Host range mutant of human immunodeficiency virus type 1: modification of cell tropism by a single point mutation at the neutralization epitope in the env gene.

We have isolated a variant of human immunodeficiency virus type 1 (HIV-1) which is highly infectious to fibroblastlike cells (BT cells) derived from human brain as well as CD4-positive T cells. This variant HIV-1, named HIV[GUN-1V], was obtained by infecting BT cells with a prototype HIV-1 isolate, named HIV[GUN-1WT], which is highly infectious to T cells but barely infectious to BT cells. HIV[GUN-1V] infects BT cells productively and this infection appeared to be mediated by CD4. To elucidate the viral gene responsible for the host range difference between the variant and prototype HIV-1s, we cloned and analyzed the provirus genomes of the two viruses. Examination of the infectivities of BT cells by various recombinant viruses and analyses of the nucleotide sequences of HIV[GUN-1V] and HIV[GUN-1WT] showed that a single nucleotide exchange was responsible for their difference in infectivity of BT cells: HIV[GUN-1V] contains a thymine residue instead of the cytosine residue in HIV[GUN-1WT] at position 931 of the env coding sequence. Replacement of cytosine by thymine at this position of the env coding sequence of the HIV[GUN-1WT] genome induced the ability to infect BT cells. The base exchange at this position was expected to change amino acid 311 of the envelope glycoprotein, gp120, from proline to serine, which is located in a variable region containing type-specific immunodominant epitopes. Thus, HIV[GUN-1V] acquired a wider host range than HIV[GUN-1WT] by a single point mutation in the env gene.     

Determination of octopine by pre-column fluorescence derivatization using benzoin

A sensitive fluorimetric method for the determination of octopine, a member of opine family, is presented. The method is based on the formation of a fluorescent derivative of octopine with benzoin and the separation by high performance liquid chromatography using a reversed-phase column (Kaseisorb LC ODS-300) within 20 min. The octopine derivative is completely separated from other guanidino compounds including arginine which is generally very high in marine invertebrates. This method gives higher sensitivity, 5 pmol minimum detection, and better reproducibility than the electrophoresis method and colorimetric method.     

Vasoactive intestinal peptide stimulates ciliary motility in rabbit tracheal epithelium: modulation by neutral endopeptidase.

We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP.     

Three Japanese families with members carrying C7 silent allele (C7*Q0). Possibility for an association between C7*Q0 and C6*B

Three Japanese families with members carrying C7 silent allele(s) (C7*Q0) are presented. C6 types in the family members were also examined, and it was found that C7*Q0 was transmitted from a parent to offsprings as a haplotype, C7*Q0-C6*B. In another study of C6 types in sera from 3 volunteer blood donors with homozygous C7 deficiencies, the C6 phenotypes were found to be C6 B (homozygote). It seems remarkable that C7*Q0 can be associated with C6*B.     

Chemical modification by diethylpyrocarbonate of an essential histidine residue in 3-ketovalidoxylamine A C-N lyase

3-Ketovalidoxylamine A C-N lyase of Flavobacterium saccharophilum is a monomeric protein with a molecular weight of 36,000. Amino acid analysis revealed that the enzyme contains 5 histidine residues and no cysteine residue. The enzyme was inactivated by diethylpyrocarbonate (DEP) following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with hydroxylamine, the enzyme activity was completely restored. The difference absorption spectrum of the modified versus native enzyme exhibited a prominent peak around 240 nm, but there was no absorbance change above 270 nm. The pH-dependence of inactivation suggested the involvement of an amino acid residue having a pKa of 6.8. These results indicate that the inactivation is due to the modification of histidine residues. Substrates of the lyase, p-nitrophenyl-3-ketovalidamine, p-nitrophenyl-alpha-D-3-ketoglucoside, and methyl-alpha-D-3-ketoglucoside, protected the enzyme against the inactivation, suggesting that the modification occurred at or near the active site. Although several histidine residues were modified by DEP, a plot of log (reciprocal of the half-time of inactivation) versus log (concentration of DEP) suggested that one histidine residue has an essential role in catalysis.     

Immunohistochemical studies of the serotonergic supraependymal plexus in the mammalian ventricular system, with special reference to the characteristic reticular ramification

Distributional and morphological features, especially characteristics of the ramification of serotonin-containing supraependymal fibers (SEF), were studied in the ventricular systems of mammals (mouse, rat, guinea pig, rabbit, cat, dog, monkey) by means of a modified peroxidase antiperoxidase technique, using antiserotonin antiserum prepared in our laboratory. SEF were present in all ventricular systems, except on the third ventricle floor and in the choroid plexus. The density of SEF was higher in the smaller species. In the rat, light- and scanning electron microscopical SEF were almost completely abolished 1 week after intraventricular administration of 5,6-dihydroxytryptamine. Ramification of SEF was complicated; the SEF formed a true network with frequent anastomosing. In the ventricular system of rats rendered hydrocephalic by kaolin administration, the mode of axonal branching in the supraependymal plexus could best be analyzed by the scanning electron microscope because the meshes of the plexus were spread out.     

1,2-α-D-Mannosidase from a wood-rotting basidiomycete, Pycnoporus sanguineus

An acidic α-D-mannosidase has been isolated from the culture filtrate of a wood-rotting Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme determined. The extracellular mannosidase was homogeneous on PAGE at pH 9.4. The M_r as determined by SDS-polyacrylamide disc gel electrophoresis was 64000, and the pI was pH 4.7 using electrofocusing. The purified enzyme had a pH optimum of 4.5 with Baker's yeast mannan and had no activity towards p-nitrophenyl-α-mannoside. The K_m and k_cat values for Manα1-2Man at pH 4.5 and 30° were 0.9 mM and 1.9 sec. the enzyme had no activity towards Manα1-3Manα1-2Man, and it cleaved specifically the 1,2-α-linked side chain of yeast α-mannan, producing free α-D-mannose.