乳酸菌的抗冷冻性及耐受机理

乳酸菌发酵剂在工业生产过程中,会受到冷冻的刺激,如真空冷冻干燥及后期的低温保藏,此外,发酵乳制品的保藏和干酪的成熟过程也都在低温中进行。这些均会对乳酸菌发酵剂及发酵乳制品质量产生一定的影响。因此,掌握乳酸菌在冷冻条件下的反应机理有助于优化发酵剂和发酵乳制品在工业生产中的冷冻、发酵和贮藏条件,从而提高产品质量和生产效益。本文对乳酸菌的抗冷冻性及机理进行了分析,并对发酵剂的保护提出具体措施。     

Human Cytochrome P450 2E1 Structures with Fatty Acid Analogs Reveal a Previously Unobserved Binding Mode

Human microsomal cytochrome P450 (CYP) 2E1 is widely known for its ability to oxidize >70 different, mostly compact, low molecular weight drugs and other xenobiotic compounds. In addition CYP2E1 oxidizes much larger C9–C20 fatty acids that can serve as endogenous signaling molecules. Previously structures of CYP2E1 with small molecules revealed a small, compact CYP2E1 active site, which would be insufficient to accommodate medium and long chain fatty acids without conformational changes in the protein. In the current work we have determined how CYP2E1 can accommodate a series of fatty acid analogs by cocrystallizing CYP2E1 with ω-imidazolyl-octanoic fatty acid, ω-imidazolyl-decanoic fatty acid, and ω-imidazolyl-dodecanoic fatty acid. In each structure direct coordination of the imidazole nitrogen to the heme iron mimics the position required for native fatty acid substrates to yield the ω-1 hydroxylated metabolites that predominate experimentally. In each case rotation of a single Phe²⁹⁸ side chain merges the active site with an adjacent void, significantly altering the active site size and topology to accommodate fatty acids. The binding of these fatty acid ligands is directly opposite the channel to the protein surface and the binding observed for fatty acids in the bacterial cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium. Instead of the BM3-like binding mode in the CYP2E1 channel, these structures reveal interactions between the fatty acid carboxylates and several residues in the F, G, and B′ helices at successive distances from the active site.     

墨兰菌根的结构及酸性磷酸酶定位研究

利用光学显微镜、电子显微镜及细胞化学方法,对墨兰菌根的结构和酸性磷酸酶定位进行了初步研究。结果表明墨兰具有典型的兰科植物根结构,发现该兰花的根的外皮层不具薄壁通道细胞,菌根真菌通过破坏部分根被和外皮层细胞而侵入根的皮层细胞并在细胞内形成菌丝结,侵入的菌丝被染菌皮层细胞质膜和电子透明物质包围,进一步被消化并聚集成衰败菌丝团块。酸性磷酸酶在染菌皮层细胞及包围菌丝的皮层细胞质膜和衰败菌丝细胞壁上有强烈的酶反应,衰败菌丝周围分布有许多单层膜的含酶小泡,它们可相互愈合形成大的含酶泡或与包围菌丝的质膜融合,类似于兰科植物共生原球茎中观察到的现象。说明皮层细胞可主动释放水解酶参与对菌丝的消化     

Purification and partial characterization of an acid β-fructosidase from sweet-pepper (Capsicum annuum L.) fruit

The present study describes the biochemical characteristics of an acid -fructosidase (EC 3.2.1.26) purified from the fruit of sweet pepper (Capsicum annuum L.). The soluble form, which constitutes more than 95% of the total activity at pH 4.5, hydrolyzes sucrose, raffinose, and stachyose. Its pH and temperature optima are 4.5 and 55 °C, respectively. Metal cations such as Ag⁺ and Hg²⁺ strongly inhibit its activity, suggesting the presence of at least one sulfhydryl group at the catalytic site. After purification of the enzyme by means of ammonium sulfate fractionation, gel chromatography (diethyl-aminoethyl-Sephacel, hydroxylapatite, concanavalin A-Sepharose), and preparative gel electrophoresis, the purified enzyme was shown to be a 42 kDa glycoprotein interacting specifically with concanavalin A. After complete chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight of the constitutive polypeptide was estimated to be 39 kDa. The enzyme glycans were characterized using both affino- and immunodetection. The enzyme has at least two N-linked oligosaccharide sidechains, one of the high-mannose type, and the other of the complex type. The high-mannose glycan has a low molecular weight (1 kDa), and is responsible for the interaction between the enzyme and concanavalin A. The complex-type glycan has an estimated molecular weight of 2 kDa. It contains one 1 2-linked xylose residue, probably one fucose residue 1 3-linked to the chitobiose unit, and no terminal galactose residue. The two glycans, associated to the 39 kDa polypeptide, constitute the acid -fructosidase of the sweet-pepper fruit.Abbreviations F -fructosidase - ConA concanavalin A - DEAE diethylaminoethyl - DTNB dithionitrobenzoic acid - endo F endo--N-acetylglucosamidase F - endo H endo--N-acetylglucosamidase H - NEM N-ethylmaleimide - PCMB parachloromercurobenzoate - PNGase glycopeptide-N-glycosidase - TFMS trifluoromethane sulfonic acid This work was partly supported by a grant from the Commission Permanente de Coopération Franco-Québécoise to L. Faye, and S. Yelle. D. Michaud was a recipient of a graduate scholarship from the Natural Science and Engineering Research Council of Canada.     

Proteinaceous Inhibitor Versus Fructose as Modulators of Pteris deflexa Invertase Activity

An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer M r 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had β -fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with K m of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.     

Proteome changes in the myocardium of experimental chronic diabetes and hypertension: role of PPARα in the associated hypertrophy

Diabetes with or without the presence of hypertension damages the heart. However, there is currently a lack of information about these associated pathologies and the alteration of linked proteins. For these reasons, we were interested in the potential synergistic interaction of diabetes and hypertension in the heart, focusing on the proteome characterization of the pathological phenotypes and the associated hypertrophic response. We treated normotensive and spontaneously hypertensive (SHR) rats with either streptozotocin or vehicle. After 22 weeks, type-I diabetic (DM1), SHR, SHR/DM1 and control left-ventricles were studied using proteomic approaches. Proteomics revealed that long-term DM1, SHR and SHR/DM1 rats exhibited 24, 53 and 53 altered proteins in the myocardia, respectively. DM1 myocardium showed over-expression of apoptotic and cytoskeleton proteins, and down-regulation of anti-apoptotic and mitochondrial metabolic enzymes. In both SHR and SHR/DM1 these changes were exacerbated and free fatty-acid (FFA) ß-oxidation enzymes were additionally decreased. Furthermore, SHR/DM1 hearts exhibited a misbalance of specific pro-hypertrophic, anti-apoptotic and mitochondrial ATP-carrier factors, which could cause additional damage. Differential proteins were validated and then clustered into different biological pathways using bioinformatics. These studies suggested the implication of FFA-nuclear receptors and hypertrophic factors in these pathologies. Although key ß-oxidation enzymes were not stimulated in DM1 and hypertensive hearts, peroxisome proliferator-activated receptors-α (PPARα) were potentially activated for other responses. In this regard, PPARα stimulation reduced hypertrophy and pro-hypertrophic factors such as annexin-V in high-glucose and angiotensin-II induced cardiomyocytes. Thus, activation of PPARα could reflect a compensatory response to the metabolic-shifted, apoptotic and hypertrophic status of the hypertensive-diabetic cardiomyopathy.     

日立835─50型氨基酸分析仪故障排除六例

给出日立８３５－５０型氨基酸分析仪使用过程中出现的六种故障现象及判断排除方法     

Energy metabolism and ATP free-energy change of the intertidal wormSipunculus nudus below a critical temperature

The intertidal wormSipunculus nudus was exposed to various temperatures for an analysis of the integrated changes in energy and acid-base status. Animals were incubated in sea water or maintained for up to 8 days at 4 and 0°C while dwelling in the sediment. Cannulation of the animals prior to experimentation allowed the analysis of blood gas parameters ( , and pH). fell to 0 torr within 8 days at 0°C. A simultaneous reduction of ventilatory activity was derived from measurements of the pattern of coelomic fluid pressure changes associated with ventilatory movements. The increase in and an onset of anaerobic metabolism, indicated by the accumulation of end products like acetate and propionate both in the coelomic fluid and the body wall musculature, led to the development of a progressive acidosis and a deviation from the alphastat regulation of intracellular pH seen in unburied animals. The drop in intracellular pH together with the depletion of the adenylates and the phosphagen, phospho-l-arginine, reflect a significant decrease in the Gibb's free-energy change of ATP hydrolysis. These changes are interpreted to indicate lethal cold injuries, because recovery was not possible when the animals were returned to 12°C after more than 2 days of exposure to 0°C. A low critical temperature indicating the onset of cold-induced anaerobiosis is concluded to exist below 4°C owing to the insufficient response of the ventilatory system to the developing hypoxia.