L-鼠李树胶糖激酶在D-阿洛酮糖合成中的应用

D-阿洛酮糖-3-差向异构酶（D-psicose-3-epimerase,DPE）可以催化D-果糖和D-阿洛酮糖之间的可逆反应,但D-阿洛酮糖的产率较低。L-鼠李树胶糖激酶（L-rhamnulose kinase,RhaB）具有磷酸化酮糖类物质的活性,将其与DPE偶联,可以打破DPE的可逆平衡,从而提高D-阿洛酮糖的产率。在反应体系中引入多聚磷酸盐激酶（polyphosphate kinase,PPK）,可实现ATP的再生,降低反应的成本。本实验在大肠杆菌Rosetta（DE3）中分别过表达DPE、RhaB和PPK,优化其反应条件,计算产物得率。结果表明,RhaB的相对分子质量约为5.3×104,最适pH和温度为8.0和35 ℃。将DPE和RhaB偶联,最适条件如下：pH 8.5,温度35 ℃,最适金属离子Mg2+,DPE和RhaB酶量比（质量比）1∶2,产物得率为70%。将DPE、RhaB和PPK偶联,ATP浓度降为D-果糖的1/5,D-阿洛酮糖得率为50%。本研究为工业化生产D-阿洛酮糖提供理论依据。     

Construction of membrane-anchoring fusion protein of Thermococcus kodakaraensis glycerol kinase and its application to repetitive batchwise reactions

We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Thermus thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coli recombinants, whereas TkGK was released from the cells during the reaction at 70°C. In this study, TkGK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coli recombinants having these fusion proteins were incubated at 70°C for 2h, more than 80% of TkGK activity was retained in the heated E. coli cells. However, the yields of G3P production by E. coli having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. coli cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities.     

A PBS2 homologue from Debaryomyces hansenii shows a differential effect on calcofluor and polymyxin B sensitivity in Saccharomyces cerevisiae

The PBS2 gene encodes a MAP kinase kinase that plays a pivotal role in osmosensing signal-transduction pathway in the yeast Saccharomyces cerevisiae. Mutation in the PBS2 gene has a pleotropic effect. Besides being osmosensitive, pbs2 mutants show altered sensitivity to polymyxin B and calcofluor. Recent studies revealed that Pbs2p plays a different role in osmoadaptation and calcofluor sensitivity. We have isolated a gene homologous to PBS2 from the highly salt-tolerant yeast Debaryomyces hansenii by phenotypic complementation. DNA sequencing of the clone revealed that the gene encoded a protein of 683 amino acid residues. Like Pbs2p, this protein also has a proline-rich motif. Further characterization revealed that this gene could complement polymyxin B sensitivity but did not affect calcofluor sensitivity. Thus, it appeared that Pbs2p also has an independent role in these two physiological processes. The GenBank Accession No. of this sequence is AF371315.     

酵母菌产甘油激酶的发酵条件优化

筛选得到产甘油激酶（GK，EC2、7．1．30）德巴利酵母菌，对其发酵条件进行了优化，确定其最适培养基配方为葡萄糖0．1％，KCl 0．18％，酵母膏0．3％，柠檬酸0．6％，甘油0．5％。最适培养条件为接种量6％，装液量为120mL／250mL，160r／min、31℃培养25h。在最适培养基及最适条件下，甘油激酶的酶活力可达1.19U／mg。     

Curcumin attenuates diabetic nephropathy by inhibiting PKC-α and PKC-β1 activity in streptozotocin-induced type I diabetic rats

Scope : We hypothesized that curcumin, a potent anti‐oxidant, might be beneficial in ameliorating the development of diabetic nephropathy through inhibition of PKC‐α and PKC‐β1 activity‐ERK1/2 pathway. Methods and results : Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ) (55 mg/kg) in rats. Three weeks after STZ injection, rats were divided into three groups, namely, normal, diabetic and diabetic treated with curcumin at 100 mg/kg/day, p.o., for 8 wk. At 11 wk after STZ injection, diabetic rats exhibited renal dysfunction, as evidenced by reduced creatinine clearance, increased blood urea nitrogen (BUN) and proteinuria, marked increases in lipid peroxidation, NOX4 and p67phox and decrease in anti‐oxidant enzyme. All of these abnormalities were significantly reversed by curcumin. Furthermore, the high‐glucose‐induced PKC‐α and PKC‐β1 activities and phosphorylated ERK1/2 was significantly diminished by curcumin. Curcumin also attenuated the expression of TGF‐β1, CTGF, osteopontin, p300 and ECM proteins such as fibronectin and type IV collagen. The high‐glucose‐induced expression of VEGF and its receptor VEGF receptor II (flk‐1) was also ameliorated by curcumin. Conclusion : These results prove that curcumin produces dual blockade of both PKC‐α and PKC‐β1 activities, which suggests that curcumin is a potential adjuvant therapy for the prevention and treatment of diabetic nephropathy.     

Fucosylated Chondroitin Sulfate from Sea Cucumber Improves Insulin Sensitivity via Activation of PI3K/PKB Pathway

This study was to investigate the effects of fucosylated chondroitin sulfate (CHS) from sea cucumber on insulin sensitivity in skeletal muscle of type 2 diabetic mice induced by a high‐fat high‐sucrose diet (HFSD). CHS supplementation for 19 wk significantly improved insulin sensitivity by 20%, and reduced blood glucose and insulin levels. Western blotting assay showed that CHS significantly increased insulin‐stimulated glucose transporter 4 (GLUT4) translocation to 1.7‐fold, phosphorylation of phosphoinositide 3‐kinase (PI3K) at p85 to 5.0‐fold, protein kinase B (PKB) at Ser473 to 1.5‐fold, and Thr308 to 1.6‐fold in skeletal muscle. However, PI3K, PKB, and GLUT4 total proteins expression were unchangeable. In addition, qRT‐PCR analysis proved that the insulin signaling was activated by CHS treatment, showing the increased mRNA expressions of glucose uptake‐related key genes. It indicated that CHS improved insulin sensitivity by activation of PI3K/PKB signaling in skeletal muscle of type 2 diabetic mice. Identification of potential mechanism by which CHS increased insulin sensitivity might provide a new functional food or pharmaceutical application of sea cucumber.     

A putative serine/threonine protein kinase gene on chromosome III of Saccharomyces cerevisiae

We have sequenced a gene on chromosome III of Saccharomyces cerevisiae which codes for a putative serine/threonine protein kinase of 726 amino acids (calculated molecular weight 82 kDa). We have called this gene KIN82. The amino acid sequence of KIN82 is most similar to the cyclic nucleotide-dependent protein kinase subfamily and the protein kinase C subfamily. Gene disruption of KIN82 did not produce any phenotype when tested under a variety of conditons. Reduced stringency hybridizations revealed the presence of another genomic sequence with high homology to the carboxy-terminal catalytic domain of KIN82.     

猪骨骼肌肌酸激酶的提纯及性质研究(下)

<正>二、分子量的测定1、葡聚精凝胶层析测分子量表二、标准蛋白及样品的洗脱体积     

三疣梭子蟹蟹黄油的降血糖作用

为研究三疣梭子蟹蟹黄油(egg oil from Portunus trituberculatus,Pt-egg oil)对糖尿病大鼠高血糖症的改善作用及机制,本文采用注射链脲佐菌素的方法建立糖尿病大鼠模型,给予100和400 mg/kg·bw的Pt-egg oil 60 d。实验结束后,检测大鼠空腹血糖和胰岛素水平,检测葡萄糖耐受性,采用实时荧光定量PCR方法检测Pt-egg oil对糖尿病大鼠骨骼肌胰岛素信号通路关键基因m RNA表达水平,采用Western blotting法检测Pt-egg oil对糖尿病大鼠胰岛素信号通路关键蛋白表达的影响。结果显示,经Pt-egg oil饲喂后,大鼠的空腹血糖水平显著地降低了36.52%(p0.01),血清胰岛素浓度增加了40.21%(p0.01),葡萄糖耐受性显著改善,骨骼肌磷脂酰肌醇-3-羟激酶(phosphatidylinositol 3-hydroxy kinase,PI3K)、蛋白激酶B(protein kinase B,Akt)和葡萄糖转运蛋白4(glucose transporter 4,Glut4)基因m RNA分别显著增加了114.52%、83.17%和90.06%(p0.01),蛋白表达分别显著提高了1.54倍、1.61倍和1.37倍(p0.01),细胞膜上Glut4蛋白表达提高了1.27倍(p0.01)。表明Pt-egg oil能通过激活PI3K/Akt胰岛素信号通路起到降低糖尿病大鼠血糖的作用。     

A protein kinase is located in the micellar fraction of fresh pasteurized skimmed farm milk

Recombinant protein kinase CK2 from the yeast Schizosaccharomyces pombe is able to phosphorylate casein in skimmed pasteurized milk. We could incorporate up to 540 pmol of phosphate into 50 microg milk proteins, i.e., 0.26 P/mol caseins. To better understand the action of protein kinase CK2 on milk proteins, we have compared the action of rspCK2alpha on milk, and on different casein micellar subfractions isolated from milk by ultracentrifugation. In contrast to the situation observed with phosphocaseinate, alpha(s) casein was the best substrate for rspCK2alpha, whether milk or micellar fractions were used as substrates. We have characterized the protein content of different micellar fractions obtained by ultracentrifugation of cow milk using capillary zone electrophoresis. We confirm that the kappa casein content of micelles largely decreases when their size increases. In contrast, the alpha(s) casein content slightly increased with micelles size and beta casein content remained constant. All of the micellar fractions were substrates for rspCK2alpha, but a significant amount of intrinsic protein kinase activity was also found. The intrinsic protein kinase used added ATP as phosphate donor, and was only slightly sensitive to high heparin concentration. It could phosphorylate micellar casein in milk ultrafiltrate, in the absence of addition of any metallic cofactor. Its activity was only slightly affected by the addition of either MgCl2 or MnCl2. CaCl2 activated the enzyme significantly. The intrinsic kinase lost its activity with time, and could incorporate from 9 to 26% of the total phosphate incorporated in the presence of rspCK2a. Alpha(s) casein was the best substrate of the intrinsic kinase, followed by beta casein. In the presence of CaCl2, the intrinsic kinase was found to incorporate up to 470 pmol of phosphate into 50 microg of milk proteins.     

A 'marker switch' approach for targeted mutagenesis of genes in Schizosaccharomyces pombe

The completion of the Schizosaccharomyces pombe genome sequencing project has led to a dramatic acceleration of gene characterization in this system. Once a gene has been identified, the challenge then comes in using reverse genetics to generate a range of mutants in this gene of interest so that the powerful genetics and wealth of genetic backgrounds available in Sz. pombe can be exploited to study the function of the newly identified molecule. Beyond simple PCR-tagging approaches, the high frequency with which illegitimate recombination occurs in Sz. pombe has made the manipulation of some loci complex, time consuming and a process of trial and error. Here we describe a simple 'marker switch' approach that enables the rapid selection of integration events at the locus of interest from an excessive background of integration at heterologous sites. We use the generation of temperature-sensitive mutations in the plo1(+) gene to validate this approach.     

Cloning and genetic analysis of the gene encoding a new protein kinase in Saccharomyces cerevisiae

We have isolated a single gene from the yeast Saccharomyces cerevisiae encoding a potential 800 amino acid polypeptide of calculated M_r 90 098 Da. This protein consists of an N-terminal region that shares significant homology with the catalytic domains of several serine- and threonine-specific protein kinases, as well as a large, unique, C-terminal domain of unknown function. Haploid disruption mutants are viable and do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients or osmotic strength. Due to the apparent structural similarity between this kinase and the protein products of the KIN1 and KIN2 genes, we have chosen to name this new gene KIN3.     

Kefir peptides exhibit antidepressant-like activity in mice through the BDNF/TrkB pathway

Depression is a prevalent, stress-related mental disorder that can lead to serious psychiatric diseases with morbidity and high mortality. Although some functional fermented dairy drinks have promising anxiolytic and antidepressant effects, the mechanism is still not clear. To determine the antidepressant-like effect and the potential molecule mechanism of kefir peptides (KP), various behavioral tests, including the elevated plus maze test, open field test, forced swimming test, and tail suspension test, were used. Administration of 150 mg/kg KP in mice reduced the duration of immobility in the forced swimming test and tail suspension test, elevated the time spent in the open arm and center zone in the elevated plus maze test, and increased the total distance traveled, average speed, and time spent in the center zone in the open field test compared with the mock group. These results indicated that KP dramatically ameliorated the depression-like behaviors. Kefir peptides were further isolated and identified using high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry, from which 3 peptides were identified and designated KFP-1, KFP-3, and KFP-5. Among these peptides, administration of KFP-3 (15 AA residues) remarkably decreased immobility time in the forced swimming test and increased mobility time in the tail suspension test. Therefore, KFP-3 may be the major active peptide with antidepressant activity in KP. Overexpression of brain-derived neurotrophic factor, phosphorylated tropomyosin receptor kinase B, and phosphorylated ERK1/2 protein levels could be detected in the hippocampus under KP administration. Therefore, we suggest that KP improves depressive-like behaviors by activating the brain-derived neurotrophic factor–phosphorylated tropomyosin receptor kinase B signaling pathway. Kefir peptides may serve as a new type of antidepressant dairy product and may provide potent antidepressant effects for clinical use.     

保健食品中非法添加物二氢吡啶类化合物诱导的肝细胞毒性与作用机制研究

目的 探究二氢吡啶类化合物所致肝细胞毒性及分子机制。方法 以不同浓度(0、3、10、30、100μmol/L)的尼莫地平作用于人肝癌细胞HepG2 24h,测定肝损伤指标ALT、AST活力,并利用Western blot检测细胞中丝裂原活化蛋白激酶(MAPK)、氧化应激通路、凋亡蛋白、自噬相关通路蛋白的表达；以不同浓度(0、3、10、30、100μmol/L)的氯维地平、硝苯地平、尼群地平、西尼地平、阿雷地平及马尼地平作用HepG2细胞24h,利用Western blot检测细胞中自噬相关通路蛋白的表达。结果 100μmol/L的尼莫地平可上调ALT和AST活力、激活MAPK通路蛋白的表达、抑制核因子E2相关因子2(Nrf2)及下游解毒蛋白血红素加氧酶1(HO-1)的表达、增加LC3Ⅱ/LCⅠ、降低SQSTM1表达；氯维地平在3μmol/L时引起细胞自噬,其他六种二氢吡啶类化合物均在100μmol/L时使细胞发生自噬。结论 尼莫地平所致的肝细胞毒性机制中自噬的发生可能是由MAPK通路介导的,其中ERK通路促进HepG2细胞自噬的作用相对最强。此外,其他六种二氢吡啶类化合物氯维地平、硝苯地平、尼群地平、西尼地平、阿雷地平及马尼地平均可引起自噬。     

Natural flavone kaempferol suppresses chemokines expression in human monocyte THP-1 cells through MAPK pathways

There is increasing evidence that daily intake of flavonoids reduced severity and prevalence of allergic diseases. However, the mechanism of its antiinflammatory effects in allergic diseases remains uncertain. Kaempferol, which belongs to the flavone group, is a strong antioxidant among natural flavonoids and is the essential component of many beverages and vegetables. Because chemokine is one of the key mediators in allergic inflammatory process, we investigated the effect of kaempferol on chemokines expression in monocytes. Our data demonstrated that kaempferol significantly inhibited the lipopolysaccharide (LPS)-induced production of monocyte-derived chemokine (MDC), interferon gamma-induced protein 10 (IP-10), and interleukin-8 (IL-8) in THP-1 cells. Growth-related oncogene-α (GRO-α) was also suppressed at a higher concentration. We also found that kaempferol was able to suppress LPS-induced mitogen-activated protein kinase (MAPK) pathways, as well as the phosphorylation of upstream c-raf and MEK1/2. In brief, kaempferol suppressed LPS-induced T helper 1 (Th1), T helper 2 (Th2), and neutrophil-related chemokines production in monocytes might be via the MAPK pathways.     

The yal017 gene on the left arm of chromosome I of Saccharomyces cerevisiae encodes a putative serine/threonine protein kinase

The DNA sequence of a region between the LTE1 and CYS3 genes on the left arm of chromosome I from Saccharomyces cerevisiae contains an open reading frame (ORF), YAL017, corresponding to the 5·0 kb FUN31 (F unction U nknown N ow) transcribed region. The predicted protein from this ORF contains 1358 amino acid residues with a molecular weight of 152 531, and an identifiable serine/threonine protein kinase catalytic domain. When compared with other yeast protein kinases, the Ya1017p kinase most resembles the SNF1 serine/threonine protein kinase which is involved in regulating sucrose fermentation genes. The Ya1017p kinase shows highest amino acid identities with two mammalian carcinoma-related serine/threonine protein kinases; PIM-1, which shows induced expression in T-cell lymphomas; and p78A1, whose expression is lost in human pancreatic carcinomas. Gene disruption of YAL017 indicates that it is non-essential for growth on glucose.