CD4-independent, productive human immunodeficiency virus type 1 infection of hepatoma cell lines in vitro.

Five hepatoma cell lines, including CZHC/8571, PLC/PRF/5, Hep3B, HepG2, and HUH7, were inoculated with three diverse isolates of human immunodeficiency virus type 1 (HIV-1). Productive infection was noted in all hepatoma cell lines, and expression of viral p24 antigen lasted for over 3 months, but its level decreased in proportion to the number of viable cells. HIV-1 antigens were also found in the cells by immunohistochemical staining and radioimmunoprecipitation assay, as were viral RNA by in situ hybridization and HIV-1-like particles by electron microscopy. Virus yield assays were also positive on supernatant fluids collected from hepatoma cultures inoculated with HIV-1. Despite their susceptibility to infection, all five hepatoma cell lines were negative for CD4 by immunofluorescence and for CD4 mRNA by slot-blot hybridization. In addition, HIV-1 infection of hepatoma cell lines was not blocked by anti-CD4 monoclonal antibody or soluble CD4. Together, these findings clearly demonstrate that all five hepatoma cell lines were susceptible to productive infection by HIV-1 in vitro via a CD4-independent mechanism.     

CD4-independent, productive infection of a neuronal cell line by human immunodeficiency virus type 1.

One neuronal cell line (SK-N-MC) was found to be susceptible to productive infection by multiple isolates of the human immunodeficiency virus type 1 (HIV-1). Characterization of SK-N-MC cells showed that these cells are neuroectodermal in origin in that they express dopamine hydroxylase, catecholamines, neuron-specific enolase, and neurofilaments. Despite their susceptibility to HIV-1 infection, SK-N-MC cells had no detectable CD4 and this infection was not blocked by anti-CD4 monoclonal antibodies (OKT4A, Leu3A) or recombinant soluble CD4. These experiments demonstrated that certain cells of neuroectodermal origin are susceptible to infection in vitro by HIV-1 via a CD4-independent mechanism.     

Formation of intermolecular and intramolecular hydrogen bonds in histidine-binding protein J of Salmonella typhimurium upon binding L-histidine. A proton nuclear magnetic resonance study

Histidine-binding protein J of Salmonella typhimurium has been chosen as a model system for a proton nuclear magnetic resonance spectroscopic investigation of binding protein-ligand interaction. This interaction is involved in the recognition step of the osmotic shock-sensitive active transport systems. When J protein binds L-histidine, four new, low-field, exchangeable proton resonances appear in the region +7 to +12 parts per million downfield from the water proton resonance (or +11.7 to +16.7 parts per million downfield from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate). Due to their chemical shift range and other properties, they indicate the formation of both intra- and intermolecular hydrogen bonds. Experiments with 15N-labeled compounds confirm this conclusion. The specificity of the hydrogen-bond formation is demonstrated by observing the effects of substrate analogs, temperature, pH, and mutations on the exchangeable proton resonances. Proton-proton nuclear Overhauser effect measurements suggest that two of these exchangeable proton resonances (at +7.2 and +10.6 parts per million from H2O) are most likely from intramolecular hydrogen-bonded protons, while the other two (at +7.1 and +9.5 parts per million from H2O) are intermolecular hydrogen bonds. Our finding of L-histidine-induced hydrogen-bond formation in histidine-binding protein J in the solution state is an excellent demonstration of the production of specific conformational changes in a periplasmic binding protein upon binding of ligand.     

Molecular genetic, biochemical and nuclear magnetic resonance studies on the role of the tryptophan residues of glutamine-binding protein from Escherichia coli

The results of molecular genetic, biochemical and nuclear magnetic resonance studies on glutamine-binding protein of Escherichia coli suggest that the only two tryptophan residues, at positions 32 and 220, in the protein molecule are likely to be involved in (or sensitive to) interactions with the membrane-bound protein components of the glutamine transport system. It has been found that both tryptophan residues have limited motional freedom, are located away from the surface of the protein molecule and are not close to the ligand-binding site. Their presence, however, is required for the optimal transport of L-glutamine across the cytoplasmic membrane, though not essential for the ligand-binding process. The relevance of these results to the structure and function of the glutamine-binding protein in the glutamine transport system is discussed.     

Liver alcohol and aldehyde dehydrogenase isozymes in a Chinese population in Hong Kong

The distribution of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozymes in the livers of a Chinese population in Hong Kong was examined. Among the 90 livers examined, 7 were typical ADH phenotype consisting the normal beta 1-type isozymes and 83 were atypical phenotype consisting the beta 2-type isozymes. Livers of 48 subjects were of deficient type in ALDH containing ALDH-II alone and 42 were of normal type with both ALDH-I and ALDH-II. When the combination of ADH and ALDH isozymes is considered, the Chinese population in Hong Kong falls into 4 subgroups. For each group, the rates of ethanol and acetaldehyde clearance have a distinct and characteristic potential which is directly related to its particular combination of isozymes.     

Tibenelast, 5,6-diethoxybenzo(B)thiophene-2-carboxylic acid, sodium salt (LY186655), is an orally active anti-asthma compound in the guinea pig

Anaphylactic shock was induced in actively sensitized guinea pigs by free inhalation of a high dose of ovalbumin (10 mg/ml) aerosol. Tibenelast (LY186655), 5,6-diethoxybenzo(b)-thiophene-2-carboxylic acid, sodium salt, proved to be a potent orally active compound against anaphylactic shock induced by high dose antigen aerosol. When a lower aerosol challenge (0.05 mg/ml) was employed, bronchoconstriction was observed with a concomitant increase in lung resistance (RL) and a fall in dynamic compliance (Cdyn). Tibenelast at 25 mg/kg p.o. prevented these changes. Tibenelast was 10 times more potent than aminophylline by i.v. administration; normalization of pulmonary function was achieved at 1 mg/kg i.v. Tibenelast was synergistic with epinephrine. Combination of no-effect doses of epinephrine (0.025 mg/kg s.c.) and tibenelast (0.1 mg/kg i.v.) normalized pulmonary function. The oral dose response curve of tibenelast was enhanced with the co-administration of epinephrine. These data suggest that tibenelast may act at a site different from that of epinephrine, although the mechanism of action of tibenelast is unclear at present. Tibenelast may be of significant value in the treatment of asthma and other respiratory diseases.     

Cloning, sequence analysis, and expression of a cDNA encoding a plastid-localized heat shock protein in maize

We have cloned and characterized a cDNA encoding a maize (Zea mays L.) heat shock protein (HSP), HSP26. The mRNA of HSP26 is present as a single mRNA species of 1.1 kilobase pairs in size and is detectable when maize seedlings are treated at 40°C but not at 28°C. Accumulation of HSP26 mRNA was detected after 10 minutes of incubation at 40°C, reaching the maximum level after 1 hour. Comparison of the deduced amino acid sequence of maize HSP26 to other HSPs indicated a strong homology to the sequences of two nuclear encoded HSPs that are transported into the chloroplasts during heat shock: pea HSP21 and soybean HSP22. Maize HSP26 was also found to cross-react with anti-pea chloroplast HSP21 antibodies. Because of the sequence homology between maize HSP26, soybean HSP22, and pea HSP21, in vitro chloroplast protein import experiments were conducted. The in vitro synthesized maize HSP26 is specifically imported to the soluble fraction of the chloroplast and processed to a smaller polypeptide. The sequence homology and antibody cross-reactivity between maize HSP26 and pea HSP21 have allowed us to conclude that maize HSP26 is a nuclear-encoded, plastid-localized protein in maize.     

A major gibberellic Acid-induced barley aleurone cysteine proteinase which digests hordein : purification and characterization

We previously described the purification and characterization of a 37,000 M_r cysteine proteinase, designated EP-A, from gibberellic acid (GA₃)-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M_r of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M_r as determined by gradient SDS-PAGE.