers of lactic acid produced in the presence of 50 mM sodium acetate in a medium. Of 27 Lactobacillus sakei strains studied, 20 strains showed high levels of DNA-DNA similarity with L. sakei NRIC 1071(T), and were confirmed as L. sakei. The three remaining strains were identified as Lactobacillus curvatus by DNA-DNA similarity, and three other strains were included in the cluster of Lactobacillus plantarum/Lactobacillus pentosus/Lactobacillus paraplantarum and one strain in the cluster of Lactobacillus paracasei on the basis of 16S rRNA gene sequences. Of the 20 L. sakei strains, 19 strains shifted the type of stereoisomers of lactic acid produced from the DL-type to the L-type in the presence of 50 mM sodium acetate. L. curvatus strains and strains included in the cluster of L. plantarum/L. pentosus/L. paraplantarum and in the cluster of L. paracasei did not shift the type of stereoisomers of lactic acid produced. The change of the type of stereoisomers of lactic acid from the DL-type to the L-type in the presence of sodium acetate was concluded to be species-specific for L. sakei and useful for identification of strains in this species.     

Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.     

Distortion of the three-dimensional structure of the vnd/NK-2 homeodomain bound to DNA induced by an embryonically lethal A35T point mutation

The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue. These structural modifications occur, and are accompanied by a 50-fold reduction in the DNA binding affinity, even though most of the protein-DNA interactions originally seen for the wild-type homeodomain are found likewise in the threonine analogue. Alterations include torsional angle changes in the loop between helix I and helix II, and in the turn between helix II and helix III, as well as in a distortion of the usual antiparallel orientation of helix I with respect to helix II. The alteration of the position of leucine 40 in the A35T mutant is proposed to explain the observed 1.27 ppm upfield shift of the corresponding amide proton resonance relative to the value observed for the wild-type analogue. A detailed comparison of the structures of the mutant A35T and wild-type vnd/NK-2 homeodomains bound to the cognate DNA is presented. The consequences of the structural alteration of the DNA-bound A35T mutant vnd/NK-2 protein may constitute the basis of the observed early embryonic lethality.