Anticoagulant activity of synthetic hirudin peptides

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.     

The genetical control of tissue-specific peroxidases,Per-1,Per-2,Per-3,Per-4, andPer-5 in wheat

Summary Isoelectric focusing (IEF) of extracts from different tissues of hexaploid wheat cv Chinese Spring provided a method of distinguishing and identifying the four known, and one newly discovered, sets of genes encoding peroxidase isozyme production.Per-1, carried on the short arms of homoeologous group 1 chromosomes, shows a high degree of conservation and is active in coleoptile tissue.Per-2, carried on the short arms of group 2 chromosomes, shows some polymorphism and is most active in root tissue.Per-3, on the long arms of group 3 chromosomes, is highly variable and most active in embryo tissue.Per-4, carried on chromosome arms7AS,4AL, and7DS, is quite variable and most active in endosperm tissue. (The chromosome nomenclature used in this paper is that agreed to by the 7th International Wheat Genetics Symposium, where the previous designations of4A and4B were reversed.) Restriction fragment length polymorphism (RFLP)-based maps of the group 7 chromosomes were used to locatePer-A4 to a distal region of7AS. In addition, a further set of genes was identified as being active in root tissue. In wheat a single locus,Per-D5, was found on chromosome arm2DS.     

Cloned hepatitis delta virus cDNA is infectious in the chimpanzee.

A head-to-tail trimer of a full-length cDNA clone of the hepatitis delta virus (HDV) genome was examined for infectivity by direct inoculation into the liver of a chimpanzee that was already infected with hepatitis B virus. Five weeks after inoculation, a marked elevation of serum alanine aminotransferase activity was observed, followed by the appearance of high levels of HDV RNA and antigen in both liver and serum and a high level of viral particles in the serum. A transient suppression of hepatitis B virus replication was evident during the acute phase of HDV infection. Seroconversion for antibodies to delta antigen occurred 3 weeks after the onset of the disease. These results demonstrate that a typical HDV infection can be initiated by inoculation of a susceptible animal with recombinant HDV cDNA.     

Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes.

BHK and mouse L cells transfected with the cDNA for the human 46 kd mannose 6-phosphate receptor (MPR 46) secrete excessive amounts of newly synthesized mannose 6-phosphate containing polypeptides. The secretion is dependent on the amount, the recycling and the affinity for ligands of MPR 46. Incubation of transfected cells with antibodies blocking the binding site of MPR 46 reduces the secretion, and cotransfection with the cDNA for the human 300 kd mannose 6-phosphate (MPR 300) restores it to normal values. These results indicate that the two mannose 6-phosphate receptors compete for binding of newly synthesized ligands. In contrast to ligands bound to MPR 300, those bound to the MPR 46 are transported to and released at a site, e.g. early endosomes or plasma membrane, from where they can exit into the medium. Since antibodies blocking the binding site of MPR 46 reduce secretion also in non-transfected BHK and mouse L cells, at least part of the basal secretion of M6P-containing polypeptides is mediated by the endogenous MPR 46.     

Survival of genetically engineered Escherichia coli in natural soil and river water

Twelve derivatives of Escherichia coli strain HB101 which contained different sizes of plasmids ranging from 3.9 Kb to 48 Kb and encoding resistance to various antibiotics were used. When these organisms were introduced into natural river water, the population declined rapidly and by day 3, the majority (i.e. more than 99.9%) of them could no longer be detected on antibiotic-amended culture plates. If the river water was filter sterilized first, the added organisms maintained their population for up to 7 d without any significant decrease in numbers. Similar results were also observed in sterilized tap water or distilled water. This indicated that the disappearance of these organisms in the aquatic environment was caused mainly by biotic factor(s). The loss of the ability to grow in the presence of antibiotics by some of the E. coli was not observed unless they were allowed to grow in the antibiotic-free environment first. When the test organisms were added to natural silt loam, a large portion of the original population still remained viable after 16 d. There was no relationship between the percentage survival of E. coli in natural river water and the sizes of plasmid harboured. On the other hand, when these bacteria were added to natural soil, survival appeared to increase as plasmid size increased. and accepted 19 August 1989     

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.     

蔷薇属38个野生种果实的维生素含量及其与分组的关系

对蔷薇属 (Rosa) 38个野生种果实 (以下简称蔷薇果 )的经济性状进行了分析 ,并测定了 VC、VE 和胡萝卜素等重要维生素的含量。蔷薇果 VC 含量在该属种间差异很大 ,以秦岭蔷薇 (R.tsinglingensis)的含量为最高 (2 576mg/ 1 0 0 g) ,德钦蔷薇 (R.deqenensis)的含量为最低 (49mg/ 1 0 0 g)。胡萝卜素含量种间差异明显 ,以软条七蔷薇 (R.henryi)的含量为最高 (1 9.2 4 mg / 1 0 0 g) ,黄刺玫 (R.xanthina)的含量为最低 (0 .0 6 mg/ 1 0 0 g)。 VE 含量种间差异较小 ,在 1 .34 mg/ 1 0 0 g(黄刺玫 )至 3.86 mg/ 1 0 0 g(硕苞蔷薇R.bracteata)之间。对蔷薇亚属 54种野生种果实重要维生素含量的统计分析表明 ,维生素含量与分组具有一定相关性 ,尤以 VC 含量与分组的相关性最为明显 ,桂味组和小叶组 Vc含量很高 (均值都高于 1 80 0mg/ 1 0 0 g) ;合柱组、月季组、木香组和硕苞组含量很低 (均值都在 30 0 mg/ 1 0 0 g以下 ) ,芹叶组除宽刺蔷薇 (R.platyacantha) VC含量很高外 ,其余种类含量都很低 (均值为 1 90 mg/ 1 0 0 g)。胡萝卜素含量与分组也具有一定相关性 ,桂味组、芹叶组、合柱组和硕苞组的胡萝卜素含量较高 ,均值在 6 mg/ 1 0 0 g以上 ;月季组、小叶组和木香组含量较低 ,均值在 0 .4mg/ 1 0 0 g以下。V     

小鼠卵母细胞减数分裂染色体的制备

PreparationofMeioticKarytypeofMouseOocyteLiChaojunYanLeipingZhangXiranChenYifeng(BiologyDepartmentofNanjingNormalUniversity,Nanjing210097)哺乳动物的卵母细胞的减数分裂过程中存在两次自发的停滞现象,第一次是在第一次减数分裂前期的双线期,这一静止期持续很长时间,一直到动物性成熟后卵母细胞进入发有周刎,在保住腺激素的作用下,卵母细胞的第一次减数分裂才重新启动．完成第一次减数分裂后．又停滞在第二次减数分裂的中期,在椅子或化学因素刺激的作用下,完成第二次减数分裂[4]．因此,对哺乳动物的卵母细胞在一…     

RFLP-based maps of the homoeologous group-6 chromosomes of wheat and their application in the tagging of Pm12, a powdery mildew resistance gene transferred from Aegilops speltoides to wheat

Genetic maps of the homoeologous group-6 chromosomes of bread wheat, Triticum aestivum, have been constructed spanning 103 cM on 6A, 90 cM on 6B and 124 cM on 6D. These maps were transferred to a Chinese Spring (CS) x line #31 cross to locate a dominant powdery mildew resistance gene, Pm12, introgressed into line #31 from Aegilops speltoides. Pm12 was shown to lie on the short arm of translocation chromosome 6BS-6SS.6SL in line #31, but could not be mapped more precisely due to the lack of recombination between the 6S Ae. speltoides segment and chromosome 6B. Possible strategies to reduce the size of the alien segment, which probably encompasses the complete long arm and more than 82% of the short arm of chromosome 6B, are discussed.     

Visualization of Golgia apparatus as an intracellular calcium store by laser scanning confocal microscope

Using laser scanning confocal microscopy,we have found that the in cells loaded with fluo-3/AM,highest intracellular Ca^2 in the perinuclear region is associated with the Golgi apparatus.The spatiotemporal subcellular distribution of Ca^2 in living human fibroblasts exposing to calcium-free medium in response to agonists has been investigated.PDGF,which releases Ca^2 from intracellular stores by inositol(1,4,5)-trisphosphate pathway ,produced a biphasic transient rise in intracellular calcium.The initial rise was resulted from a direct release of calcium from the golgi apparatus.Calcium could be also released from and reaccumulated into the Golgi apparatus by the stimulation of thapsigargin,an inhibitor of the Ca^2 transport ATPase of intracellular calcium store,Permeablizing the plasma membrane by 10μM digitonin resulted in the calcium release from the Golgi apparatus and depletion of the internal calcium store.These results suggest that the Golgi apparatus plays a role in Ca^2 regulation in signal transduction.