Perspectives on retroviruses and the etiologic agent of AIDS

In 1911, the first retrovirus was described: the Rous sarcoma virus, an avian retrovirus. Forty years later the murine leukemic virus, a mouse retrovirus, was reported. Although many other retroviruses from non-primate species were identified during the 1960s, the first primate retrovirus was not recognized until it was isolated from a monkey tumor in 1970. The search for human retroviruses in human leukemic cells remained unsuccessful at that time. Facilitated by the discovery of T-cell growth factor, a substance used for the propagation of human leukocytes in cultures, the first human retrovirus was discovered in 1980. Soon thereafter, in 1983, another human retrovirus, human immunodeficiency virus (HIV), was reported and implicated as the etiologic agent of AIDS. The isolation and identification of HIV has stimulated much interest in the study of human retroviruses and the control of this new viral disease.     

A Ca2+-Dependent Protein Kinase Activity Associated with Serotonin Binding Protein

The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.     

Use of synthetic DNA in expression of foreign proteins

Synthetic DNAs and oligonucleotides, which can be prepared conveniently by combining chemical synthesis and enzymatic methods, have been used extensively in recombinant DNA research. Examples include total gene synthesis, probes for the isolation of specific genes from cDNA or genomic libraries, linkers containing specific restriction sites for cloning, primers for DNA and RNA sequencing, and primers for the construction of specific mutations (either deletion, insertion or point mutations) by oligonucleotide-directed site-specific mutagenesis.This article reviews recent advances in the chemical and enzymatic synthesis of oligo- and polynucleotides and the application of synthetic DNA to the expression of foreign proteins. The synthesis of genes, including structural genes and regulatory genes are reviewed. Oligonucleotide-directed site-specific mutagenesis and use of synthetic DNA to optimize foreign protein expression are also discussed.     

血液病患者的巨细胞病毒感染

用间接酶联免疫吸附实验(ELISA)对新近诊断的179例血液病患者血清巨细胞病毒(HCMV)IgM和IgA抗体进行了检测。阳性率分别为11,17％11,73％,明显高于对照人群(4,76％和3,97％),提示血液病患者由于免疫功能下降,易于发生HCMV活动性感染。     

Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates.     

Hydrodynamic and mechanical aspects of endothelial permeability

The passive transport of water through the endothelial cell layer junctions is considered from the standpoint of hydrodynamic theories based on ultrastructural information. The local geometry of tight junctions based on molecular level forces and elastic membrane properties has been modeled and leads to estimates of the hydraulic resistance of the clefts. It is shown that the large resistance measured experimentally can be accounted for in this model. The transport of large macromolecules via vesicles which diffuse across the endothelial cell has been developed, but recent experimental data do not appear to support this mechanism as a primary pathway. Fused vesicles forming an open channel appear to be rare. Leaky junctions, such as around dying endothelial cells or produced by cytoskeletal changes within the cells, may be important in control of endothelial permeability. Another kind of model is a fiber matrix model of the endocapillary layer, extending into the intercellular clefts which can also account for the molecular seiving properties of the endothelial cell layer but may produce a large resistance to water flux.     

Purification of human C3b inactivator by monoclonal-antibody affinity chromatography

Monoclonal antibody has been obtained to the human complement control protein C3b inactivator after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to Sepharose-CL-4B to give a specific affinity column, which was used to isolate C3b inactivator from human serum in 70% yield. The product was characterized by size, chain structure, amino acid analysis and proteolytic activity.     

Epidemic hemorrhagic fever in Hubei Province, The People's Republic of China: a clinical and serological study

Between July 1975 and April 1980, 71 patients were admitted to the Second Attached Hospital of Hubei Provincial Medical College in Wuchang with the diagnosis of epidemic hemorrhagic fever (EHF). The clinical course among these patients was similar to that described for patients with Korean hemorrhagic fever, and hemorrhagic fever with renal syndrome of the U.S.S.R. The overall mortality was 11.2 percent. Sera obtained from some of these patients as well as from patients admitted to the First Attached Hospital of Hubei Provincial Medical College were tested against an antigen associated with Korean hemorrhagic fever and showed exceedingly high antibody titers. We conclude that EHF in Central China represents the same or a closely related disease process as Korean hemorrhagic fever.