Involvement of a serine protease in tumour-necrosis-factor-mediated cytotoxicity

We investigated the effect of various protease inhibitors on the anti-proliferative and cytotoxic action of tumour necrosis factor (TNF) on mouse L929 fibrosarcoma cells. 1. The following serine-type protease inhibitors led to inhibition of TNF action: phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethane, N alpha-p-tosyl-L-phenylalanyl chloromethane, N alpha-p-tosyl-L-arginine methyl ester, L-leucine methyl ester, DL-phenylalanine methyl ester, N-acetyl-DL-phenylalanine-beta-naphthyl ester, p-nitrophenyl p'-guanidino-benzoate and antipain. We could not detect an effect of inhibitors specific for thiol protease on TNF. 2. Inhibition of TNF-mediated cytotoxicity was evident in both the presence and absence of actinomycin D or cycloheximide. 3. TNF itself was not found to be a protease, as it had no proteolytic activity in a sensitive colorimetric assay. [1,3-3H]Diisopropyl fluorophosphate, an effective irreversible inhibitor of serine proteases, did not bind to TNF. Pretreatment of TNF with N alpha-p-tosyl-L-lysine chloromethane did not influence its biological activity. 4. The addition of protease inhibitor to the cells at various times after TNF administration led to a gradual loss of protection, suggesting that the protease acts at a rather late stage. 5. Protease inhibitors did not influence TNF binding, internalization or metabolization. 6. No increase in supernatant protease activity or in cell-associated protease activity could be detected after treatment of L929 cells with TNF. Our results document the involvement of protease activity, acting quite late during the cytolytic and growth inhibiting processes induced by TNF.     

Sex differences in the inhibition by ATD of testosterone-activated mounting behavior in guinea pigs

Adult male and female guinea pigs from a genetically heterogeneous stock were gonadectomized and tested for mounting behavior before and during various treatments with testosterone cypionate (TC) alone or in combination with an aromatase inhibitor, 1,4,6-androstatriene-3,17-dione (ATD). ATD was implanted subdermally in Silastic capsules (either 1 or 2 in females; 2 or 3 in males). In females 2 capsules of ATD completely blocked the behavioral effects of TC, and 1 capsule was an effective blocker in 58% of the females. The blocking effect was reversed by injection of diethylstilbestrol. In males, there was no measurable effect of ATD on mounting activity even when 3 capsules were implanted. Moreover, the TC induction of higher components of male sexual behavior (intromission and ejaculation) was also not impaired by ATD. Results are interpreted as indicating that either the process of male sexual differentiation or the male genotype eliminates the requirement for aromatization in androgenic activation of sexual behavior.     

Cloning of Trametes versicolor Genes Induced by Nitrogen Starvation

We have screened a genomic library of Trametes versicolor for genes whose expression is associated with nitrogen starvation, which has been shown to induce ligninolytic activity. Using two different approaches based on differential expression, we isolated 29 clones. These were shown by restriction mapping and cross-hybridization to code for 11 distinct differentially expressed genes. Northern analysis of the kinetics of expression of these genes revealed that at least four of them have kinetics of induction that parallel kinetics of induction of ligninolytic activity.     

A monoclonal antibody that inhibits opioid binding to rat brain membranes

To understand the structure-function relationship and to probe the molecular characteristics of the purified opioid receptor, monoclonal antibodies (mab) were raised against a purified opioid receptor protein. After intensive screening of almost 1500 hybridoma cell lines, only 7 clones were shown to have very high immunoreactivity against the purified receptor. Moreover, out of these 7 clones, only 2, 3B4F11 and 3A27G, were found to inhibit the ligand binding property of the mu-opioid receptor. The mab 3B4F11 was found to inhibit 3H-diprenorphine binding to the purified receptor in a dose dependent manner with a maximal inhibition of 100% achieved with 20 micrograms of the antibody. Likewise, Fab fragments prepared from the mabs 3B4F11 inhibited 3H-diprenorphine binding to P2 membranes in a dose-dependent manner. In addition, it was found that the binding of 3H-DAGO, 3H-DPDPE and 3H-EKC was inhibited with approximately equal potency, suggesting that the Fabs prepared from the mab 3B4F11 interact with all 3 receptor types.     

Developmental rates of heterozygous and homozygous rainbow trout reared at three temperatures

The hatching distributions of rainbow trout (Salmo gairdneri) with different genotypes at eight loci are compared in two experiments with the same strain. Embryos were incubated at temperatures colder (5 and 8°C) and warmer (12°C) than normally experienced by these fish (9.5°C). At hatching, embryos were separated into five hatching groups representing the chronological order of hatching. There is no significant correlation between multilocus heterozygosity and hatching time at any temperature in either experiment. Fish in the middle of the hatching distribution had the highest average heterozygosity. In both experiments, heterozygotes at the majority of loci examined tended to hatch relatively later within the hatching distribution at 12°C than at both 5 and 8°C. Fish with different genotypes atPgm2 andCk1 showed significant differences in hatching time that were consistent between experiments.Ck1 heterozygotes hatched sooner than homozygotes at 8°C but later at 12°C.Pgm2 heterozygotes hatched later than homozygotes at all temperatures and significantly later in four of five cases. At the other loci examined, however, the relative hatching distributions of fish with particular genotypes were not significantly different or repeatable between experiments.This research was supported by National Science Foundation Grant BSR-8300039 awarded to Dr. Fred W. Allendorf. Moira M. Ferguson was supported by a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada.     

Heterokaryosis inPolyporus ostreiformis for improved yield of exo-1,4-α-d-glucosidase

An amylolytic strain ofPolyporus ostreiformis was subjected to multistep mutagenic treatment and a heterokaryotic mutant was finally selected which produced exo-1,4-α-d-glucosidase in a yield of 2.54 U/mL against the parental yield of 0.42 U/mL. The crude enzyme extract of the mutant was used to hydrolyze soluble starch, maltose and amylopectin and chromatography of the hydrolyzates revealed exo-1,4-α-D-glucosidase activity predominant in the enzyme system.     

Adaptation of Aquatic Microbial Communities to Quaternary Ammonium Compounds

The effects of long-chain (C₁₂ to C₁₈) quaternary ammonium compounds (QACs) on the density, heterotrophic activity, and biodegradation capabilities of heterotrophic bacteria were examined in situ in a lake ecosystem. Monoalkyl and dialkyl substituted QACs were tested over a range of concentrations (0.001 to 10 mg/liter) in both acute (3 h) and chronic (21 day) exposures. In general, none of the QACs tested had significant adverse effects on bacterial densities in either acute or chronic studies. However, significant decreases in bacterial heterotrophic activity were noted in acute studies at QAC concentrations from 0.1 to 10 mg/liter. Chronic exposure of lake microbial communities to a specific monoalkyl QAC resulted in an adaptive response and recovery of heterotrophic activity. No-observable-effect level in the adapted populations was >10 mg/liter. Chronic exposure also resulted in significant increases in the number and activity of bacteria capable of biodegrading the material. The increase in biodegradation capability was observed at low (microgram per liter) concentrations which are approximately the same as realistic environmental levels. In general, our studies indicated that exposure of lake microbial communities to QACs results in the development of adapted communities which are less sensitive to potential toxic effects and more active in the biodegradation of these materials.     

Genetic and developmental characterization of the aldox-2 locus of Drosophila melanogaster

The aldox-2 locus in Drosophila melanogaster has been shown to affect differentially three molybdoenzymes, aldehyde oxidase, pyridoxal oxidase, and xanthine dehydrogenase. These effects are most obvious at times surrounding the pupal-adult boundary, when the normal organism accumulates large amounts of these enzymes in their active form. This locus has been more precisely mapped genetically to 2-82.9 +/- 2.1, with complete concordance between the effects of all recombinant chromosomes on all three enzymes. The cytogenetic location has also been determined to be between 52E and 54E8, with the likelihood that it lies within the region 54B1-54E8. The aldox-2 mutant allele has no visible phenotype and is completely recessive for enzyme effects at all stages tested. Segmental duplication of this region, including the aldox-2+ allele, has no apparent effect on the visible phenotype or the enzymatic activity. The mutant aldox-2 allele has no effect on the developmental expression of two unrelated enzymes, 6-phosphogluconate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. The effects of this locus on aldehyde oxidase, xanthine dehydrogenase, and pyridoxal oxidase suggest that this locus may code for a product involved in the synthesis of the molybdenum cofactor common to these enzymes.     

Direct transesterification of all classes of lipids in a one-step reaction

Conventional techniques for the determination of fatty acid composition of lipids require solvent extraction, purification, hydrolysis, and derivatization procedures that are both lengthy and cumbersome. A 1-hr direct transesterification procedure carried out in methanol-benzene 4:1 with acetyl chloride circumvented all these steps and was applicable for analysis of both simple (triglycerides) and complex lipids (cholesteryl esters, phospholipids, and sphingomyelin). Recoveries (greater than 95%) of standards unaffected by the presence of 5% water and 200 mg of silica suggested that the technique could be used for the quantitative analysis of total fatty acids as well as of fatty acids in classes of lipids separated on silica from biological samples. When compared to the Folch procedure, the technique led to a 20.1% increase in total fatty acids for plasma, 3.9% for feces, 7.4% for bile, and 9.7% for rat liver. We therefore conclude that this one-step direct transesterification procedure is superior to currently used methods, not only because of its simplicity and speed, but also because of its added precision.