Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus.

A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.     

Cleavage of growth hormone by rabbit liver plasmalemma enhances binding

Several studies have shown that proteolytic cleavage can enhance the biological activity of the growth hormone (GH) molecule. It seemed possible, therefore, that proteolytic modification of GH might be a normal function of GH-target tissues. Plasmalemma-enriched fractions isolated from rabbit liver were found to contain a proteinase(s) which cleaves the large disulfide loop of human and rat GH. The proteolytic activity was specific to plasmalemma-enriched fractions in that much lower activities were observed in microsomal-enriched fractions prepared from the same livers. The plasmalemmal proteinase(s) may be a trypsin-like enzyme because proteolytic activity was decreased by two serine proteinase inhibitors. Inhibition by unlabeled human GH of 125I-GH binding to receptors did not prevent cleavage of the tracer; therefore, hormone-receptor interaction was not required for cleavage of the GH molecule. In binding studies, cleaved GH associated more readily than did intact hormone with rabbit liver receptors. These studies suggest that plasmalemma-enriched fractions prepared from rabbit liver contain a proteinase which cleaves the GH molecule in a highly specific manner. Moreover, it is unlikely that inactivation of GH is the function of this limited proteolysis because cleaved hormone is bound preferentially by at least a subset of receptors in rabbit liver.     

Isolation and sequence of a cDNA clone that contains the entire coding region for chicken smooth-muscle alpha-tropomyosin

We have constructed a cDNA-expression library of approximately 100,000 members from embryonic chicken smooth-muscle mRNA using the plasmid-expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, we have identified and isolated clones encoding smooth-muscle tropomyosin. Plasmid pSMT-10 (approximately 1100 base pairs) was found to hybrid-select mRNA for smooth-muscle alpha-tropomyosin. DNA-sequence analysis revealed that pSMT-10 contained the entire coding region for alpha-tropomyosin and portions of the 5'- and 3'-untranslated regions. Comparison of the derived amino acid sequence of smooth-muscle alpha-tropomyosin with known skeletal-muscle (rabbit and chicken) and platelet (equine) sequences revealed extensive homology between the various proteins. The smooth-muscle tropomyosin shows the greatest sequence divergence from the skeletal-muscle tropomyosins at the COOH-terminal region. In contrast, the smooth-muscle tropomyosin is most homologous to the platelet tropomyosin at the COOH-terminal end. The relationship of the various tropomyosin sequences to function (e.g. interactions with troponin) are considered.     

Abscisic acid induction of cloned cotton late embryogenesis-abundant (Lea) mRNAs

Summary Earlier studies found that cotton (Gossypium hirsutum L.) cotyledons contain several mRNAs which are more abundant during late embryogenesis than in mid-embryogenesis or early germination. They are here termed Late embryogenesis-abundant mRNAs, encoded by Lea loci. Complementary DNA clones for 18 such mRNA sequences, defined at a hybridization criterion of T_m-15°C, were identified in a mature embryo cDNA library by differential cDNA hybridization. At a lower hybridization criterion, some sequence homology was found within several of these cloned Lea mRNA sequences. Each Lea mRNA sequence comprises 0.04–1.3% of mature embryo poly(A)⁺ mRNA, a level ten-fold to several hundred-fold higher than in young embryo or 24 h seedling poly(A)⁺ mRNA. Of 18 Lea mRNA sequences examined in cultured young embryos, the level of at least 13 are specifically increased by exogenous abscisic acid (ABA), several to a level near that in normal mature embryos. However, the abundance of several of the sequences does not appear to be significantly modulated by ABA. The LEA polypeptides encoded by 10 Lea mRNA sequences were identified by hybrid-arrested translation. They include most of the late embryogenesis-abundant, ABA-inducible, polypeptides previously identified. Preliminary results suggest that many of the individual Lea mRNA sequences are transcribed from 1–3 genes in each of cotton's two subgenomes.     

Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading

We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of soil water status on critical phosphorus concentrations inStylosanthes hamata cv. Verano

Summary The effect of soil water status on the critical phosphorus concentration (CPC) determined in apices and whole tops ofStylosanthes hamata cv. Verano was investigated in a glasshouse trial. The species was grown with six rates of P and three ranges of soil water potential and was harvested at 10 and 14 weeks after germination. The CPC of both whole tops and apices decliced between the two harvests. At the first harvest the CPC of both whole tops and apices increased as the soil water potential decreased but at the second harvest there was no effect of soil water potential on CPC. It is suggested that at the earlier harvest water stress was delaying physiological development, resulting in a CPC characteristic of chronologically younger tissue, but that by the second harvest the decline in CPC with age had ceased for all water treatments.     

Comparison of the time-mortality response of Heliothis zea to 14 isolates of Heliothis nuclear polyhedrosis virus

Twelve singly embedded isolates (SEV) and two multiply embedded isolates (MEV) of nuclear polyhedrosis viruses from Heliothis larvae were compared by time-mortality assays in neonate H. zea larvae. The isolates could be separated into six groups based on differences in the 50% survival time (ST₅₀) values. Isolates with identical restriction endonuclease (REN) profiles did not differ significantly in their ST₅₀ values, whereas isolates with several different REN cleavage sites also had significantly different ST₅₀ values. With the exception of one isolate from India, the singly embedded isolates acted faster than the multiply embedded isolates.     

Monoclonal antibodies to leucine enkephalin

Monoclonal antibodies to leucine enkephalin have been produced after fusion of mouse myeloma cells with spleen cells from hyper-immune mice. Hybrid clones 2D1 and SL1 were characterised using radioimmunoassay and an enzyme-linked immunosorbent assay. The antibody 2D1 was of low affinity and showed a maximum sensitivity of 0.1ng. The antibody binds equally well to the sulphated leucine enkephalin and to methionine enkephalin. It does not cross-react with dynorphin, methionine enkephalin-arg-phe or oxidised methionine enkephalin. The hybrid clone SL1 appears to be specific for leucine enkephalin. Preliminary immunocytochemical studies have shown that both antibodies bind specifically to leucine enkephalin in defined areas of the central nervous system.