In vitro protein degradation measured by differential loss of labeled methionine and 3-methylhistidine: The effect of insulin

A new technique for studying the effect of insulin on protein degradation is reported. The method is based on measuring the parallel release of a reutilizable and a nonreutilizable amino acid from muscle protein. Animals are prelabeled in vivo with [Me-³H]methionine which labels both the nonreutilizable 3-methylhistidine and the reutilizable methionine of tissue protein. The data presented show that insulin has only a trivial effect on the loss of 3-methylhistidine from muscle protein, while it substantially diminishes the efflux of methionine. The analysis of muscle protein confirms the observation that insulin causes the reincorporation of methionine and has a minimum effect on the loss of 3-methylhistidine. This supports the view that the major inhibitory effect of insulin on gluconeogenesis is the diversion of the flow of amino acids away from the gluconeogenesis pathway back toward protein synthesis.     

A new streptomycete and a new polyene antibiotic,acmycin

A new antifungal antibiotic named acmycin was isolated from a soil streptomycete. Detailed comparative taxonomic studies showed that the organism differed from three related species of streptomycetes. The organism was referred to asStreptomyces sp. AC₂. The isolated antibiotic appears to be of polyene nature.     

Collagenolytic activity of a soil actinomycete

A soil actinomycete hydrolyzed collagen extracted from bovine Achilles tendon, calf skin, carp swim-bladder and rat tail tendon. Glucose, mannose, aspartic acid and asparagine increased its collagenolytic activity which was optimum at 28 °C and at pH 7.2 – 7.5. Metallic ions, NaEDTA, cysteine, 4-chloromercuribenzoate, glutathione and sodium azide were inhibitory.     

On the Nature of the Interaction Between Serotonin and Serotonin Binding Protein: Effect of Nucleotides, Ions, and Sulfhydryl Reagents

Abstract: Rat brain serotonin binding protein (SBP) was found to have essential -S-S and -SH groups. Both reduction of the disulfide bond by dithiothreitol or mercaptoethanol and modification of -SH group(s) by Ellman reagent or alkylating agents caused loss of binding capacity. In contrast, formation of a mixed disulfide bond with sodium metabisulfite did not affect the binding capacity. Serotonin in the presence of Fe²⁺ and phosphate was found to bind to either an -SH group or to a site in very close proximity. Addition of serotonin protected -SH groups from modification by Ellman reagent and from denaturation of protein upon storage. Lipids that enhance binding of serotonin to SBP also protected -SH groups from modification. Nucleotides were found to be strong inhibitors of the binding of serotonin to SBP. The inhibitory effect of nucleotides was due to their chelating properties and not to their ability to phosphorylate the protein or to bind directly to it. Inhibition by nucleotides and other chelators was reversible. Binding capacity was fully restored after removal of the chelator by molecular sieve chromatography and addition of Fe²⁺. The ionic environment had a marked effect on the binding: intracellular ions such as K⁺ were found to enhance the binding, and extracellular ions such as Na⁺ and Ca²⁺ inhibited the binding. Based on these data and our previous studies, we suggest that SBP is an intracellular protein that acts as a storage protein. Consistent with our data is formation of a complex of SBP-S-Fe-S that in a hydrophobic surrounding could bind up to four molecules of serotonin in coordination bond with Fe²⁺ and thereby reduce the osmotic pressure within a storage vesicle. Extracellular ionic conditions that favor the dissociation of the complex would free the amine to interact with its receptor or the presynaptic reuptake carrier.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

Dryinid parasitoids of rice leafhoppers and planthoppers in the Philippines II. Rearing techniques

Three new techniques of rearing dryinids parasitising rice hoppers were developed, namely, laboratory rearing technique for detailed observations, device for transporting and rearing field-collected hoppers to estimate percentage parasitism and a breeding technique.

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Résumé Trois nouvelles techniques d’élevage de dryiinides parasites des cicadelles du riz ont été mises au point. L’une pour l’élevage en laboratoire en vue d’observations précises, un système pour le transport et l’élevage de cicadelles récoltées dans la nature afin d’évaluer le pourcentage de parasitisme et une technique d’élevage permanent.

     

Morphology,serology and biochemical characters of phage CVX-5, isolated from a patient with colitis

Electronmicroscopic observations indicate that bacteriophage CVX-5 has an angular head with long spiral tail which is noncontractile, possibly having 2--3 tail fibres attached at the distal part of the tail. This phage is antigenically unrelated to any of the T-phages. Inhibition of phage CVX-5 multiplication by mitomycin C and incorporation of 3H-thymidine into this phage indicate that phage CVX-5 is a DNA phage.