Kinetics of the reconstitution of hemoglobin from semihemoglobins α and β with heme

Kinetics of the reconstitution of hemoglobin from semihemoglobins and with hemin dicyanide have been investigated using three kinds of stopped-flow technique (Soret absorption, fluorescence quenching of tryptophan, and Soret CD). The semihemoglobins and are occupied by heme in the and chains, respectively, the other chain being heme-free. Based on the kinetic results, the following scheme for the reconstitution is proposed; First, hemin dicyanide enters the pocket-like site of the apo chains. Second, in semihemoglobin , the CN-ligand in the fifth coordination position of iron is replaced by the imidazole ring of the proximal His immediately after the heme insertion. In contrast, semihemoglobin changes its conformation after the heme insertion, and this is followed by the ligand replacement. Finally, the partial structure changes induced by the ligand replacement propagate onto the whole molecule and the final conformation is attained. The results indicate that semihemoglobin retains a more rigid and organized structure, and more closely approaches its final structure than does semihemoglobin . Correspondence to: Y. Kawamura-Konishi     

Long-term feeding on powdered food causes hyperglycemia and signs of systemic illness in mice

Aims

Dietary habits are crucial factors affecting metabolic homeostasis. However, few animal experiments have addressed the effects of long-term feeding with soft food on parameters reflecting systemic health.

Main methods

Using mice, we compared the effects of short (3 days) and long (17 weeks from weaning) feeding periods between powdered food and normal pellet food on the levels of blood glucose, serum levels of insulin, catecholamines, and corticosterone, blood pressure, and/or social interaction behaviors. In addition, the effects of a human glucagon-like peptide-1 analog, liraglutide (a new drug with protective effects against neuronal and cardiovascular diseases), were compared between the powder and pellet groups.

Key finding

(i) Powdered food, even for such a short period, resulted in a greater glycemic response than pellet food, consistent with powdered food being more easily digested and absorbed. (ii) Long-term feeding on powdered food induced hyperglycemia and related systemic signs of illness, including increases in serum adrenaline, noradrenaline, and corticosterone, higher blood pressures (especially diastolic), and increased social interaction behaviors. (iii) Liraglutide, when administered subcutaneously for the last 2 weeks of the 17-week period of feeding, improved these changes (including those in social interaction behaviors).

Significance

The hyperglycemia associated with long-term powdered-food feeding may lead to certain systemic illness signs, such as elevations of blood glucose, hypertension, and abnormal behaviors in mice. Mastication of food of adequate hardness may be very important for the maintenance of systemic (physical and mental) health, possibly via reduction in the levels of blood glucose and/or adrenal stress hormones (catecholamines and glucocorticoids).     

The effect of bacteria on interspecific relationships between the euryhaline rotifer Brachionus rotundiformis and the harpacticoid copepod Tigriopus japonicus

Inconsistent results have been obtained on the population growth of Brachionus rotundiformis and Tigriopus japonicus, when results from single-species and two-species mixed cultures are compared. Bacteria growth was not regulated in these experiments, which could be the cause for this. In order to test this possibility, we conducted similar experiments under axenic and synxenic (with presence of one species of bacteria) conditions. The population growth of B. rotundiformis was suppressed by the presence of T. japonicus in axenic cultures. T. japonicus could not persist in axenic cultures, but its population increased when grown in synxenic cultures. T. japonicus used RT bacteria strain as a food source, while these bacteria were toxic to B. rotundiformis. These results suggest that bacteria can modify the interspecific relationship between B. rotundiformis and T. japonicus.     

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A λgt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5′-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.     

Pterygodermatites nycticebi (Nematoda: Rictulariidae): accidental detection of encapsulated third-stage larvae in the tissue of a white-fronted marmoset

Twin, white-fronted marmosets (Callithrix geoffroyi) born and raised in a zoo in Japan died at 7 mo of age. Several encapsulated nematode larvae were detected in the intestinal wall, as well as a few in the mesenteric lymph nodes of 1 of the twins. In the other marmoset, no encapsulated nematode larva was detected in the organs, but many adult Pterygodermatites nycticebi were found in the intestinal lumen. In the past 5 yr, 5 primates kept in the same zoo, i.e., 1 squirrel monkey (Saimiri sciureus), 2 Pygmy marmosets (Cebuella pygmaea), 1 Senegal galago (Galago senegalensis), and 1 cotton-top tamarin (Saguinus oedipus), died from heavy infestation with the same nematode. A few migrating larvae of the rictulariid were also identified histologically in the intestinal wall and liver of the cotton-top tamarin. Although no other primate currently held in the same zoo was infected with the rictulariid, German cockroaches (Blattella germanica) collected with traps near marmoset cages had encapsulated P. nycticebi larvae, indicating latent perpetuation of the life cycle of this rictulariid species in the zoo premises. Our results indicated that encapsulation or migration of third-stage larvae of P. nycticebi might occur accidentally in the organs of callithrichid primates.     

A granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by carrageenin-induced inflammatory cells of mice

Seven days after subcutaneous injection of 2% carrageenin solution in mice, the induced inflammatory tissue was isolated and treated with 0.1% trypsin. When the dispersed cells were incubated in a nutrient medium containing 5--20% calf serum, the cells grew adhering to the culture-dish surface and colony-stimulating factor (CSF) was accumulated in the medium gradually. Addition of lipopolysaccharide (Escherichia coli endotoxin) in the cell culture did not enhance the CSF production. It was shown by isoelectrofocusing that the majority of the produced CSF was an acidic molecule (pI = 3.8), while the treatment of this CSF with neuraminidase yielded a less acidic CSF species (pI = 5.1). Upon gel-filtration chromatography in the presence of 6 M guanidine HCl, the CSF exhibited an apparent molecular weight of 42,000. On the other hand, polyacrylamide gel-electrophoresis in the presence of 0.1% sodium dodecyl sulfate gave a molecular weight estimate of 65,000. Microscopic examination of the bone marrow cell culture showed that about one-third of the colonies were granulocytic. Addition of prostaglandin E₁(PGE₁) in the bone marrow cell culture significantly inhibited the action of the CSF, while prostaglandin D₂ was less inhibitory than PGE₁. The result suggests that the cells isolated from the inflammatory tissue are capable of producing CSF, which may have a role for proliferation and maturation of the mononuclear phagocytes at the site of inflammation.     

The terminal oxidase of Photobacterium phosphoreum. A novel cytochrome

The terminal oxidase of Photobacterium phosphoreum has been purified to the electrophoretically homogeneous state and some of its properties have been studied.The enzyme catalyses oxidation of ascorbate in the presence of phenazine methosulphate or N,N,N′,N′-tetramethyl-p-phenylenediamine. The reaction is inhibited by cyanide. Nitrite at comparatively high concentrations inhibits the enzyme, but the enzyme does not catalyse nitrite reduction with ascorbate plus the electron mediator as the electron donor.The enzyme shows the absorption peaks at 632, 565, 534 and 436 nm in the reduced form. It has two kinds of haems: protohaem and haem d. Namely, the enzyme is a ‘cytochrome bd’-type oxidase; a novel cytochrome.     

Regulation of Lck and Fyn tyrosine kinase activities by transmembrane protein tyrosine phosphatase leukocyte common antigen-related molecule

Leukocyte common antigen-related molecule (LAR) is a receptor-like protein tyrosine phosphatase (PTPase) with two PTPase domains. In the present study, we detected the expression of LAR in the brain, kidney, and thymus of mice using anti-LAR PTPase domain subunit monoclonal antibody (mAb) YU1. In the thymus, LAR was expressed on CD4(-)CD8(-) and CD4(-)CD8(low) thymocytes. The development of thymocytes in CD45 knockout mice is blocked partially in the maturation of CD4(-)CD8(-) to CD4(+)CD8(+). We postulated that LAR regulates Lck and Fyn in the immature thymocytes. Transfection of wild-type LAR activated extracellular signal-regulated kinase signal transduction pathway in CD45-deficient Jurkat cells stimulated with anti-CD3 mAb. LAR mutants, with Cys to Ser mutation in the catalytic center of PTPase D1, bound to tyrosine-phosphorylated Lck and Fyn, and LAR PTPase domain 2 was tyrosine phosphorylated by Fyn tyrosine kinase. The phosphorylated LAR was associated with Fyn Src homology 2 domain. Moreover, LAR dephosphorylated phosphorylated tyrosine residues in both the COOH terminus and kinase domain of Fyn in vitro. Our results indicate that Lck and Fyn would be substrates of LAR in immature thymocytes and that each LAR PTPase domain plays distinct functional roles in phosphorylation and dephosphorylation.