Selective degradation of insulin within rat liver endosomes

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.     

Cleavage of dengue virus NS1-NS2A requires an octapeptide sequence at the C terminus of NS1.

The length of amino acid sequence at the NS1-NS2A juncture of dengue virus that is required for specific cleavage effected by the cis-acting function of NS2A was identified by deletion analysis. Recombinant DNA sequences of NS1-NS2A, each containing a deletion in NS1 followed by a sequence of 3 to 20 amino acids at the C terminus of NS1 preceding the cleavage site, were constructed and expressed with vaccinia virus as a vector. The NS1 product of recombinant vaccinia virus-infected cells was immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The occurrence of cleavage between NS1 and NS2A was indicated by the appearance of shortened NS1. Failure to cleave this site yielded a large NS1-NS2A fusion protein. This analysis indicated that a minimum length of eight amino acids at the NS1 C terminus preceding the NS1-NS2A juncture is required for cleavage to take place. Comparison of this eight-amino-acid sequence of the NS1 C terminus of dengue type 4 virus with the analogous sequences of 12 other flaviviruses suggests that the consensus cleavage site sequence is as follows: (table; see text)     

Dengue virus-specific cross-reactive CD8+ human cytotoxic T lymphocytes

Stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, CD8+, class I-restricted cytotoxic T lymphocytes (CTL) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. These CTL lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the E gene or several nonstructural dengue virus type 4 genes. These results demonstrate that both dengue virus structural and nonstructural proteins are targets for the cytotoxic T-cell-mediated immune response to dengue virus and suggest that serotype-cross-reactive CD8+ CTL may be important mediators of viral clearance and of virus-induced immunopathology during secondary dengue virus infections.     

Endothelium-dependent basilar and aortic vascular responses in normotensive and coarctation hypertensive rats

The responsiveness of acetylcholine (ACh), nitroglycerin (NG) and norepinephrine (NE) (aorta only) in both basilar arteries (BA) and thoracic aortic (TA) rings from coarctation hypertensive rats (CHR) were studied and compared to their sham-operated normotensive control rats (SNR). The effects of these agents were also evaluated in TA or BA with and without endothelium from naive normotensive rats (NNR). Blood pressure (BP) and plasma renin activity (PRA) of CHR were significantly higher than their time-matched SNR. Endothelium removal from TA of NNR significantly enhanced NE and NG sensitivity and reduced the maximum ACh relaxation. Removal of BA endothelium of NNR abolished ACh-induced relaxation but had no effect on NG-induced relaxation. In BA from CHR at any stage of hypertension studied, the sensitivity and maximum relaxation induced by ACh or NG were not significantly different than their respective time-matched SNR. ACh sensitivity of TA did not change in 1 Day CHR but decreased in 4 and 14 Day CHR. NG sensitivity increased, did not change and decreased in 1, 4 and 14 Day CHR, respectively. NE sensitivity increased in all stages of hypertension. These data suggest that in coarctation-induced hypertension there is a complex progression of events in TA which is modulated by different mechanisms as evidenced by the changes in the effects of NE, ACh and NG at various stages of hypertension. The results also suggest that the vascular endothelium of TA but not of BA may provide an acute protective mechanism to counteract the imbalance created by the increased sensitivity of smooth muscle cells to contractile agonists in the early stage of hypertension. However, persistent hypertension appears to override this mechanism.     

几种哺乳动物精子顶体膜囊泡形成的研究

在猪、绵羊、地鼠这几种哺乳动物的精子体外获能后,在顶体反应的超显微结构中观察到:精子顶体膜囊泡化呈现多种形态,但囊泡都是由精子顶体的双层外膜多位点自我融合而形成的,质膜并不参与囊泡化,这一结果与前人报道的不同。     

Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors.

A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible.     

小麦幼苗根系镉螯合素

从经Cd~(2+)处理的小麦幼苗根系中分离得到一种镉结合复合物(Cd-BC)。通过SephadexG75,DEAE-52柱层析纯化,鉴定了此复合物性质:(1)紫外吸收光谱在255～265 tim间有一个“肩”,A_(250)/A_(280)>1;(2)在Sephadex G75柱层析上的表观分子量约为10kD,但在SDS-聚丙烯酰胺凝胶电泳上呈现的条带紧接着前沿,分子量非常小;(3)氨基酸组分分析,约90％的氨基酸残基为Glu/Gln,Cys和Gly,三者比例约为4:4:1。结果说明小麦幼苗根系Cd-BC是寡聚肽,是植物镉螯合素(Cd-PCs)的聚合体。     

Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection.

A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied.