Attachment to an endogenous laminin-like protein initiates sprouting by leech neurons

Leech neurons in culture sprout rapidly when attached to extracts from connective tissue surrounding the nervous system. Laminin-like molecules that promote sprouting have now been isolated from this extracellular matrix. Two mAbs have been prepared that react on immunoblots with a approximately equal to 220- and a approximately equal to 340-kD polypeptide, respectively. These antibodies have been used to purify molecules with cross-shaped structures in the electron microscope. The molecules, of approximately equal to 10(3) kD on nonreducing SDS gels, have subunits of approximately equal to 340, 220, and 160-180 kD. Attachment to the laminin-like molecules was sufficient to initiate sprouting by single isolated leech neurons in defined medium. This demonstrates directly a function for a laminin-related invertebrate protein. The mAbs directed against the approximately equal to 220-kD chains of the laminin-like leech molecule labeled basement membrane extracellular matrix in leech ganglia and nerves. A polyclonal antiserum against the approximately equal to 220-kD polypeptide inhibited neurite outgrowth. Vertebrate laminin did not mediate the sprouting of leech neurons; similarly, the leech molecule was an inert substrate for vertebrate neurons. Although some traits of structure, function, and distribution are conserved between vertebrate laminin and the invertebrate molecule, our results suggest that the functional domains differ.     

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Summary Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproductbility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

Histochemical localization of IGF-I and IGF-II mRNA in the rat between birth and adulthood

We describe the postnatal ontogeny and localization of insulin-like growth factors I and II (IGF-I and -II) in the rat. We have used oligodeoxyribonucleotide probes for in situ hybridization (hybridization histochemistry) and for Northern blotting. IGF-II mRNA is strongly expressed in liver, skeletal muscle, perichondrium, leptomeninges and choroid plexus of the newborn. Demonstrable levels fall dramatically in the liver at 18-20 days postnatally but persist for longer periods in muscle and remain undiminished throughout life in the pia/choroid plexus, indicating that different control mechanisms operate in these tissues. IGF-I mRNA is predominantly found in the liver. Its level in this organ rises well before levels of IGF-II fall. This suggests that distinct factors govern the expression of IGF-I and -II genes.     

Field inversion gel electrophoresis in denaturing polyacrylamide gels.

The velocities of single stranded DNA molecules in denaturing polyacrylamide gels during symmetric and asymmetric field inversion were measured at different pulse times and gel concentrations. Under the conditions chosen in our study, pulse times as short as a few milliseconds lead to a retardation of DNA molecules larger than 400 bases. We found that a field inversion with an electric field in the forward direction of about double the strength of that applied in the backward direction is a good compromise between the degree of retardation, the temperature control requirements and the run time of the gel.     

Interaction of a cellular 57-kilodalton protein with the internal translation initiation site of foot-and-mouth disease virus.

A cellular 57-kDa protein (p57) that binds specifically to the internal translation initiation site in the 5' untranslated region of foot-and-mouth disease virus RNA was detected in cell extracts of different mammalian species by UV cross-linking. The protein binds to two distinct sites of the translation control region which have as the only common sequence a UUUC motif. The first binding site consists of a conserved hairpin structure, whereas the second binding site contains an essential pyrimidine-rich region without obvious secondary structure. Competition experiments indicate that the complexes with the two binding sites were formed by a single p57 species. The protein binds also to the 5' untranslated region of other picornaviruses. Results from footprint analyses with foot-and-mouth disease RNA suggest the participation of additional cellular factors in the translation initiation complex.     

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated

Summary The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 by encoding a protein of 2076 amino acids and 229980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS -subunit. The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3 end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 by and encodes a protein of 2051 amino acids and 228667 Da molecular weight.     

Potentiating effect of caffeine on the teratogenicity of acetazolamide in C57BL/6J mice

Pregnant C57BL/6J mice were treated with 0 or 50 mg of caffeine (CAFF) per kg, and 0, 200 mg/kg (L) or 1,000 mg/kg (H) of acetazolamide (ACZM) during day 9 of gestation (9DPC). Individual fetuses were examined for gross morphological abnormalities and skeletal variations. The increase in fetal malformations seen, especially right forelimb electrodactyly, was augmented at both dose levels of acetazolamide by concomitant exposure to caffeine. Both frequency and severity of ectrodactyly were potentiated by caffeine. Skeletal examination revealed a reduction of the number of ossified cervical and caudal vertebral centra among litters exposed to ACZM at either dose. In either case (ACZM-H, ACZM-L) that effect was augmented by co-administration of CAFF. The first cervical vertebra (C1) appeared to provide the most sensitive index of teratogenic exposure. This study provides evidence that a subteratogenic dose of caffeine can potentiate the teratogenic effect of acetazolamide in C57BL/6J mice when dams are treated on day 9 of gestation. In addition, skeletal examination provided evidence that simultaneous treatment with both agents delayed fetal development. Many litters exposed to ACZM or both agents displayed a reduction in skeletal ossification even in the absence of gross morphological abnormalities, suggesting that ossification can be used as an indicator of prenatal exposure to potentially harmful substances in the C57BL/6 mouse strain.     

Identification of novel genes specifically expressed in Chlamydomonas reinhardtii zygotes

The maturation of zygotes formed by the fusion of two gametes is the essential part of the diploid phase of the Chlamydomonas reinhardtii sexual life cycle and results in mature zygotes competent to germinate. To understand the molecular mechanisms underlying zygote maturation and the attainment of competence for germination we isolated genomic clones representing three different genes that are specifically expressed in Chlamydomonas reinhardtii zygotes. Accumulation of the RNAs started more than 24 h after mating, setting these genes apart from genes expressed in young zygotes [9]. Upon light-induced germination of zygotes, the mRNAs disappeared. The patterns of RNA accumulation and disappearance were gene-specific and suggested a function of these genes in maturation and/or in initial steps of germination.     

Changes in soil microflora associated with control of Sclerotium Rolfsii by Furfuraldehyde

The efficacy of 2‐furfuraldehyde for control of Sclerotium rolfsii was studied in laboratory and greenhouse experiments. Mycelial growth of the fungus was reduced proportionally with concentrations of 0.1–0.5 ml furfuraldehyde l^‐1 agar medium, and viability of sclerotia diminished on exposure to 2‐furfuraldehyde vapours. Detectable populations of bacteria and fungi, including Trichoderma spp., were reduced significantly (9=0.05) when furfuraldehyde was added to the agar used for soil dilution plates of untreated soil. Repeated treatments of natural soil with the fumigant significantly increased populations of Trichoderma spp. and bacteria, but diminished numbers of actinomycetes. Increasing dosages applied to soil artificially infested with S. rolfsii caused a reduction of disease on lentil, Lens culinaris. Results indicate that the compound, when applied to field soil, changes the composition of soil microflora and has potential for integrated control of S. rolfsii.     

A Comparison of the Efficacy of Nuclear Polyhedrosis and Granulosis Viruses in Spray and Bait Formulations for the Control of Agrotis segetum (Lepidoptera: Noctuidae) in Maize

Agrotis segetum nuclear polyhedrosis virus (AsNPV) and granulosis virus (AsGV), propagated in laboratory cultures of A. segetum in England and A. ipsilon in Spain, respectively, were applied to plots of maize plants at the one‐ to four‐leaf stage of growth. Plots were arranged in a 6 x 6 Latin square design and infested with second‐instar A. segetum larvae (the common cutworm). Each virus was applied in separate treatments by two application methods; as an aqueous spray containing 0.1% Agral as a wetting agent, and as a bran bait. The NPV was applied at a rate of 4 X 10¹² polyhedra/ha, and the GV at 4 X 10¹³ granules/ha. Soil and plants were sampled for larvae on three occasions following virus treatment: 24 h, 4 days and 11 days. The larvae were reared on diet in the laboratory, until death or pupation, to examine the rate and level of viral infection. Infection data showed 87.5% and 91% NPV infection and 12.5% and 55% GV infection in spray and bait treatments, respectively, in larvae sampled 24 h after treatment. In larvae sampled 4 days after treatment, the results were 78% and 100% NPV infection, and 13% and 6% GV infection. A total of only six larvae were retrieved on day 11. In both treatments larvae infected with AsNPV died significantly more rapidly and at an earlier instar than those infected with AsGV, indicating that AsNPV appears to have better potential as a control agent for A. segetum.