MICROTUBULES ASSOCIATED WITH SIMULTANEOUS CYTOKINESIS OF COENOCYTIC MICROSPOROCYTES

Microtubule arrays associated with simultaneous cytokinesis in the coenocytic microsporocytes of Lonicera japonica and Impatiens sultani were studied by indirect immunofluorescence. The future division planes are not predicted prior to meiosis by either a preprophase band of microtubules or cytoplasmic lobing. Cleavage planes appear to be determined by position of the four haploid nuclei and the development of postmeiotic microtubule systems. Perpendicular second division spindles in Lonicera result in tetrahedrally arranged tetrads while parallel spindles in Impatiens result in tetragonal arrangement. Immediately following meiosis bands of microtubules, the secondary spindles, develop between both sister and nonsister nuclei. These arrays give way to systems of microtubules that radiate equally from each of the four nuclei in the coenocytic sporocyte. Simultaneous cytokinesis is initiated by centripetal wall deposition at the periphery of the sporocyte and proceeds along planes marked by interaction of the opposing arrays of nuclear-based microtubules.     

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.     

Chromosome distribution of intracisternal A-particle sequences in the Syrian hamster and mouse

Metaphase chromosomes of Syrian hamster and BALB/c mice were hybridized in situ with radiolabeled probes derived from cloned intracisternal A-particle (IAP) genes of the corresponding species. The DNAs of these species are known to contain about 900 and 1,000 copies, respectively, of the retrovirus-like IAP sequence elements per haploid genome. Multiple IAP sequences were found on all chromosomes of both hamster and mouse. In the hamster, more than half of the IAP sequences were located in regions of non-centromeric constitutive heterochromatin, at an average concentration per unit chromosome length 5 times greater than in the euchromatic regions. The other dispersed sequences showed marked local variations in concentration along the chromosome lengths; both discrete foci and large grain clusters were observed as well as regions apparently lacking IAP sequences. Within the resolution of the techniques, IAP sequences appeared to be more evenly distributed over the mouse chromosomes; however, some prominent variations in concentration were seen. The number of potentially active IAP genes in the Syrian hamster, and by extension in the mouse, may be restricted by the preferential location of IAP sequences in genetically inert regions of the genome.     

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.