Effects of chitin and its soluble derivatives on survival of Vibrio cholerae O1 at low temperature

Chitin concentrations greater than 0.04% (wt/wt) protected cholera vibrios against killing at low temperature. This protective effect was detected with both the soluble form of chitin, glycol chitin, and the insoluble particulate form of chitin. Some amino acids or peptides also showed the same protective effect.     

Regulation of redox states of plasma proteins by metabolism and transport of glutathione and related compounds

Glutathione is one of the most abundant naturally occurring thiols in living organisms and is synthesized in its reduced from (GSH). GSH has been known to play a fundamental role in cellular events in different cells and tissues, including protection of organisms against oxidative stress. The two peptide linkages of GSH are sequentially degraded by -glutamyltransferase and peptidases that hydrolyze the cysteinylglycine bond; all these enzymes are localized on the outer surface of cell membranes. The turnover of GSH in animals can be understood on the basis of the following three factors: (1) synthesis of GSH occurs exclusively intracellularly, while its degradation occurs predominantly extracellularly; (2) plasma membranes of many tissues and cells have secretory transport systems for GSH and its derivatives; (3) levels of the transferase, a key enzyme for GSH degradation, differ from one tissue to another. Thus, GSH released from tissues with low transferase activity (such as the liver) must be transferred for its rapid turnover to tissues with high enzyme activity (such as the kidney). Further studies on the states of thiol compounds transported via the circulation should be relevant to the understanding of the full scope and physiological significance of the interorgan cooperation of GSH metabolism. Many enzymes and proteins have free SH and disulfide groups within molecules. Function, stability, and in vivo fate of these macromolecules could be affected significantly by their redox state. Although cells and tissues have enzymic defense mechanisms against oxidative stress, the mechanism by which the homeostasis of the redox state of extracellular compartments (such as plasma, urine, bile, etc.) is maintained remains obscure. Plasma mercaptoalbumin (M-Alb) has 17 disulfide bonds and one free cysteinyl residue (Cys-34). This free thiol group can form mixed disulfides with low-molecular weight compounds, such as GSH and cysteine, to generate nonmercaptoalbumin (NM-Alb). Thus, when titrated by several different thiol reagents, less than 1 mole of free SH group (0.4–0.7) was usually detected per mole albumin. The ratio of M-Alb to NM-Alb in plasma samples varies significantly from one sample to another. Many plasma proteins in nonalbumin fractions also formed mixed disulfides with GSH and cysteine. The extent of mixed disulfide formation and the ratio of M-Alb to NM-Alb appeared to change markedly, depending on the redox state of the organisms. The present paper describes the mode of interorgan metabolism and transport of GSH and related compounds, the mechanism by which the redox state of albumin and other plasma proteins is controlled, and their biological significance in healthy and diseased conditions in normal and analbuminemic mutant rats.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B

Using synthetic leumorphin, we obtained antisera for leumorphin and set up two radioimmunoassays (RIAs) with different specificities. Gel exclusion chromatography coupled with the two RIAs showed the existence of a considerable amount of leumorphin-like peptide in water extracts from porcine neurointermediate pituitaries. Reverse phase high performance liquid chromatography revealed that leumorphin-like peptide in the water extracts was indistinguishable from synthetic leumorphin. These results along with potent opioid activity of leumorphin indicate that leumorphin is a novel endogenous opioid peptide derived from preproenkephalin B.     

Optical properties of lysozyme. pH and saccharide binding difference spectra.

Difference spectra associated with changes in pH and with binding of saccharides have been recorded for hen egg white (HEW) lysozyme, turkey egg white (TEW) lysozyme, and for the derivatives of the hen protein in which Tre-62 or Trp-108 had been oxidized specifically to oxindolealanine to give the Oxa-62 or Oxa-108-proteins. Identical pH difference spectra were obtained for HEW, TEW, and Oxa-62-lysozymes. Oxidation of Trp-108 is reflected in both the high and low pH (pH 7 versus 5 and pH 2 versus 5) difference spectra. The magnitude of the low pH difference spectrum is enhanced by binding of saccharide for HEW and Oxa-62-lysozymes but not for TEW lysozyme. The shapes and magnitudes of saccharide binding difference spectra are affected by oxidation of residues 62 or 108. These results can be interpreted in terms of the perturbations responsible for the lysozyme difference spectra. The pH 7 versus 5 difference spectrum results from perturbation by Glu-35 of Trp-108 and another tryptophan, probably Trp-63. Perturbation of Trp-108 and one or more other tryptophan residues by several carboxylate groups is responsible for the low pH difference spectra of the unliganded HEW and TEW lysozyme molecules. Perturbation of Trp-108 makes a principal contribution to the saccharide-binding difference spectrum. Perturbation of the Oxa-108 chromophore by ionization of Glu-35 or by saccharide binding produces absorbance changes in the 250 to 265 nm region.     

A simple one-step purification of human α1-proteinase inhibitor by immunoadsorbent column chromatography

A one-step purification of human α₁-proteinase inhibitor was described using the rabbit anti-α₁-proteinase inhibitor antibody coupled to CNBr-activated Sepharose 4B. The elution of α₁-proteinase inhibitor from the immunoadsorbent column using 0.1 M Na₂CO₃/0.5 M NaCl solution gave an 85% yield. The properties of eluted α₁-proteinase inhibitor were identical with that of α₁-proteinase inhibitor that was purified by the conventional method. In addition, the specific activity of purified α₁-proteinase inhibitor was more than 93% of that of the theoretical value.     

Micropropagation of ‘Yamatoimo’ Chinese yam (Dioscorea opposita) from immature leaves

A micropropagation system for Yamatoimo Chinese yam (Dioscorea opposita Thunb.) was developed. Immature leaves collected from virus-free plants growing in the greenhouse were cultured on MS medium supplemented with 8.9 M benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. After 2–3 months, multiple buds that were clumps of green-colored bulbous structures including adventitious buds and meristematic regions 2–3 mm in diameter were formed on immature leaves. Transplanting clusters of multiple buds to fresh MS medium supplemented with 0.11 M -naphthaleneacetic acid (NAA), 0.89 M BA and 6% (w/v) sucrose was effective for inducing shoot formation, leading to plantlet formation. After 6 months, a large number of microtubers, about 3–7 mm diameter, were obtained.Abbreviations NAA -naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Legionella pneumophila virulence conserved after multiple single-colony passage on agar

Multiple passage ofLegionella pneumophila on either supplemented Mueller-Hinton or charcoal yeast extract agar by the conventional batch passing technique results in loss of virulence. In this studyL. pneumophila virulence was maintained after multiple passage on buffered charcoal yeast extract (BCYE ) agar by a single-colony transfer technique. Virulence was determined by assessing the growth ofL. pneumophila for thioglycolate-induced susceptible A/J mouse macrophages in vitro and infectivity for susceptible A/J mice in vivo. Passage of the virulentL. pneumophila, 30 times on BCYE agar by the single-colony transfer procedure still resulted in virulence, as compared with the nonpassaged parental bacteria, both in vitro and in vivo. Lethality for susceptible, A/J mice by systemic infection was comparable for the 30th colony-passaged bacteria and the parentalL. pneumophila. These results show that theL. pneumophila phenotype associated with intracellular growth in macrophages or infectivity for susceptible mice is stable following passage by the single-colony transfer procedure on BCYE agar.     

Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons.

The effect of the fluorescent Ca2+ indicator dye Fura-2 on Ca2+ dynamics was studied in proximal apical dendrites of neocortical layer V and hippocampal CA1 pyramidal neurons in rat brain slices using somatic whole-cell recording and a charge-coupled device camera. A single action potential evoked a transient increase of intradendritic calcium concentration ([Ca2+]i) that was reduced in size and prolonged when the Fura-2 concentration was increased from 20 to 250 microM. Extrapolation to zero Fura-2 concentration suggests that "physiological" transients at 37 degrees C have large amplitudes (150-300 nM) and fast decays (time constant < 100 ms). Assuming a homogeneous compartment model for the dendrite, 0.5-1% of the total Ca2+ entering during an action potential was estimated to remain free. Washout of cytoplasmic Ca2+ buffers was not detectable, suggesting that they are relatively immobile. During trains of action potentials, [Ca2+]i increased and rapidly reached a steady state (time constant < 200 ms), fluctuating around a plateau level which depended linearly on the action potential frequency. Thus, the mean dendritic [Ca2+]i encodes the action potential frequency during physiological patterns of electrical activity and may regulate Ca(2+)-dependent dendritic functions in an activity-dependent way.     

Localization of a DNA topoisomerase II to mitochondria inDictyostelium discoideum: Deletion mutant analysis and mitochondrial targeting signal presequence

DNA topoisomerase II ofDictyostelium discoideum (TopA), the gene (topA) encoding which we cloned, was shown to have an additional N-terminal region which contains a putative mitochondrial targeting signal presequence. We constructed overexpression mutants which expressed the wild-type or the N-terminally deleted enzyme, and examined its localization by immunofluorescence microscopy and proteinase K digestion experiment. These experiments revealed that the enzyme is located in the mitochondria by virtue of the additional N-terminal region. Furthermore, in the cell extract depleted the enzyme by immunoprecipitation, nuclear DNA topoisomerase II activity was not decreased. These results confirmed that TopA is located in the mitochondria, even through its amino acid sequence is highly similar to those of nuclear type topoisomerase II of other organisms. Thus, this report is the first to establish the location of the mitochondrial targeting signal presequence in DNA topoisomerase II and in proteins ofD. discoideum directly by analyzing deletion mutants. Tsukuba Advanced Research Alliance (TARA researcher for the Sakabe project)