Inhibition of HIV replication by naphthalenemonosulfonic acid derivatives and a bis naphthalenedisulfonic acid compound

Several naphthalenemonosulfonic acid analogs and a bis naphthalenedisulfonic acid have been evaluated for anti-HIV activity in assays using H9 and MOLT-3 cells. Among the naphthalenemonosulfonic acids, a 4-amino-5-hydroxy compound and a 4,5-diamino compound showed low anti-HIV activity (upto 50% inhibition) at non-toxic doses. The bis naphthalenedisulfonic acid compound demonstrated significant suppression of HIV-1 antigen expression as measured by monoclonal antibodies to p17 (95%), p24 (94%) and syncytia inhibition (82%) at a dose of 20 micrograms/ml that was non-toxic to the host cells. The bis naphthalenedisulfonic acid analog represents a new class of compounds which may be effective in the treatment of HIV infected patients. The structure activity relationship and a probable mode of action of these compounds is discussed.     

Superovulation of beef heifers with Folltropin: A new FSH preparation containing reduced LH activity

The optimum superovulatory dose of Folltropin was determined and compared with a standard 28 mg dose of FSH-P in beef heifers. In Experiment 1, mean numbers of corpora lutea (CL) did not differ among the groups treated with 10, 20, 30 or 40 mg Folltropin or FSH-P, and the mean CL number was reduced (P<0.05) only in the 5 mg Folltropin group. Mean numbers of ova/embryos recovered, fertilized and transferable were greater (P<0.05) for the 10, 20 and 30 mg Folltropin groups than for the 5 mg group. The 40 mg Folltropin group and the FSH-P group were intermediate. The percentage of fertilized and transferable embryos did not differ over the dosages used in this experiment. In Experiment 2, mean numbers of CL were greater for the 9, 18 and 36 mg Folltropin groups than for the 4.5 mg group, with the 9 mg group being lower than the 36 mg group (P<0.05). The 18 mg group was intermediate and did not differ. Mean numbers of ova/embryos recovered and fertilized ova were greater for the 9, 18 and 36 mg groups (P<0.05) than for the 4.5 mg group. The percent of fertilized and mean number and percentage of transferable embryos did not differ among treatments. We conclude that Folltropin may be a satisfactory superovulatory replacement for FSH-P and that a dose of 18 to 20 mg Folltropin may be within the optimum superovulatory dosage range for beef heifers. Dosages of Folltropin of more than twice the optimum did not result in deterioration of ova/embryo quality.     

Selective suppression of the catalytic activity of cDNA-expressed cytochrome P4502B1 toward polycyclic hydrocarbons in the microsomal membrane: modification of this effect by specific amino acid substitutions.

Human hepatoma HEPG2 cells were infected with recombinant vaccinia virus vectors containing cDNAs encoding both known and variant rat cytochromes P450 (CYP). CYP2B1 and CYP2B2 cytochromes were equally well expressed (110-140 pmol/mg of microsomal protein) and catalyzed metabolism of 7,12-dimethylbenz[a]anthracene (DMBA). Their regioselectivity for DMBA metabolism paralleled that of the respective purified rat liver enzymes and reproduced previously reported regioselective differences between CYP2B1 and CYP2B2 [Wilson et al. (1984) Carcinogenesis 5, 1475-1483]. CYP2A1 and CYP2A2 expressed in HEPG2 microsomes exhibited nearly equal DMBA-metabolizing activities that closely matched that of purified CYP2A1. Although purified rat liver CYP2B1 was 3 times more active than purified rat liver CYP2B2, the expressed recombinant microsomal CYP2B1 (rCYP2B1) was 20 times less active than rCYP2B2, where activity matched that of the purified cytochrome. Microsomal suppression of rCYP2B1 catalytic activity was also observed for benzo[a]pyrene. Specific amino acid substitutions at equivalent positions of the completely homologous NH2-terminal halves of rCYP2B1 and rCYP2B2 changed this suppression effect. Thus, a L58----F, I114----F double mutant exhibited 3 times the normal activity for rCYP2B1 while remaining inhibitory for rCYP2B2. The single substitutions produced very different effects. The L58----F substitution prevented expression of rCYP2B1, while the I114----F substitution was inhibitory for both rCYP2B1 and rCYP2B2 (40 and 70%). A single E282----V mutation produced a stimulation of rCYP2B1 activity comparable to that of the L58----F, I114----F double substitution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of cytochrome P450s CYP2A1 and CYP2A2: influence of the distal helix on the kinetics of testosterone hydroxylation.

Cytochrome P450s CYP2A1 and CYP2A2 exhibit 88% sequence similarity, yet CYP2A1 metabolizes testosterone almost exclusively (90%) at the 7 alpha-position, whereas CYP2A2 forms several metabolites, with 15 alpha-hydroxytestosterone as a major metabolite. One of the regions with relatively low sequence homology corresponds by sequence alignment to the I and J helices of P450cam. Since this region is known to be part of the active site for P450cam, 26 single point and two double point mutants were prepared where the amino acid for one form was substituted with that of the other. Mutant and wild-type enzymes were expressed in Hep G2 cells using the vaccinia virus vector. Analysis of testosterone regioselectivity revealed that 25 of the mutants show the same regioselectivity as the parent wild-type enzymes and three are inactive, suggesting that no single amino acid in this region is totally responsible for the different selectivities of CYP2A1 and CYP2A2. Kinetic analysis of the CYP2A1 mutants showed that four of the mutants with changes near the conserved oxygen-binding region had Km values with much higher and Vmax values much lower than those of the wild-type enzyme and one mutant had a Vmax value twice as high as that of the wild-type enzyme. Deuterium isotope effects on 7 alpha-hydroxxylation were used to determine changes in the rate of reduction and estimate the relative amount of excess water formation. Changes in reduction rates and the amount of water produced are not sufficient to account for the differences in Vmax values, suggesting that the amount of hydrogen peroxide released is a primary determinant for changes in Vmax.     

Compensation of respiratory alkalosis induced after acclimation to simulated altitude

Conscious intact rats previously acclimated for 3 wk to barometric pressure of 370-380 Torr (3WHx) were made alkalotic for 3 h by a decrease in inspired O2 fraction from 0.10 to 0.075 at ambient barometric pressure (730-740 Torr). Controls were normoxic littermates (Nx) in which inspired O2 fraction was lowered from approximately 0.21 to 0.10 for 3 h. Arterial PCO2 decreased progressively and similarly in both groups (65-70% of control at 15 min). Initially, arterial pH increased less in 3WHx (0.09 +/- 0.004 vs. 0.15 +/- 0.008). As hypocapnia continued, delta[HCO3-]/delta pH (mmol.l-1.pH) became more negative in Nx, from -15.2 +/- 2.5 at 15 min to -37.0 +/- 2.9 at 3 h, indicating nonrespiratory compensation of alkalosis. In 3WHx, delta[HCO3-]/delta pH did not change during alkalosis. Cumulative renal excretion of base (mueq/100 g) during alkalosis increased by 73.2 +/- 11.1 in Nx and 25.4 +/- 7.3 in 3WHx. This difference was mainly due to a larger increase in HCO3- excretion in Nx. The data suggest that the smaller compensation of hypocapnic alkalosis in 3WHx is partly due to the smaller increase in renal base excretion. Because base availability limits renal base excretion, the smaller renal response of 3WHx may be secondary to the low plasma HCO3- concentration that accompanies altitude acclimation.     

Cloning of Choriocarcinoma Cells Shows That Invasion Correlates with Expression and Activation of Gelatinase A

Implantation and placental development are dependent upon trophoblast invasion of the endometrium. While the villous trophoblast does not display invasive behavior, the extravillous cytotrophoblast is highly invasive. By cloning BeWo choriocarcinoma cells, we have isolated two distinct clones that share similarities with villous and extravillous cytotrophoblasts. When cultured at the surface of a type I collagen gel, BeWo MC-1 cells were not invasive, whereas BeWo MC-2 cells rapidly invaded this matrix. When injected subcutaneously in nude mice, BeWo MC-1 cells developed a localized tumor and BeWo MC-2 cells developed larger tumors with micrometastases. Gelatinase A expression and minute amounts of gelatinase B were detected in the parental cell line and in both clones. However, the parental and the BeWo MC-2 cells secreted 5- to 10-fold more gelatinase A than the BeWo MC-1 cells. Laminin and matrigel stimulated the production of gelatinase A in BeWo MC-2 cells. Type I collagen promoted the conversion of the 72-kDa progelatinase A in an active enzyme only in parental BeWo and in BeWo MC-2 cells. These clones provide an interesting model for studying the complex mechanisms regulating implantation as well as the controlled invasiveness during implantation compared to tumor invasion.     

Clean technology in aquaculture — a production without waste products?

A method has been developed for the determination of H₂S and FeS in sediments. FeS is converted into H₂S which is flushed from the samples directly into an excess of chlorine bleach, NaC1O or KClO with some Zn²⁺ added. Either the excess can be titrated back potentiometrically with As₂O₃, or the sulphate formed can be measured colorimetrically. The precision is primarily controlled by the homogeneity of the sediment suspensions and can be better than 99%.     

Studies of the Inheritance of Human Ribosomal DNA Variants Detected in Two-Dimensional Separations of Genomic Restriction Fragments

We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separations of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in ~20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions.