Identification, cloning, and characterization of a Lactococcus lactis branched-chain alpha-keto acid decarboxylase involved in flavor formation

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain alpha-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3' terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain alpha-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.     

Xenopus Cep57 is a novel kinetochore component involved in microtubule attachment

For chromosomes to congress and segregate during cell division, kinetochores must form stable attachments with spindle microtubules. We find that the centrosome protein, xCep57, localizes to kinetochores and interacts with the kinetochore proteins Zwint, Mis12, and CLIP-170. Immunodepletion of xCep57 from egg extracts yields weakened and elongated bipolar spindles which fail to align chromosomes. In the absence of xCep57, tension is lost between sister kinetochores, and spindle microtubules are no longer resistant to low doses of nocodazole. xCep57 inhibition on isolated mitotic chromosomes inhibits kinetochore-microtubule binding in vitro. xCep57 also interacts with gamma-tubulin. In xCep57 immunodepleted extracts, sperm centrosomes nucleate with normal kinetics, but are unable maintain microtubule anchorage. This characterization places xCep57 in a novel class of proteins required for stable microtubule attachments at the kinetochore and at the centrosome and suggests that the mechanism of microtubule binding at these two places is mechanistically similar.     

Genes and proteins involved in bacterial magnetic particle formation

Magnetic bacteria synthesize intracellular magnetosomes that impart a cellular swimming behaviour referred to as magnetotaxis. The magnetic structures aligned in chains are postulated to function as biological compass needles allowing the bacterium to migrate along redox gradients through the Earth's geomagnetic field lines. Despite the discovery of this unique group of microorganisms 28 years ago, the mechanisms of magnetic crystal biomineralization have yet to be fully elucidated. This review describes the current knowledge of the genes and proteins involved in magnetite formation in magnetic bacteria and the biotechnological applications of biomagnetites in the interdisciplinary fields of nanobiotechnology, medicine and environmental management.     

Construction of a branched chain at C-3 of a hexopyranoside. Synthesis of miharamycin sugar moiety analogs

Synthesis of the conveniently protected epimer at C-3' of the miharamycin sugar moiety was accomplished starting from the corresponding 3,3'-spiroepoxide. Reaction of the epoxide with lithium cyanide, followed by hydrolysis and spontaneous cyclization, afforded the intermediate deoxylactone methyl 4,6-O-benzylidene-3-C-(carboxymethyl)-alpha-D-glucopyranoside-3',2-lacto ne (8). Stereoselective hydroxylation with MoO5 x py x HMPA, reduction with lithium aluminum hydride and cyclization with diethyl azodicarboxylate-triphenylphosphine gave the target molecule methyl 2,3'-anhydro-4,6-O-benzylidene-3-C-[(R)-1,2-dihydroxyethyl]-alpha -D-glucopyranoside (5). Direct reduction of 8 gave other analogs having no C-3' hydroxyl group together with having a C-3' hydroxyl group (hemiacetal). In addition, C-3' epimers were also synthesized through C-3', C-3' dihydroxy analogs. Wittig reaction of an appropriate ketosugar with [(ethoxycarbonyl)methylene]triphenylphosphorane leading to a 7:3 Z/E mixture, followed by hydroxylation with osmium tetroxide, reduction and cyclization afforded the target molecule 5 and the miharamycin sugar moiety methyl 2,3'-anhydro-4,6-O-benzylidene-3-C-[(S)-1,2-dihydroxyethyl]-alpha -D-glucopyranoside. Examination of X-ray data for 5 and its NMR spectroscopy data allowed us to explain a contradiction reported in the literature.     

暗褐网柄牛肝菌菌核形成相关基因分析

【背景】暗褐网柄牛肝菌(Phlebopus portentosus)是第一个能够人工栽培的食用牛肝菌,人工栽培过程中,不同菌株会形成数量不等的菌核。【目的】探明不同菌株产核差异机制。【方法】采集多菌核(JH1)、寡菌核(JH2)菌株的成熟菌核及无菌核(JH3)菌株培养相同时间的菌丝体进行转录组测序,分析差异表达基因对菌核形成的作用和功能。【结果】KEGG富集分析显示,JH2 vs. JH1互比,苯丙氨酸、酪氨酸和色氨酸的生物合成,精氨酸和脯氨酸代谢,半胱氨酸及蛋氨酸代谢显著富集;JH3 vs. JH1互比,乙醛酸和二羧酸代谢显著富集;JH3 vs. JH2互比,谷胱甘肽、乙醛酸和二羧酸代谢显著富集。菌核形成相关基因分析显示,从JH2 vs. JH1、JH3 vs. JH1和JH3 vs. JH2的差异表达基因中分别筛选到69、118和82条与信号转导、感知刺激、防御、碳水化合物活性酶等有关的基因,其中碳水化合物活性酶基因数量最多。三个比较组共有的碳水化合物活性酶基因在JH1中的表达量高于JH2、JH3,表明JH1更能充分利用底物营养以形成更多菌核。【结论】本研究从转录组水平初步分析了暗...     

Vitamin K2 and geranylgeraniol, its side chain component, inhibited osteoclast formation in a different manner

We comparatively examined the mechanism by which vitamin K(2) (Menatetrenone, MK4) and its side chain component, geranylgeraniol (GGO), inhibited osteoclast formation in the co-culture system of stromal cells with spleen cells. Both MK4 and GGO inhibited osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)). MK4, but not GGO, inhibited cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in the co-culture system. To elucidate the precise mechanism of the inhibitory effect of GGO on osteoclast formation, the co-cultured cells were stimulated with PGE(2). GGO, but not MK4, inhibited osteoclast formation via suppression of the receptor activator of NF-kappaB ligand (RANKL) expression. Moreover, GGO abolished the disruption of osteoclastic actin rings induced by nitrogen-containing bisphosphonate (N-BP), whereas MK4 did not affect it at all. These data suggest that MK4 inhibited osteoclast formation independently of GGO, and that MK4, but not GGO, has no competitive action on the anti-osteoporotic effect of N-BP.     

LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism.     

Genes involved in formation of structured multicellular communities by Bacillus subtilis

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.     

Characterization of pneumolysin from Streptococcus pneumoniae,interacting with carbohydrate moiety and cholesterol as a component of cell membrane

The cytolytic mechanism of cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target cell membrane. Membrane cholesterol was thought to serve as the common receptor for these toxins, but not all CDCs require cholesterol for binding. One member of this toxin family, pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae, and the mechanism via which PLY binds to its putative receptor or cholesterol on the cell membrane is still poorly understood. Here, we demonstrated that PLY interacted with carbohydrate moiety and cholesterol as a component of the cell membrane, using the inhibitory effect of hemolytic activity. The hemolytic activity of PLY was inhibited by cholesterol-MβCD, which is in a 3β configuration at the C3-hydroxy group, but is not in a 3α-configuration. In the interaction between PLY and carbohydrate moiety, the mannose showed a dose-dependent increase in the inhibition of PLY hemolytic activity. The binding ability of mannose with truncated PLYs, as determined by the pull-down assay, showed that mannose might favor binding to domain 4 rather than domains 1–3. These studies provide a new model for the mechanism of cellular recognition by PLY, as well as a foundation for future investigations into whether non-sterol molecules can serve as receptors for other members of the CDC family of toxins.     

51-kd protein, a component of microtubule-organizing granules in the mitotic apparatus involved in aster formation in vitro

Mitotic apparatuses (MAs) isolated from sea urchin metaphase eggs were chilled on ice to depolymerize microtubules, homogenized, and incubated with tubulin. This caused formation of many small asters with microtubules focusing on granules which were probably fragments of the centrosome. The aster-forming protein components of the granules in the homogenized MAs were solubilized in 0.5 M KCl containing 50% glycerol. After dialysis against low-ionic-strength buffer solution, proteins congregated to form granular assembly capable of initiating aster formation. Phosphocellulose column chromatography enabled the separation of the aster-forming protein fraction which contained a 51,000 molecular weight protein (51-kd protein) as a major component. The protein fraction possessing the aster-forming activity was also prepared from methaphase whole egg homogenate, and the elution profile of the 51-kd protein on phosphocellulose column also coincided with that of the aster-forming activity. The granular assembly reconstituted from the phosphocellulose fraction formed asters whose microtubules show the same growth rate and length distribution as those of asters reconstructed from the granules in the homogenized MAs. Anti-51-kd protein antibody that was raised in rabbit and affinity-purified stained the center of asters which were reconstructed either from the granules in the homogenized MAs or from the granular assembly reconstituted from the phosphocellulose fraction. These results suggest that the 51-kd protein is a component in the aster-forming activity of the centrosomal component in vitro.     

The intracellular localization of the microvillus 110K protein,a component considered to be involved in side-on membrane attachment of F-actin

The microfilament bundle of intestinal epithelial cell microvilli is known to contain four major associated proteins in addition to actin. Of particular interest is a polypeptide of molecular weight 110000 (110K protein), since it is assumed to provide the lateral attachment of the bundle to the inner side of the plasma membrane. 110K protein was purified by SDS gel electrophoresis and used to elicit antibodies. Antigen-affinity-purified IgGs were used to study the intracellular organization of 110K protein by immunocytochemical procedures. The results are consistent with the proposed membrane attachment function for the 110K protein. It is absent from the terminal web level and restricted to that part of the core filament bundle which underlies the plasma membrane of the microvilli.     

Genes involved in meiosis and sporulation of a yeast

Summary 300 mutants blocked in meiosis or sporulation were isolated in Schizosaccharomyces pombe and grouped by complementation tests, linkage studies and cytological observation. In total, 5 genes pertaining to meiosis and 18 genes necessary for sporulation could be identified. In addition, a gene participating in dissolution of the separating walls during cell fusion was detected.     

MsmE, a lipoprotein involved in sugar transport in Streptococcus mutans.

Metabolic labelling by [14C]palmitic acid showed that growth of Streptococcus mutans LT11 in raffinose, an inducer of the msm operon, resulted in increased production of a 45-kDa lipoprotein corresponding to MsmE, which is believed to be a sugar-binding protein. MsmE was also labelled when an msmE clone was expressed in Escherichia coli. The presence of a lipid anchor on MsmE provides a likely explanation of how the sugar-binding protein component of the msm binding protein-dependent multiple sugar transport system is retained at the cell surface.     

Phosphatidylinositol is involved in the membrane attachment of NCAM-120, the smallest component of the neural cell adhesion molecule.

The rodent neural cell adhesion molecule (NCAM) consists of three glycoproteins with Mr of 180,000, 140,000 and 120,000. The Mr 120,000 protein (NCAM-120) has been shown to exist in membrane-bound and soluble forms but the nature of its membrane association and release has remained obscure. We show here that phosphatidylinositol-specific phospholipase C (PI-PLC), but not a phospholipase C of different specificity, releases a substantial proportion of NCAM-120 from brain membranes and solubilizes almost quantitatively NCAM-120 present at the surface of C6 astroglial cells. The PI-PLC effect was highly selective since only one other protein species was detectably released from C6 cells. These results suggest that NCAM-120 is held in the membrane by covalently bound phosphatidylinositol or a closely related lipid in a way similar to several other surface proteins from eukaryotic cells. The presence of NCAM in a form which can be released from the cell surface by a highly selective mechanism raises additional possibilities for modulation and control of cell--cell adhesion.     

Homologues of isomeric dideoxynucleosides as potential antiviral agents: synthesis of isodideoxynucleosides with a furanethanol sugar moiety.

The synthesis of a homologues series of compounds related to (R, S)-isodideoxynucleosides has been completed by coupling a variety of natural purine and pyrimidine bases with a modified sugar intermediate. This sugar precursor was prepared regiospecifically and stereospecifically from D-glucose.     

Synthesis of the sugar moiety of TIME-EA4, a glycopeptide isolated from silkworm diapause eggs

We describe the efficient synthesis of the tetrasaccharide, 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,4-di-O-acetyl-3-O-allyl-beta-D-mannopyranosyl-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl azide, which is the protected form of the sugar unit of TIME-EA4 that is isolated from the diapausing eggs of the silkworm, Bombyx mori. The beta-linked D-mannoside of the tetrasaccharide was obtained using the conventional oxidation-reduction method for inversion of the configuration at the C-2 hydroxyl group of beta-D-glucoside. The reduction was effected with NaBH(4) in a methanolic solution in a ratio of 98:2 in favor of the beta-D-mannoside that was obtained in 87% yield.     

Pore formation and function of phosphoporin PhoE of Escherichia coli are determined by the core sugar moiety of lipopolysaccharide

The lipid matrix of the outer membrane of Gram-negative bacteria is an asymmetric bilayer composed of a phospholipid inner leaflet and a lipopolysaccharide outer leaflet. Incorporated into this lipid matrix are, among other macromolecules, the porins, which have a sieve-like function for the transport or exclusion of hydrophilic substances. It is known that a reduced amount of porins is found in the outer membrane of rough mutants as compared with wild-type bacteria. This observation was discussed to be caused by a reduced number of insertion sites in the former. We performed electrical measurements on reconstituted planar bilayers composed of lipopolysaccharide on one side and a phospholipid mixture on the other side using lipopolysaccharide from various rough mutant strains of Salmonella enterica serovar Minnesota. We found that pore formation by PhoE trimers that were added to the phospholipid side of the bilayers increased with the increasing length of the lipopolysaccharide core sugar moiety. These results allow us to conclude that the length of the sugar moiety of lipopolysaccharide is the parameter governing pore formation and that no particular insertion sites are required. Furthermore, we found that the voltage gating of the porin channels is strongly dependent on the composition of the lipid matrix.     

An assessment of preferential attachment as a mechanism for human sexual network formation

Recent research into the properties of human sexual-contact networks has suggested that the degree distribution of the contact graph exhibits power-law scaling. One notable property of this power-law scaling is that the epidemic threshold for the population disappears when the scaling exponent rho is in the range 2 < rho < or = 3. This property is of fundamental significance for the control of sexually transmitted diseases (STDs) such as HIV/AIDS since it implies that an STD can persist regardless of its transmissibility. A stochastic process, known as preferential attachment, that yields one form of power-law scaling has been suggested to underlie the scaling of sexual degree distributions. The limiting distribution of this preferential attachment process is the Yule distribution, which we fit using maximum likelihood to local network data from samples of three populations: (i) the Rakai district, Uganda; (ii) Sweden; and (iii) the USA. For all local networks but one, our interval estimates of the scaling parameters are in the range where epidemic thresholds exist. The estimate of the exponent for male networks in the USA is close to 3, but the preferential attachment model is a very poor fit to these data. We conclude that the epidemic thresholds implied by this model exist in both single-sex and two-sex epidemic model formulations. A strong conclusion that we derive from these results is that public health interventions aimed at reducing the transmissibility of STD pathogens, such as implementing condom use or high-activity anti-retroviral therapy, have the potential to bring a population below the epidemic transition, even in populations exhibiting large degrees of behavioural heterogeneity.