Utilization of cephalothin by Yersinia enterocolitica as sole carbon and energy source

Two Yersinia enterocolitica strains were able to utilize the products of cephalothin degradation. The utilization of these products was shown by an increase of oxygen uptake by Y. enterocolitica with cephalothin as the only substrate, and by the growth of both strains with the hydrolysis products of cephalothin as sole energy and carbon sources. Nuclear magnetic resonance analysis of the cephalothin degradation reaction demonstrated the progressive disappearance of hydrolysis products. However, the products of benzylpenicillin degradation could not be utilized by Y. enterocolitica.     

Wounding Inhibits Protein Synthesis yet Stimulates Polysome Formation in Aged, Excised Pea Epicotyls

Wounding of aged, previously-excised pea epicotyl segments byremoval of the basal 1–2 mm resulted in a rapid (beginningwithin 15 min) recruitment of monosomes on to polysomes andan even more rapid (maximal between 6–12 min) inhibitionof protein synthesis in the remaining tissue. This inhibitionof protein synthesis in vivo did not appear to be an artefactcaused by the removal of highly active tissue (e.g., callus,contaminating bacteria), since wounds inflicted at a site distantfrom the region analyzed still elicited the response, and proteinsynthesis in the 1–2 mm slices (normally discarded) wasinhibited even more strongly than it was in the remaining tissue.The proportion of radioactive methionine in nascent chains (boundto polysomes) increased, while the production of completed polypeptidesdecreased, after wounding. Cycloheximide, a known inhibitorof the ribosome translocation/release process mimicked someof the effects of wounding. We interpret the results to indicatethat the initial effect of wounding is to inhibit translationby inhibiting the ribosome translocation/release process, whereasthe subsequent recovery in protein synthesis is brought aboutpartly by a recovery in ribosome translocation/release and partlyby enhanced initiation. ¹ Present address: Harvard-MIT Division of Health Science andTechnology, MIT, Cambridge, Massachusetts 02139, U.S.A. ² Present address: Institute of Agricultural Environment Control,College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama790, Japan. (Received May 26, 1986; Accepted August 4, 1986)     

Rapid Isolation of the 7S-Nerve Growth Factor Complex and Its Subunits from Murine Submaxillary Glands and Saliva

7S-Nerve growth factor (NGF) and its alpha, beta-NGF, and gamma subunits have been purified from murine submaxillary glands and saliva by a combination of gel filtration on rigid polyvinyl gels, reversed-phase liquid chromatography on short alkyl chain supports (C4 columns), and ion-exchange chromatography on silica-based carboxymethyl columns. This technique is superior to previously used methods in that it is much more rapid and allows the purification of larger quantities of polypeptide from the same amount of starting material. Beta-NGF prepared with this method elicits the outgrowth of fibers of cells of a pheochromocytoma cell line (PC 12) in vitro, indicating that the biological activity is not impaired by the organic solvents and strong acids utilized for its isolation.     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Phylogenetic Survey of Proteins Related to Synapsin I and Biochemical Analysis of Four Such Proteins from Fish Brain

A phylogenetic survey of proteins immunologically related to Synapsin I, a major synaptic vesicle-associated phosphoprotein in mammals was carried out. Proteins antigenically related to Synapsin I were found by use of radioimmunoassay and other radioimmunochemical techniques in the nervous systems of several vertebrate and invertebrate species, which included birds, reptiles, amphibians, fish, echinoderms, arthropods, and mollusks. Four proteins present in fish brain, antigenically related to Synapsin I, were further studied and found to resemble mammalian Synapsin I in several respects. Like Synapsin I, the fish proteins were present in high amounts in nervous tissue, were enriched in synaptosomal fractions of brain where they were substrates for endogenous protein kinases, were acid extractable, and were sensitive to digestion by collagenase. In addition, two-dimensional peptide-mapping analysis revealed some homology between major phosphopeptide fragments of Synapsin I and the fish proteins. The results indicate that proteins related to Synapsin I are wide-spread in the animal kingdom.     

A PIF-dependent recombinogenic signal in the mitochondrial DNA of yeast

From their recombination properties, tandem rho^- mutants of the mitochondrial genome of Saccharomyces cerevisiae were divided into two categories. In crosses between PIF-independent rho^- and rho⁺ strains, the recombination frequency is low and similar in PIF/pif and pif/pif diploids. In crosses between PIF-dependent rho^- and rho⁺ strains, the recombination frequency is stimulated 10-50 times in PIF/pif diploids and is drastically decreased in pif/pif diploids. These results suggest that a recombinogenic signal is present in the mitochondrial (mt) DNA of PIF-dependent rho^- clones. This signal is not recognized in pif mutants. Sequence analysis of a series of small (<300 bp) overlapping tandem rho^- genomes located in the ery region of the 21S rRNA gene led us to identify an essential element of this signal within a 41-bp A+T sequence exhibiting over 26 bp a perfect dyad symmetry. However the recombinogenic signal is not sequence-specific since the sequence described above does not characterize PIF-dependent rho^- clones located in the oli1 region. Our results rather suggest that the recombinogenic signal is related to the topology of rho^- DNA. Denaturated sites in the double helix or cruciform structures elicited by local negative supercoiling might be preferred sites of the initiation of recombination.     

Vasoactive intestinal peptide (VIP) binding to solubilized material from rat liver plasma membranes

A non-ionic detergent such as Lubrol-PX extracts in soluble form the VIP-binding structures of rat liver plasma membranes. Detergent-solubitized proteins bind specifically [¹²⁵I]VIP and the complex tracer-protein is identified by the use of Sepharose 6B columns. The interaction is only possible in the absence of detergent (below 0.001%) and is inhibited by native peptide. A molecular weight of about 80,000 was estimated for VIP-binding proteins by reference to a series of globular markers of proteins. Binding to VIP soluble proteins is specific and dependent on time as studied by the Hummel and Dreyer (Biochim. Biophys. Acta 63:530–532, 1962) assay.