Phosphofructokinase activities within the order Spirochaetales and the characterisation of the pyrophosphate-dependent phosphofructokinase from Spirochaeta thermophila

The subtype of phosphofructokinase activity, either ATP-, ADP- or pyrophosphate-dependent, present in members of three genera from the Spirochaetales was investigated. The individual species/strains examined included Spirochaeta alkalica, S. asiatica, S. halophila, S. isovalerica, S. litoralis, S. zuelzerae, S. thermophila, two thermophilic spirochetes, Treponema bryantii, T. denticola, paragraph signT. pectinovorum, Leptospira biflexa and L. interrogans. All of the Spirochaeta strains, regardless of their phenotype, possessed primarily a pyrophosphate-dependent phosphofructokinase. In contrast, T. bryantii, T. denticola and L. biflexa had predominantly an ATP-dependent activity, whereas no activity was detected in T. pectinovorum or paragraph signL. interrogans. The results suggest that pyrophosphate-dependent phosphofructokinase activity may be a reliable phenotypic marker for the genus Spirochaeta and that there are potentially interesting differences in how the catabolism of saccharides is controlled among members of genera within the Spirochaetales. The pyrophosphate-dependent phosphofructokinase from S. thermophila strain RI 19.B1 was purified (303-fold) to homogeneity and biochemically characterised. The S. thermophila enzyme displayed hyperbolic kinetics with respect to both the forward and reverse cosubstrates and was not significantly affected by traditional activators or inhibitors of phosphofructokinase. The biochemical characterisation represents the first spirochete phosphofructokinase to be described.     

梅毒疫苗的研究进展

梅毒是由梅毒螺旋体(Tp)感染所引起的性传播疾病。梅毒感染可引发机体多系统慢性持续性损害,可垂直传播,还可明显增加感染和传播艾滋病的危险性。尽管付出相当的努力,但Tp不同寻常的生物学特征及当前梅毒疫苗方法学的不足严重阻碍了梅毒疫苗的研究进程,迄今为止,国内外仍然没有切实有效的疫苗能预防Tp感染。简要介绍了梅毒感染后机体免疫应答特点,综述了梅毒疫苗的类型及疫苗发展中可能存在的障碍,并进一步提出了今后梅毒疫苗设计的新方向。     

Kinetic analysis of PPi-dependent phosphofructokinase from Porphyromonas gingivalis

We have previously cloned the gene encoding a pyrophosphate-dependent phosphofructokinase (PFK), designated PgPFK, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In this study, recombinant PgPFK was purified to homogeneity, and biochemically characterized. The apparent K(m) value for fructose 6-phosphate was 2.2 mM, which was approximately 20 times higher than that for fructose 1,6-bisphosphate. The value was significantly greater than any other described PFKs, except for Amycolatopsis methanolica PFK which is proposed to function as a fructose 1,6 bisphosphatase (FBPase). The PgPFK appears to serves as FBPase in this organism. We postulate that this may lead to the gluconeogenic pathways to synthesize the lipopolysaccharides and/or glycoconjugates essential for cell viability.     

Two 16S-23S ribosomal DNA intergenic regions in different Treponema pallidum subspecies contain tRNA genes

Abstract The 16S-23S intergenic spacers of Treponema pallidum subspecies pallidum , Nichols strain, and Treponema pallidum subspecies pertenue , Gauthier strain, have been cloned, characterized and sequenced. Isoleucine and alanine tRNA genes have been identified within the 16S-23S intergenic regions on separate alleles of 293 and 303 bases, respectively. The two alleles are present in both T.p. pallidum and T.p. pertenue , and show no sequence differences between the bacterial subspecies. The ile-tRNA and ala-tRNA genes show 65% and 84% sequence identity, respectively, with the homologous genes of the related spirochete, Borrelia burgdorferi .     

现用梅毒血清试验临床应用价值评估

通过检测梅毒患者正规治疗前、后血清抗体变化,评价梅毒实验室各种血清学试验方法对梅毒的诊断和随访的临床意义.本文采用梅毒螺旋体(TP)暗视野显微镜检查、快速血浆反应素(RPR)试验、TP明胶凝集试验(TPPA)和TP-IgM酶联免疫吸附试验(TP-IgM ELISA)等方法对135例梅毒患者正规治疗前、后的血清进行检测....     

化学发光微粒子免疫法和梅毒螺旋体颗粒凝集试验检测梅毒抗体的比较

目的评价化学发光微粒子免疫法（chemiluminescence microparticle immunoassay, CMIA）检测临床血清标本梅毒螺旋体抗体的敏感性和特异性。方法用梅毒螺旋体颗粒凝集试验（TVeponema Pallidum particie agglutination test,TPPA）法作为对照标准,采用CMIA法检测2012年11月到12月1200例住院患者的血清标本,并用卡方检验评价两种检测方法对同一个样本的化验结果的一致性。结果1200例血清标本中用CMIA法检出阳性率为11.3%,TPPA法检出阳性率为10. 9% ,以TPPA为标准,CMIA法敏感性为96. 9%,特异性为99. 2%,其中CMIA法检测血清S/CO值〉 4. 00的110例,用TPPA确认107例阳性,阳性预测值（PPV）为97.3%;CMIA法S/C0值在1.0-9.0,TPPA可出现阴性结果。结论CMIA法可替代TPPA法进行梅毒螺旋体抗体检测,对于CMIA法检测S/C0值1. 0-4.0的需进一步复检。     

Expression and sequence analysis of a Treponema pallidum gene, tpn38(b), encoding an exported protein with homology to T. pallidum and Borrelia burgdorferi proteins

Abstract An Escherichia coli clone containing recombinant plasmid C19 was identified from a Treponema pallidum genomic DNA library by in situ immunoassay. E. coli maxicells containing pC19 synthesized a treponemal protein doublet of 39.2 and 38.2 kDa, designated TpN38(b). Pulse-chase and protein processing studies showed that TpN38(b) is synthesized with a cleavable amino-terminal signal peptide. A 2.0-kb fragment of pC19 containing the tpn38(b) gene was subcloned and sequenced. The tpn38(b) gene is 1029 nucleotides long and encodes a protein of 343 amino acids with a calculated molecular mass of 37.9 kDa. The deduced amino acid sequence of TpN38(b) has homology with the T. pallidum TpN35 lipoprotein and the Borrelia burgdorferi BmpA, BmpB, BmpC, and BmpD proteins.     

腾冲嗜热厌氧菌6-磷酸果糖激酶的克隆、表达及其生物活性

6_磷酸果糖激酶(PFK)是糖酵解途径一个关键酶。基于腾冲嗜热厌氧菌基因组中的注释,基因TTE1816可能是PFK的一种,但是,它是否确有生物活性还必须有实验数据的支持。腾冲嗜热厌氧菌在最适温度培养后,提取细菌全蛋白,并采用双向电泳将可溶性蛋白质分离,然后运用质谱鉴定若干染色斑点。实验表明,TTE1816在高温条件下能够表达蛋白质。将TTE1816基因体外克隆至细菌表达载体,并在BL_21大肠杆菌中表达为可溶性蛋白。酶动力学实验表明,重组蛋白TTE1816具有PFK的催化活性,最适反应温度在60℃。它还能够催化葡萄糖、果糖、甘露糖和6_磷酸葡萄糖的磷酸化反应。另外,在高底物浓度和酶浓度的条件下,TTE1816还表现果糖二磷酸酶的特性。结果证明,TTE1816是腾冲嗜热厌氧菌中PFK家族的一个新成员。     

Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice

We examined the effects of lactate on the enzymatic activity of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) in various mouse tissues. Our results showed that lactate inhibited PFK activity in all the analyzed tissues. This inhibitory effect was observed in skeletal muscle even in the presence of insulin. Lactate directly inhibited the phosphorylation of PFK tyrosine residues in skeletal muscle, an important mechanism of the enzyme activation. Moreover, lactate indirectly inhibited HK activity, which resulted from its cellular redistribution, here attributed to alterations of HK structure. PK activity was not affected by lactate. The activity of HK and PFK is directly related to glucose metabolism. Thus, it is conceivable that lactate exposure can induce inhibition of glucose consumption in tissues.     

梅毒螺旋体Tp92蛋白B细胞表位的预测与鉴定

【目的】预测和鉴定梅毒螺旋体(Tp)Tp92蛋白的B细胞表位,为深入探讨这些表位在梅毒表位疫苗中的作用奠定基础。【方法】采用Mobyle、ABCpred和IEDB在线软件综合分析预测Tp92蛋白的B细胞表位,人工合成6条表位多肽,以梅毒患者/感染兔血清(同时设健康人/兔血清对照)为标本,用间接ELISA法鉴定预测Tp92蛋白B细胞表位的免疫反应性。【结果】软件预测显示,Tp92蛋白的P1(24-39AA)、P2(332-347AA)、P3(520-536AA)、P4(575-588AA)、P5(103-118AA)、P6(694-712AA)氨基酸序列可能为其B细胞表位。间接ELISA分析表明,预测的P1、P3、P5和P6均与梅毒患者/感染兔血清呈阳性反应,而与健康人/兔血清不反应。【结论】本研究初步得出以下结论:P1、P3、P5和P6均为Tp92蛋白潜在的特异性B细胞表位,尤其是P3和P6免疫反应性最强。     

免疫-PCR法检测梅毒螺旋体特异性抗体

以梅毒螺旋体重组蛋白为抗原,应用免疫-PCR方法检测梅毒螺旋体抗体,并同常规ELISA法进行比较,探讨免疫-PCR方法检测梅毒螺旋体特异性抗体的可行性。结果免疫-PCR法敏感性是常规ELISA法的104倍,阳性检出率高于ELISA法;对照血清标本梅毒螺旋体抗体检测为阴性。表明免疫-PCR方法具有较高敏感性和特异性,有一定的临床推广价值,对梅毒患者的早期诊断及时治疗等具有重要意义。     

梅毒血清抗体检测与临床的关系分析

目的探讨梅毒血清抗体检测与临床的相关性。方法对我院10710例进行梅毒血清抗体检测者,根据性别、年龄的不同比较它们之间的关系,并进行分析结果判断与临床的相关性。结果不同性别患者梅毒血清抗体检测无显著性差异（P〉0.05）,而不同年龄患者梅毒血清抗体检测有显著性差异（P〈0.01）。结论由于患者自身原因、试剂原因及HIV感染等因素,造成梅毒血清抗体试验有一定的假阳性和假阴性,因此对梅毒血清抗体试验结果应结合病史及临床情况进行综合分析判断。     

Filamentous actin and its associated binding proteins are the stimulatory site for 6-phosphofructo-1-kinase association within the membrane of human erythrocytes

Glycolytic enzymes reversibly associate with the human erythrocyte membrane (EM) as part of their regulatory mechanism. The site for this association has been described as the amino terminus of band 3, a transmembrane anion transporter. Binding of glycolytic enzymes to this site is recognized to inhibit glycolysis, since binding inhibits the catalytic activity of these enzymes, including the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK). However, the existence of a putative stimulatory site for glycolytic enzymes within the EM has been proposed. PFK has been described as able to reversibly associate with other proteins, such as microtubules, which inhibit the enzyme, and filamentous actin, which activates the enzyme. Here, it is demonstrated that PFK also binds to actin filaments and its associated binding proteins in the protein meshwork that forms the erythrocyte cytoskeleton. Through fluorescence resonance energy transfer experiments using either confocal microscopy or fluorescence spectroscopy, we show that, within the EM, PFK and actin filaments containing its associated binding proteins are located close enough to propose binding between them. Moreover, specifically blocking PFK binding to band 3 results in an association of the enzyme with the EM that increases the enzyme's catalytic activity. Conversely, disruption of the association between PFK and actin filaments containing its associated binding proteins potentiates the inhibitory action of the EM on the enzyme. Furthermore, it is shown that insulin signaling increases the association of PFK to actin filaments and its associated binding proteins, revealing that this event may play a role on the stimulatory effects of insulin on erythrocyte glycolysis. In summary, the present work presents evidence that filamentous actin and its associated binding proteins are the stimulatory site for PFK within the EM.     

三种方法检测梅毒螺旋体抗体的比较

应用ELISA法、TRUST法和TPPA法分别检测梅毒患者血清标本中梅毒螺旋体IgG抗体,比较3种方法的敏感性和特异性,选择一种适合于梅毒螺旋体抗体检测的高敏感性和高特异性的血清学检测方法。