精脒与疟原虫抗氯喹关系的研究

抑制伯氏疟原虫感染RBC中50％精脒量,抗氯喹株需要喂服400mg/kg氯喹,而敏感的为20mg/kg,相差至少20倍。两株感染鼠同样喂服氟喹20mg/kg和400mg/kg一次,3小时后,在全血和感染RBC中的氯喹量,低剂量组接近,400mg/kg组抗氯喹的更高,而积聚在疟原虫中的氯喹量则抗氯喹的低于敏感。抗氯喹感染RBC中的精脒,78‰集中在疟原虫,故认为精脒量的提高,可能与氯喹进入疟原虫产生竞争性抑制。MGBG抑制抗氯喹鼠中的疟原虫生长,精脒能逆转其作用。     

伯氏疟原虫氯喹敏感株和抗性株感染红细胞精脒合成酶体内观察

给伯氏疟原虫氯喹敏感株(CS)和抗氯喹株(CR)感染鼠腹腔注入~3H-丁二胺(0.74MB_q/鼠),4h后用荧光法与液闪法测定CS、CR感染RBC中产生的~3H-精脒量,以示精脒合成酶的活力(以dpra/10~9感染RBC表示),并观察了氯喹对酶活力的影响。两株感染RBC的酶活力在治疗前接近,CS为59 987.9±17403(16),CR为53818.4±15565(13)。氯喹治疗20h后CS与CR分别为20033±3260(8)与65304±20176(11)即CS酶活力降低66.6％,CR的活力不变,推测氯喹对两株疟原虫感染RBC精脒抑制的差异点是在精眯合成酶环节。     

丝孢酵母高甲硫氨酸突变株的选育及营养调控

以丝孢酵母(Trichosporon Behr)ST851为原始菌株,经紫外线诱变,在含乙硫氨酸的双层平板上筛选到多株抗乙硫氨酸突变株。其中ST851-10株抗乙硫氨酸浓度达到350μg/ml,其菌体蛋白质含量由40.5％提高到44.3％,菌体甲硫氨酸含量由20.45mg/g-DCW增加到29.32mg/g-DCW。在以苹果渣为碳源、尿素为氮源、硫酸镁作硫源的最适培养条件下,固态发酵24h后,蛋白质和甲硫氨酸含量较原始菌株分别提高了15.8％和44.9％。培养基中C/N值低有利于甲硫氨酸的合成,C/N值高则适合于菌体生长。在苹果渣固态发酵过程中,适当补加氮源既有利于菌体生长和甲硫氨酸的合成,又可起到调节培养基pH值的作用。     

特异性IgG抗体在异种/同种异株疟原虫感染治愈后P.y17XL再感染中的保护作用

为探讨特异性IgG抗体在异种/同种异株疟原虫治愈后再感染过程中的作用,用不同种/株疟原虫感染BALB/c小鼠,经治愈后用P.y17XL再感染。通过计数红细胞感染率和检测再感染后血清中特异性IgG抗体的水平变化,发现P.y17XNL感染治愈组小鼠几乎不出现原虫血症,其余异种疟原虫感染治愈组小鼠出现了不同程度的虫血症发生时相延迟和水平降低,部分生存率有所延长;不同虫种/株感染治愈后P.y17XL再感染的小鼠IFN-γ水平均明显低于同时间点P.y17XL初次感染的小鼠;P.v、P.y17XNL感染治愈小鼠血清中P.y17XL特异性IgG抗体水平出现显著增加(P<0.05),且以IgG1亚类升高为主。表明特异性IgG抗体可在宿主抗同源疟原虫再感染中发挥着重要作用,而对异种疟原虫再感染保护性有限。     

疫苗

甲型流感病毒低温适应重组疫苗株基因组中ts缺损的定位〔英」/MeaveQ。、。E…IAeta Virol一1984,28(3)一204~21〔译自DBA,1984,3(1了),84一08111〕852424一卯一,本文研究了亲代流感病毒基因组中以及低温适应株A/Leningrad/134/17/57或A/Leningrad/134/47/57与甲型流感病毒流行株H]Nl和H3NZ的重组病毒基因组中的温度敏感(ts)表型及ts突变的定位。流行株HINI和H3NZ具有ts表型,并在i或2个基因中含有ts突变。在40℃克隆纯化3次后,ts表型丢失。具有不同ts表型的2个低温适应株,在编码非糖蛋白的3个基因(A/Lenin-grad/134八7/57于25oC…     

尾静脉注射和鼻腔接种禽流感H5N1亚型病毒对血细胞的影响

[目的]Balb/c小鼠通过尾静脉注射和鼻腔接种禽流感H5N1亚型病毒造疾病模型,观察其血细胞变化。[方法]以禽流感H5N1亚型病毒通过静脉和鼻腔接种Balb/c小鼠,分别在0~7天和0~11天取血进行血细胞计数和分类,同时对于静脉接种方法进行了0~8小时的短时间监控。[结果]静脉接种途径在接种病毒后2小时就能显著的降低小鼠的白细胞总数、粒细胞及淋巴细胞数,白细胞总数及淋巴细胞数在第3天、粒细胞数在第2天就恢复正常而后均显著升高;而鼻腔接种仅在1~3天淋巴细胞有所降低,而后恢复正常;1~6天白细胞总数及粒细胞数显著升高,而后恢复正常。[结论]从血细胞变化而言,静脉接种相比鼻腔接种方法要好。     

5-~(131)碘尿嘧啶在细菌脱氧核糖核酸复制研究中的应用

本文报导了一个用5-~(131)碘尿嘧啶代替[~3H]-胸腺嘧啶进行细菌DNA复制研究的新方法。通过对放射性参入产物的碱(KOH)水解和酶(DNase和RNase)水解的实验证明:5-~(131)碘尿嘧啶与[~3H]-胸腺嘧啶一样能特异地参入大肠杆菌胸腺嘧啶缺陷变异株的DNA,而不能参入其RNA。对氨基酸饥饿同步化的大肠杆菌15T~-,当应用5-~(131)碘尿嘧啶的参入来测定复加氨基酸后的DNA复制起步时间时,获得的结果与使用[~3H]-胸腺嘧啶脱氧核苷参入的结果相一致。天然的DNA碱基成分胸腺嘧啶强烈地竞争性地抑制5-~(131)碘尿嘧啶的参入能力,而天然的RNA碱基成分尿嘧啶则影响很小。此外,5-碘尿嘧啶对大肠杆菌胸腺嘧啶缺陷变异株的生长有抑制作用,但这种抑制要在加入5-碘尿嘧啶后2小时才明显地产生,而5-碘尿嘧啶对大肠杆菌野生株的生长没有影响。实验结果表明:在细菌DNA复制的研究中,5-~(131)碘尿嘧啶参入的方法与使用[~3H]-胸腺嘧啶参入的方法有同样的可靠性,其优点是由于~(131)碘辐射γ射线,故放射性参入样品的制备和测定都比较简便,因而,它比[~3H]-胸腺嘧啶更适合于有关临床和实验室的使用。     

玉米赤霉烯酮单克隆抗体和免疫酶技术研究

采用蛋白质连接技术合成玉米赤霉烯酮抗原,免疫Balb/c鼠,通过淋巴细胞杂交瘤技术建立六株分泌抗玉米赤霉烯酮的单克隆抗体杂交瘤细胞株。间接酶联免疫吸附试验测定细胞上清抗体效价为1:2084(4H8)、1:256(6H9、4H3、2H5、2C8)、1:16(3F10);腹水抗体效价为10~9(4H3、4H8)、10~8(2H5)、10~7(6H9)、10~5(3H10)。竞争间接酶联免疫吸附试验测定六株单克隆抗体对玉米赤霉烯酮的敏感度为0.3—0.8ng/ml。六株抗体与玉米亦霉烯醇的交叉反应率为1.3—9.0％。六株单克隆抗体均属IgG类。细胞体外传代培养和冻存复苏后分泌抗体稳定。纯化抗体在37℃保存12天稳定,-30℃保存90天抗体滴度不变。用该抗体建立竞争间接酶联免疫吸附试验检测掺合玉米赤霉烯酮的玉米、小麦、饲料,平均回收率分别为105％、90％、103％,平均批间变异系数为5.8％、2.8％、6.8％,批内变异系数为3.8％、12.7％、15.7％。样品中玉米赤霉烯酮掺合量与竞争间接酶联免疫吸附试验检出量有良好相关性(r≥0.9996)。     

国内不同地区恶性疟原虫分离株间几种生物学特性的比较研究

本文报道海南岛与苏皖地区恶性疟原虫分离株在体外连续培养中生长特征、药敏性、同工酶谱、表面抗原等方面的比较研究。海南岛两株原虫适应体外培养的速度及增殖率均明显高于苏皖两株原虫,但后者产生配子体的能力较强。体外微量药效测定显示,海南岛两株均表现对氯喹有高度抗性,而苏皖两株则均为敏感株,此外不同原虫分离株在同工酶谱及单克隆抗体间接荧光试验中亦可显示明显的株间差异。文章对恶性疟原虫种内不同虫株间生物学特性的差异进行了讨论。     

糖皮质激素受体的光亲和标记

利用[~3H]去炎松缩酮([~3H]TA)进行了糖皮质激素受体(GCR)的光亲和标记。[~3H]TA和GCR共价交联的证据有:(1)光照后,10％的三氯醋酸不能使[~3H]TA和GCR解离;(2)SDS-聚丙烯酰胺凝胶电泳时,有单一的放射活性峰;(3)用地塞米松保护后,GCR不被标记。采用本方法测定的GCR分子量为94000±3000 dalton,该方法可用于GCR分子量的测定、提纯和配基结合区的分析。     

Hypoxanthine Transport and Metabolism in the Central Nervous System

The mechanisms by which hypoxanthine, the principal purine in plasma and CSF, enters and leaves rabbit brain, choroid plexus, and CSF were investigated in the isolated choroid plexus in vitro and by injecting [14C]hypoxanthine intraventricularly and [3H]hypoxanthine intravenously. The isolated choroid plexus accumulated and extensively metabolized [14C]hypoxanthine; however, 14C was readily released from choroid plexus principally as [14C]-hypoxanthine. After infusion of [3H]hypoxanthine intravenously, [3H]hypoxanthine entered CSF and brain slowly and was converted in brain to nucleotides. Fewer than 5% of the acid-soluble purine nucleotides in brain entered rabbit brain from plasma hypoxanthine (and inosine) per 24 h. After intraventricular injection of [14C]hypoxanthine, the [14C]hypoxanthine was cleared from the CSF into the blood or accumulated by brain and largely converted into 14C-nucleotides. Little [14C]xanthine and no [14C]uric acid or allantoin were formed. These studies show that brain, unlike most other tissues, rapidly recycles hypoxanthine and converts it into purine nucleotides, and not unsalvageable purines.     

Modification of yeast ribosomal proteins. Methylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins uniformly labelled in vivo with [methyl-3H]methionine and [1-14C]methionine revealed that four ribosomal proteins are methylated, i.e. proteins S31, S32, L15 and L41. Lysine and arginine appear to be the predominant acceptors of the methyl groups. The degree of methylation ranges from 0.09 to 0.20 methyl group per modified ribosomal protein species.     

Plasmodium chabaudi: association of reversal of chloroquine resistance with increased accumulation of chloroquine in resistant parasites.

The effects of tricyclic antidepressants, desipramine and imipramine, and phenothiazines, chlorpromazine and trifluoperazine, on chloroquine (CQ)-resistant and CQ-sensitive lines of P. chabaudi were examined in vivo. In mice that received daily injections of these drugs the growth of CQ-resistant and CQ-sensitive parasites was unaffected or affected very slightly, if at all. A combination of CQ and each drug suppressed the growth of CQ-resistant parasites in a dose-dependent manner. In addition, in CQ-sensitive parasites each drug also increased the susceptibility to CQ. Measurements of CQ levels by high-performance liquid chromatography showed that CQ accumulated in sensitive parasites to more than twice the level in resistant parasites at 2 to 4 hr after an injection of CQ. Verapamil and desipramine substantially increased CQ levels in both CQ-resistant and CQ-sensitive parasites. These results suggest that not only Ca2+ antagonists but tricyclic antidepressants reverse CQ resistance in CQ-resistant parasites and enhance the inhibitory effect in sensitive parasites by increasing CQ levels in those parasites. The effects of Ca2+ antagonists, tricyclic antidepressants, and phenothiazines on a pyrimethamine-resistant line of P. chabaudi were also studied. None of the Ca2+ antagonists (verapamil, nicardipine, and diltiazem) affected the growth of the parasite in combination with 20 mg/kg pyrimethamine. Tricyclic antidepressants and phenothiazines suppressed pyrimethamine-resistant parasites to some extent. However, the extent of this suppression was less pronounced as compared with that of suppression of CQ resistance by the same drugs.     

Glutathione is involved in the antimalarial action of chloroquine and its modulation affects drug sensitivity of human and murine species of Plasmodium

Ferriprotoporphyrin IX (FP) is released inside the food vacuole of the malaria parasite during the digestion of host cell hemoglobin. FP is detoxified by its biomineralization to hemozoin. This process is effectively inhibited by chloroquine (CQ) and amodiaquine (AQ). Undegraded FP accumulates in the membrane fraction and inhibits enzymes of infected cells in parallel with parasite killing. FP is demonstrably degraded by reduced glutathione (GSH) in a radical-mediated mechanism. This degradation is inhibited by CQ and AQ in a competitive manner, thus explaining the ability of increased GSH levels in Plasmodium falciparum-infected cells to increase resistance to CQ and vice versa, and to render Plasmodium berghei that were selected for CQ resistance in vivo sensitive to the CQ when glutathione synthesis is inhibited. Some over-the-counter drugs that are known to reduce GSH in body tissues when used in excess were found to enhance the antimalarial action of CQ and AQ in mice infected either with P. berghei or Plasmodium vinckei. In contrast, N-acetyl-cysteine which is expected to increase the cellular levels of GSH, antagonized the action of CQ. These results suggest that some over-the-counter drugs can be used in combination with some antimalarials to which the parasite has become resistant.     

Synthesis and content of polyamines in bloodstream Trypanosma brucei

The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.     

Comparison of the inhibitory effects of diverse amino acids and amino acid analogs on 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity in isolated epidermal cells

At a concentration of 1.25 mM, 14 amino acids were capable of inhibiting the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in isolated epidermal cells. The greatest percentages of inhibition of TPA-induced epidermal ornithine decarboxylase activity were as follows: cysteine, 98%; tryptophan, 74%; methionine, 64%; phenylalanine, 51%; glycine, 44%; asparagine, 43%; glutamic acid, 42%; leucine, 40%; and arginine, 39%. These amino acid treatments did not alter the time- and concentration-response curves for induction of ornithine decarboxylase activity by TPA. Moreover, there was no difference between the rates at which [3H]arginine, [3H]leucine, [3H]phenylalanine, [3H]methionine, [3H]tryptophan and [14C]cysteine were taken up by freshly isolated epidermal cells or incorporated into epidermal proteins. Arginine, phenylalanine and methionine inhibited the induction of ornithine decarboxylase activity by the tumor promoter to degrees comparable to those elicited by their analogs canavanine and homoarginine, beta-2-thienyl-DL-alanine, and ethionine, respectively. These amino acids and amino acid analogs did not alter the overall rate of protein synthesis. In contrast, both the amino acids and their analogs increased the rates of proteolysis in isolated epidermal cells, an effect which correlated well with the abilities of these different compounds to inhibit TPA-induced ornithine decarboxylase activity. Moreover, both methionine and phenylalanine decreased the half-life and increased the rate of heat denaturation of the TPA-induced enzyme, a result identical to that obtained after treatment with the analogs ethionine and beta-2-thienyl-DL-alanine, respectively. Taken together, these results suggest that millimolar concentrations of exogenous amino acids might induce the synthesis of abnormal proteins and nonfunctional enzymes. Therefore, it is speculated that the uptake of unbalanced amounts of amino acids into the epidermal target cells might alter the stability and the ultrastructure of the TPA-stimulated enzyme just as the amino acid analogs do.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

Significance of adenylic acid catabolites for mouse thymus regeneration]

The distribution of [8-14C]adenylic acid catabolites in mouse thymocytes in cortisone-resistant and total thymocyte population has been studied. The accumulation of labelled catabolites in a form of hypoxanthine was found preferentially in cortisone resistant thymocytes but not in total population. This accumulation considerably grows after incubation of cortisone resistant thymocytes with non-peptide mitogenic factor. The excretion of labelled hypoxanthine into medium was also observed. In order to investigate a significance of hypoxanthine accumulation cortisone resistant thymocytes were incubated in the presence of hypoxanthine range concentration and [14C]thymidine incorporation into thymocyte DNA was determined. It was found that [14C]thymidine incorporation into thymocyte DNA increases after incubation in presence of 0.5-5.0 micrograms of hypoxanthine or 0.0005-5.0 micrograms of uric acid. It has been concluded that stimulation of [14C]thymidine incorporation and thymocyte proliferation by non-peptide mitogenic factor is caused by hypoxanthine accumulation.     

Biosynthesis of 3-dimethylsulfoniopropionate in Wollastonia biflora (L.) DC. Evidence that S-methylmethionine is an intermediate.

The compatible solute 3-dimethylsulfoniopropionate (DMSP) is accumulated by certain salt-tolerant flowering plants and marine algae. It is the major biogenic precursor of dimethylsulfide, an important sulfur-containing trace gas in the atmosphere. DMSP biosynthesis was investigated in Wollastonia biflora (L.) DC. [= Wedelia biflora (L.) DC., Melanthera biflora (L.) Wild, Asteraceae]. After characterizing DMSP and glycine betaine accumulation in three diverse genotypes, a glycine betaine-free genotype was chosen for radiotracer and stable isotope-labeling studies. In discs from young leaves, label from [U-14C]methionine was readily incorporated into the dimethylsulfide and acrylate moieties of DMSP. This establishes that DMSP is derived from methionine by deamination, decarboxylation, oxidation, and methylation steps, without indicating their order. Five lines of evidence indicated that methylation is the first step in the sequence, not the last. (a) In pulse-chase experiments with [14C]methionine, S-methylmethionine (SMM) had the labeling pattern expected of a pathway intermediate, whereas 3-methylthiopropionate (MTP) did not. (b) [14C]SMM was efficiently converted to DMSP but [14C]MTP was not. (c) The addition of unlabeled SMM, but not of MTP, reduced the synthesis of [14C]DMSP from [14C]methionine. (d) The dimethylsulfide group of [13CH3,C2H3]SMM was incorporated as a unit into DMSP. (e) When [C2H3,C2H3]SMM was given together with [13CH3]methionine, the main product was [C2H3,C2H3]DMSP, not [13CH3,C2H3]DMSP or [13CH3,13CH3]DMSP. The stable isotope labeling results also show that the SMM cycle does not operate at a high level in W. biflora leaves.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast.

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-14C]hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-14C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-14C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-14C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-14C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-3H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the 'salvage' pathways and de novo synthesis of purines and pyrimidines.