乙型肝炎病毒（HBV）核心基因的变异及分析

对１８份不同类型的乙型肝炎病毒感染者血清及４份肝癌组织,用套式ＰＣＲ扩增ＰｒｅＣ／Ｃ基因,结果１５份标本可供进行核苷酸序列分析。１２例血清标本在Ｃ区均出现点突变并导致氨基酸改变。１份肝癌患者的血清及癌组织与另１份癌组织中获得的Ｃ基因克隆有２１３－３２４个核苷酸缺失。     

乙型肝炎病毒前S2抗原决定簇与乙型肝炎病毒核心抗原的基因融合与表达

乙肝前Ｓ２（ＨＢＶ ＰｒｅＳ２）肽段由５５个氨基酸组成,其Ｎ端肽段含Ｔｈ和Ｂ细胞抗原决定簇。我们将化学合成的ＰｒｅＳ２ ｅｐｉｔｏｐｅ（１２０－１４５）基因不同位点进行融合,融合基因在大肠杆菌中获得表达,并对融合蛋白进行了纯化。经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ实验表明,融合蛋白具有ＰｒｅＳ２和ＨＢｃＡｇ两者的抗原性。此外,研究还表明,强启动子能使表达水平有一定的提高。     

含不同长度前C序列的乙型肝炎病毒核心抗原基因在重组痘苗病毒中的表达

核酸序列分析表明,乙型肝炎(乙肝)病毒核心抗原基因(C基因)编码区内存在两个ATG。目前认为第二个ATG为乙肝病毒C抗原的起始密码子,两个ATG之间的序列称为前C序列,共87bp。Uy、Rutter、Roossinck及景新等的研究表明,含前C序列时,C基因在大肠杆菌中的表达是形成具有HBeAg活性的P25~e膜蛋白,在哺乳动物细胞中的表达则是形成分泌性的HBeAg;不含前C序列时,在大肠杆菌和哺乳动物细胞中均形成P21~e     

慢性乙型肝炎患者血清HBV基因分型

为了解长春市慢性乙型肝炎患者血清中的乙型肝炎病毒(HBV)基因型情况及其与临床特点的相关性,应用型特异性引物进行巢式PCR方法对长春市69例慢性乙型肝炎患者血清HBV进行基因分型检测。在69例血清标本中,B型10例(占14.5%);C型41例(占59.4%);B C混合型8例(占11.6%);未分型的患者共10例(占14.5%)。C基因型患者的HBV-DNA定量、HBeAg阳性率明显高于B基因型患者(HBV-DNA:P<0.01;HBeAg:χ2=3.98,P<0.05),C基因型患者肝功检查指标谷丙转氨酶(ALT)和总胆红素(TB IL)均较B基因型患者高(P<0.01)。长春地区存在HBV B基因型、C基因型、B C混合基因型及未分型,C基因型为优势基因,引起的肝脏活动性炎症较B基因型明显。     

乙型肝炎病毒与乙型肝炎疫苗

乙型肝炎已成为严重威胁人类健康的世界性疾病之一,我国也是高流行区。现在对本病的认识与研究逐渐深入,已取得了很多研究成果。本文仅就乙型肝炎的病原(乙型肝炎病毒)和与疫苗有关的两个问题简述如下: (一)乙型肝炎病毒乙型肝炎病毒(HBV)属DNA病毒,该病毒与其相关抗原颗粒在电镜下分三种不同形态:直径为22nm小球型颗粒、直径为22nm长为200nm的管状颗粒和直径为42nm的大球型颗粒(又称Dane颗粒)。一般认为Dane颗粒是完整的病毒。这三种形态病毒颗粒的衣膜均由表面抗原(HBsAg)组成,不含核酸。Dane颗粒衣膜内为27nm的核心,核心部分包括核心抗原(HBcAg)和e抗原(HBeAg)、内源性DNA模板及相应的DNA聚合酶。据研究HBV进入肝细胞浆脱去衣膜,其环形     

乙型肝炎病毒X基因研究的新进展

本文对近３年有关乙型肝炎病毒（ＨＢＶ）Ｘ基因研究的国内外主要文献进行了综述,重点介绍了Ｘ蛋白的结构对其反式激活作用的影响、Ｘ蛋白与信号传导的关系、Ｘ蛋白对抑癌基因Ｐ５３和转化生长因子ＴＧＦ－β１功能的影响,以及Ｘ基因转基因小鼠模型的建立和反义核酸对该模型发展演进的阻抑作用。对Ｘ蛋白的组织、血清中的检测和相应单克隆抗体核素导向治疗也作了简要介绍。     

乙肝病毒X基因编码氨基酸的变异分析

本文对乙肝病毒感染者中X基因部分区段及其编码的氨基酸变异情况进行了分析。采用巢氏PCR扩增X基因部分区段,对有特异性条带的扩增产物进行序列测定,测序结果用相关软件进行处理,翻译成蛋白质后与文献中已经公布的和疾病相关的变异位点进行比对并进行统计。在90份样本中,扩增出X基因的占42份,与此同时,测序成功的为28份。翻译为氨基酸之后,在X蛋白的C端存在着不同程度的变异,导致X蛋白整个读码框架的改变,主要表现为X蛋白的反式激活能力丢失,这种丢失会造成C末端靶动凋亡与N末端抗凋亡能力的平衡被改变甚至被破坏。     

乙型肝炎病毒DNA在患者血清及肝组织中存在状态的研究

对29例肝炎,1例尸检肝组织和血清中乙型肝炎病毒的DNA(HBV DNA)进行了研究,发现HBsAg( )/HBeAg( )患者中,有9/17(52.94％)血清HBV DNA阳性;HBsAg( )/抗-HBe( )患者中,2/6(33.33％)也为阳性。从30例肝组织中提取DNA经琼脂糖电泳,Southern吸印转移及分子杂交试验结果表明,27例HBV DNA阳性,全部有游离型HBV DNA。27例中有5例经用标记pBR322探针杂交排除非特异杂交带后,在高分子量区有HBV DNA特异的杂交带,提示有HBV DNA整合。     

Separation of poly(ADP-ribosylated) nuclear proteins by polyacrylamide gel electrophoresis at acidic pH and low temperature

A method for the extraction and electrophoresis of poly(ADP-ribosylated) nuclear proteins is described. An extraction method using lithium dodecyl sulfate as detergent at pH 2.4 and room temperature is shown to fully extract nuclear proteins under conditions where full stability of protein-linked polymer is ensured. The polyacrylamide gel electrophoresis is performed again under conditions where full stability is ensured. This work provides a technique whereby misinterpretation of relative ADP ribosylation of nuclear proteins can be avoided.     

High-pressure liquid chromatography separation and determination of rat liver folates

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H₄PteGlu), 5-methyl-H₄PteGlu, 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5,10-methenyl-H₄PteGlu, 5,10-methylene-H₄PteGlu, H₂PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H₄PteGlu and 5-methyl-H₄PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H₄PteGlu and 5-formyl-H₄PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H₂PteGlu, or 5,10-methylene-H₄PteGlu was detected in rat liver samples under our conditions. Distribution of ¹⁴C derived from a previous [¹⁴C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H₄PteGlu, 5-methyl-H₄PteGlu, and 10-formyl-H₄PteGlu in studies dealing with the folate system was emphasized.     

Temperature dependence of binding of hyaluronate oligomer to bovine nasal proteoglycan

An ultrafiltration technique was used to study the temperature coefficient of the association constant K for 1:1 binding of proteoglycan to a hyaluronate oligosaccharide fraction containing an average of about 16 monosaccharide units. The proteoglycan was concentrated during the filtration experiment in order to provide minimal disturbance of the equilibrium in the retained solution. Analytical results calculated from assay of ³H-labeled hyaluronate in the filtrate fractions were extrapolated back to initial equilibrium cell conditions. At 10 °C values of K obtained in this way from ultrafiltration agreed within experimental error with those from equilibrium dialysis. Apparent K values obtained with both techniques tended to decrease somewhat with increasing proteoglycan concentration, due probably in part to excluded volume effects. Values of K obtained by ultrafiltration over the temperature range 5 to 40 °C were used to estimate the enthalpy of binding ΔH° as ?17.5 (±1.5) kcal mol^?1 and the entropy of binding ΔS° as ?50 (±5) cal K^?1 mol^?1 (based on a 1 μm standard state). The dilute solution value of K at 37 °C is sufficiently large to suggest that most of the proteoglycan monomers having a binding site are complexed under tissue conditions.     

Enzymic hydration of benzene oxide: Assay and properties

A radiometric assay for epoxide hydratase using [¹⁴C]benzene oxide as substrate has been developed. The reaction product trans-1,2-[¹⁴C]dihydroxy-1,2-dihydrobenzene (benzene dihydrodiol) was separated from the other components by simple extraction of the unreacted substrate and phenol (a rearrangement product) into a mixture of light petroleum and diethyl ether followed by extraction of the benzene dihydrodiol into ethyl acetate. The product was then estimated by scintillation counting. Using this assay the enzymic hydration of benzene oxide and the possible existence of a microsomal epoxide hydratase with a greater specificity toward benzene oxide were reinvestigated. The sequence of activities of microsomes from various organs was liver > kidney > lung > skin, the pH optimum of enzymic benzene oxide hydration was about pH 9.0, which is similar to that of styrene oxide hydration and both activities were equally stable when liver microsomal fractions were stored. The effect of low molecular weight inhibitors upon the hydration of styrene and benzene oxide by liver microsomes was similar in some cases and dissimilar in others. However, all the dissimilarities could be explained without recourse to the hypothesis of the existence of a separate benzene oxide hydratase. During enzyme purification studies the activity toward benzene oxide was inhibited by the detergent used (cutscum) but was recovered when the detergent was removed. Solubilization without significant loss of activity was successful using sodium cholate. This allowed immunoprecipitation studies, which were performed using monospecific antiserum raised against homogeneous epoxide hydratase. The dose-response curves of the extent of precipitation of activity with increasing amounts of added antiserum were indistinguishable for benzene oxide and styrene oxide as substrate. At high antiserum concentrations precipitation was complete with both substrates. The findings, taken together, indicate the presence in rat liver microsomes of a single epoxide hydratase catalyzing the hydration of both styrene and benzene oxide or the presence of enzymes so closely related that these cannot be distinguished by any of the criteria tested.     

Effect of the oral hypoglycemic agent 2-tetradecylglycidic acid on fatty acid oxidation in suckling rats in vivo and in perfused liver

The hypoglycemic agent, 2-tetradecylglycidic acid (TDGA), administered in vivo lowered the concentration of plasma glucose and ketone bodies but raised the concentration of liver and plasma triglycerides in 10-day-old suckling rats. Phospholipid and cholesterol content of the plasma and liver were unaffected by drug treatment. TDGA inhibited the in vivo oxidation of [1-¹⁴C]palmitate but not that of [1-¹⁴C]decanoate. In suckling rat liver perfusion, TDGA totally inhibited ketone body formation from palmitate and depressed ketone body production from decanoate by 20%. Liver ATP and ADP content in the presence of TDGA decreased although this was probably a reflection of the increased triglyceride content of the liver since the $\text{ATP}\text{ADP}$ was the same as control livers. The results are discussed in relation to the diet and to the inhibition of carnitine acyl transferase in suckling rats.     

Detergent-induced distinctions between fluoride- and vanadate-stimulated adenylate cyclases and their responses to guanine nucleotides

The influence of detergents on fluoride- and vanadate-stimulated adenylate cyclases was investigated with enzyme from liver and adipocyte plasma membranes. Stimulation of the adipocyte cyclase by Na3VO4 was maximal (sixfold) at 3 mM, was not additive with fluoride stimulation, and was readily reversed by washing of the membranes. Vanadate stimulation of the hepatic cyclase was specifically blocked by catechol, which had no effect on basal activity or on fluoride- or glucagon-stimulated activities. The hepatic enzyme, stimulated by fluoride ion, guanyl-5'-yl-(beta,gamma-imino)diphosphate (GPP(NH)P), or GPP(NH)P and glucagon, was inhibited by vanadate with 50% inhibition seen with 2 to 6 mM vanadate. The fluoride-activated adipocyte adenylate cyclase was inhibited by guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) more potently than by GPP(NH)P, with 50% inhibition being seen with 10 nM GTP gamma S or 100 nM GPP(NH)P. These nucleotides also inhibited the vanadate-stimulated enzyme, but with one-third the potency seen with the fluoride-activated cyclase. Dispersion of the adipocyte cyclase by Lubrol-PX into a 30,000g supernatant fraction caused no change in activation of the enzyme by fluoride, but reduced vanadate-stimulated activity 80%. By comparison, this treatment enhanced stimulation by GPP(NH)P twofold and by GTP gamma S threefold. More importantly, perhaps, the treatment with detergent blocked inhibition of the basal enzyme by GTP, blocked inhibition of fluoride- and vanadate-stimulated cyclases by GTP, GPP(NH)P, or GTP gamma S, and rendered vanadate-stimulated activity sensitive to enhancement by guanine nucleotides. The data indicate differences in the actions of vanadate and fluoride, made evident by the influence of guanine nucleotides and detergent treatment. The observations would be consistent with the idea that the effects of vandate may be due to the formation of GDP X V on the enzyme. The data strongly suggest that treatment of adenylate cyclase with Lubrol-PX causes a functional blockade in the guanine nucleotide-dependent inhibitory regulation (mediated by Ni), thereby allowing activation by the stimulatory guanine nucleotide-dependent regulatory component (Ns).