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.     

Nanosecond dynamics of the mouse acetylcholinesterase cys69-cys96 omega loop

The paradox of high substrate turnover occurring within the confines of a deep, narrow gorge through which acetylcholine must traverse to reach the catalytic site of acetylcholinesterase has suggested the existence of transient gorge enlargements that would enhance substrate accessibility. To establish a foundation for the experimental study of transient fluctuations in structure, site-directed labeling in conjunction with time-resolved fluorescence anisotropy were utilized to assess the possible involvement of the omega loop (Omega loop), a segment that forms the outer wall of the gorge. Specifically, the flexibility of three residues (L76C, E81C, and E84C) in the Cys69-Cys96 Omega loop and one residue (Y124C) across the gorge from the Omega loop were studied in the absence and presence of two inhibitors of different size, fasciculin and huperzine. Additionally, to validate the approach molecular dynamics was employed to simulate anisotropy decay of the side chains. The results show that the Omega loop residues are significantly more mobile than the non-loop residue facing the interior of the gorge. Moreover, fasciculin, which binds at the mouth of the gorge, well removed from the active site, decreases the mobility of 5-((((2-acetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid reporter groups attached to L76C and Y124C but increases the mobility of the reporter groups attached to E81C and E84C. Huperzine, which binds at the base of active-site gorge, has no effect on the mobility of reporter groups attached to L76C and Y124C but increases the mobility of the reporter groups attached to E81C and E84C. Besides showing that fluctuations of the Omega loop residues are not tightly coupled, the results indicate that residues in the Omega loop exhibit distinctive conformational fluctuations and therefore are likely to contribute to transient gorge enlargements in the non-liganded enzyme.     

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.     

Characterization of the S272A,D site-directed mutations of O-acetylserine sulfhydrylase: involvement of the pyridine ring in the alpha,beta-elimination reaction

O-Acetylserine sulfhydrylase catalyzes the synthesis of l-cysteine from O-acetyl-l-serine (OAS) and inorganic bisulfide. An anti-E2 mechanism has been proposed for the OASS-catalyzed elimination of acetate from OAS (Tai, C.-H., and Cook, P. F. (2001) Acc. Chem. Res. 34, 49-59). Site-directed mutagenesis was used to change S272 to alanine, which would be expected to eliminate the hydrogen bond to N1 of PLP or to aspartate, which would be expected to enhance the hydrogen-bonding interaction. Both mutant enzymes catalyze the overall reaction and are in fact still good enzymes, consistent with the proposed anti-E2 mechanism. Data suggest that hydrogen bonding to the pyridine ring does not play a significant role in the alpha,beta-elimination reaction catalyzed by OASS-A. The V/K(OAS), which reflects the first half-reaction, is identical to the wild-type enzyme in the case of the S272D mutant enzyme and is decreased by only a factor of 3 in the case of the S272A mutant enzyme. In the case of the alanine mutation, and to a lesser extent the aspartate mutation, a decrease in the rate of the elimination is compensated by an increase in affinity for OAS, leading to the observed second-order rate constant, V/K. The decrease in the rate of the elimination is proposed to result from a change in the orientation of the bound cofactor, as might be expected since one of the ligands that determines the position of the bound PLP has been changed. Consistent with a change in the orientation of the cofactor are the results from a number of the spectral probes. The visible CD data for the internal Schiff base have a molar ellipticity 50% that of wild-type enzyme, and the alpha-aminoacrylate intermediate has a molar ellipticity 25% that of wild-type enzyme. The alpha-aminoacrylate intermediate can be formed from l-cysteine and l-serine with the S272A,D mutant enzymes, but not with the wild-type enzyme, and taken together with the increased K(d) for the serine external Schiff base is consistent with a change in cofactor orientation in the active site. The long wavelength fluorescence emission for the S272A mutant enzyme, attributed to F?rster resonance energy transfer (McClure, G. D., Jr., and Cook, P. F. (1994) Biochemistry 33, 1647-1683) has an intensity near zero, as compared to significant fluorescence for the wild-type enzyme.     

Selective formation of Sed5p-containing SNARE complexes is mediated by combinatorial binding interactions

Sed5p is the only syntaxin family member required for protein transport through the yeast Golgi and it is known to bind up to nine other soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in vivo. We describe in vitro binding experiments in which we identify ternary and quaternary Sed5p-containing SNARE complexes. The formation of SNARE complexes among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p and is syntaxin-selective. In addition, Sed5p-containing SNARE complexes form selectively and this selectivity is mediated by Sed5p-containing intermediates that discriminate among subsequent binding partners. Although many of these SNAREs have overlapping distributions in vivo, the SNAREs that form complexes with Sed5p in vitro reflect their functionally distinct locales. Although SNARE-SNARE interactions are promiscuous and a single SNARE protein is often found in more than one complex, both the biochemical as well as genetic analyses reported here suggest that this is not a result of nonselective direct substitution of one SNARE for another. Rather our data are consistent with the existence of multiple (perhaps parallel) trafficking pathways where Sed5p-containing SNARE complexes play overlapping and/or distinct functional roles.     

Localization of the C-terminus of rat glutathione S-transferase A1-1: crystal structure of mutants W21F and W21F/F220Y

Twelve C-terminal residues of human glutathione S-transferase A1-1 form a helix in the presence of glutathione-conjugate, or substrate alone, and partly cover the active site. According to X-ray structures, the helix is disordered in the absence of glutathione, but it is not known if it is helical and delocalized, or in a random-coil conformation. Mutation to a tyrosine of residue 220 within this helix was previously shown to affect the pK(a) of Tyr-9 at the active site, in the apo form of the enzyme, and it was proposed that an on-face hydrogen bond between Tyr-220 and Tyr-9 provided a means for affecting this pK(a). In the current study, X-ray structures of the W21F and of the C-terminal mutation, W21F/F220Y, with glutathione sulfonate bound, show that the C-terminal helix is disordered (or delocalized) in the W21F crystal but is visible and ordered in a novel location, a crystal packing crevice, in one of three monomers in the W21F/F220Y crystal, and the proposed hydrogen bond is not formed. Fluorescence spectroscopy studies using an engineered F222W mutant show that the C-terminus remains delocalized in the absence of glutathione or when only the glutathione binding site is occupied, but is ordered and localized in the presence of substrate or conjugate, consistent with these and previous crystallographic studies. Proteins 2001;42:192-200.     

血管紧张素Ⅱ对模拟缺血心室肌细胞L-型钙通道的影响

实验研究了血管紧张素II（AngⅡ)对模拟缺血心室肌细胞L－型钙离子通道的作用,用胶原酶酶解法急性分离豚鼠心室肌细胞,以全细胞膜片钳方法记录心室肌细胞的L－型钙电流（ICa L.)。采用低氧,无糖,高乳酸和酸中毒综合方式模拟缺血液灌流,造成心室肌细胞的模拟缺血,并在缺血的基础上继续用含100mmol/A AngⅡ灌流细胞,观察AngⅡ对模拟缺血心室肌细胞钙离子通道的影响,实验结果显示,模拟缺血时ICa.L峰值电流明显减小,最大激活电压为0mV,AngⅡ能抵抗模拟缺血对ICa,L的抑制效应,使ICa,L峰值电流增大,并使最大激活电压左移至－10mV。     

Auto/cross-regulation of Hoxb3 expression in posterior hindbrain and spinal cord

The complex and dynamic pattern of Hoxb3 expression in the developing hindbrain and the associated neural crest of mouse embryos is controlled by three separate cis-regulatory elements: element I (region A), element IIIa, and the r5 enhancer (element IVa). We have examined the cis-regulatory element IIIa by transgenic and mutational analysis to determine the upstream trans-acting factors and mechanisms that are involved in controlling the expression of the mouse Hoxb3 gene in the anterior spinal cord and hindbrain up to the r5/r6 boundary, as well as the associated neural crest which migrate to the third and posterior branchial arches and to the gut. By deletion analysis, we have identified the sequence requirements within a 482-bp element III482. Two Hox binding sites are identified in element III482 and we have shown that in vitro both Hoxb3 and Hoxb4 proteins can interact with these Hox binding sites, suggesting that auto/cross-regulation is required for establishing the expression of Hoxb3 in the neural tube domain. Interestingly, we have identified a novel GCCAGGC sequence motif within element III482, which is also required to direct gene expression to a subset of the expression domains except for rhombomere 6 and the associated neural crest migrating to the third and posterior branchial arches. Element III482 can direct a higher level of reporter gene expression in r6, which led us to investigate whether kreisler is involved in regulating Hoxb3 expression in r6 through this element. However, our transgenic and mutational analysis has demonstrated that, although kreisler binding sites are present, they are not required for the establishment or maintenance of reporter gene expression in r6. Our results have provided evidence that the expression of Hoxb3 in the neural tube up to the r5/r6 boundary is auto/cross-regulated by Hox genes and expression of Hoxb3 in r6 does not require kreisler.