Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.     

Immunoblotting of mite aeroallergens collected with an indoor Burkard air sampler

We examined the kinetics of airborne levels of mite allergen particles in a house by combined use of an indoor Burkard air sampler and immunoblotting. Airborne mite allergens collected on the Burkard sampling tape were transferred onto a nitrocellulose membrane, reacted with mouse monoclonal anti-mite allergen (Der pI) antibody, then treated with alkaline phosphatase conjugated anti-mouse IgG. Finally, the blotted allergen on the membrane was reacted with BCIP/NBT phosphatase, and purple spots visible by the naked eye were produced. The shape of the spots was observed under a microscope, and the spot area was measured by an image processor. This technique might be useful for analyzing the behavior of airborne allergen particles in indoor environments.     

Histological Observations on Initiation and Morphogenesis in Immature and Mature Embryo Derived Callus of Barley (Hordeum vulgare L.)

Callus was induced from immature and mature embryos of barley(cv. Haruna Nijo) on Murashige and Skoog medium containing 2mg l^-1 2,4-D and 5 mg l^-1 picloram, respectively. Paraffin sections(10 µm thick) were prepared for histology during callusinitiation and plant regeneration. Meristems were regeneratedfrom nodular compact callus (NC) derived from scutellar epidermisin immature embryos, whereas they were regenerated from NC derivedfrom epidermal cells of leaf or coleoptile bases in mature embryos.Regardless of the explant source, regeneration was predominantlythrough organogenesis, although regeneration through somaticembryogenesis infrequently occurred. Thus, the callus inducedfrom immature and mature embryos of barley was regarded as 'nodularcompact' rather than 'embryogenic'.Copyright 1995, 1999 AcademicPress Barley, callus, Hordeum vulgare, histology, immature embryo, mature embryo, regeneration     

Cold-sensitive conditional mutations in Era, an essential Escherichia coli GTPase, isolated by localized random polymerase chain reaction mutagenesis

Abstract Conditional cold-sensitive mutations in Era, an essential Escherichia coli GTPase, were isolated. Localized random polymerase chain reaction (PCR) mutagenesis employing Taq and T7 DNA polymerases under error prone amplification conditions was exploited to generate mutations in the era gene. A plasmid exchange technique was used to identify conditional cold-sensitive mutations in Era that give rise to defective cell growth below 30 °C. Three recessive missense mutations in Era, N26S, A156D, and E200K, were isolated. All three mutations are located at residues conserved in Era homologues from Streptococcus mutans and Coxiella burnetii .     

Production of monoclonal antibodies and development of an ELISA for solamargine

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against solamargine was produced by fusing splenocytes immunized with a solamargine-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. Extensive cross-reaction of anti-solamargine antibodies against solasonine appeared. Aglycone of solamargine, solasodine cross-reacted with anti-solamargine antibodies resulting in a 43.8% cross-reaction. Insignificant cross-reaction appeared with tomatine (2.06%). The full measuring range of the assay extends from 57.5 pmol ml^–1 to 11.5 nmol ml^–1 of solamargine.     

Cell-Free Synthesis of a Putative Precursor of Polygalacturonase in Tomato Fruits

By using a monospecific anti-polygalacturonase-2 antibody, a54K-dalton polypeptide was detected in in vitro translationproducts by a wheat germ cell-free translation system programmedwith polyadenylated RNA from ripe tomato pericarp tissue. Thisputative precursor of polygalacturonase was about 9K daltonslarger in molecular weight than polygalacturonase-2. (Received December 12, 1983; Accepted May 17, 1984)     

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.