A Simple Colorimetrie Assay for Aspartate Aminotransferase

γ-Glutamylmethylamide (γ-GMA) synthetase was detected in crude extracts of Methylophaga sp. AA-30, but neither methylamine dehydrogenase nor N-methylglutamate dehydrogenase was observed. A large amount of γ-GMA was accumulated in the cells when the growth on methanol-methylamine was inhibited with iodoacetate, but the accumulation was not observed in the cells grown on methanol-(NH₄)₂SO₄. It is thought that γ-GMA is a metabolic intermediate of the methylamine-dissimilating pathway in the bacterium. In addition, γ-GMA-dissimilating enzymes were found in methylamine-grown cells. The enzymes, which consisted of H protein and L protein, required α-ketoglutaric acid, Mg²⁺ or Mn²⁺, and ammonia as a cofactor. Although the enzyme catalyzed the formation of glutamate from γ-GMA, it did not catalyze the formation of N-methylglutamate. Consequently, in this bacterium, methylamine seems to be metabolized through a different pathway from the N-methylglutamate pathway.     

Extra tyrosine in the carbohydrate-binding module of Irpex lacteus Xyn10B enhances its cellulose-binding ability

The xylanase (Xyn10B) that strongly adsorbs on microcrystalline cellulose was isolated from Driselase. The Xyn10B contains a Carbohydrate-binding module family 1 (CBM1) (IrpCBM_Xyn10B) at N-terminus. The canonical essential aromatic residues required for cellulose binding were conserved in IrpCBM_Xyn10B; however, its adsorption ability was markedly higher than that typically observed for the CBM1 of an endoglucanase from Trametes hirsuta (ThCBM_EG1). An analysis of the CBM-GFP fusion proteins revealed that the binding capacity to cellulose (7.8 μmol/g) and distribution coefficient (2.0 L/μmol) of IrpCBM_Xyn10B-GFP were twofold higher than those of ThCBM_EG1-GFP (3.4 μmol/g and 1.2 L/μmol, respectively), used as a reference structure. Besides the canonical aromatic residues (W24-Y50-Y51) of typical CBM1-containing proteins, IrpCBM_Xyn10B had an additional aromatic residue (Y52). The mutation of Y52 to Ser (IrpCBM_Y52S-GFP) reduced these adsorption parameters to 4.4 μmol/g and 1.5 L/μmol, which were similar to those of ThCBM_EG1-GFP. These results indicate that Y52 plays a crucial role in strong cellulose binding.     

The affinity of hemoglobin for oxygen affects ventilatory responses in mutant mice with Presbyterian hemoglobinopathy

The purpose of this study was to test whether chronically enhanced O2 delivery to tissues, without arterial hyperoxia, can change acute ventilatory responses to hypercapnia and hypoxia. The effects of decreased hemoglobin (Hb)-O2 affinity on ventilatory responses during hypercapnia (0, 5, 7, and 9% CO2 in O2) and hypoxia (10 and 15% O2 in N2) were assessed in mutant mice expressing Hb Presbyterian (mutation in the beta-globin gene, beta108 Asn --> Lys). O2 consumption during normoxia, measured via open-circuit methods, was significantly higher in the mutant mice than in wild-type mice. Respiratory measurements were conducted with a whole body, unrestrained, single-chamber plethysmograph under conscious conditions. During hypercapnia, there was no difference between the slopes of the hypercapnic ventilatory responses, whereas minute ventilation at the same levels of arterial PCO2 was lower in the Presbyterian mice than in the wild-type mice. During both hypoxic exposures, ventilatory responses were blunted in the mutant mice compared with responses in the wild-type mice. The effects of brief hyperoxia exposure (100% O2) after 10% hypoxia on ventilation were examined in anesthetized, spontaneously breathing mice with a double-chamber plethysmograph. No significant difference was found in ventilatory responses to brief hypoxia between both groups of mice, indicating possible involvement of central mechanisms in blunted ventilatory responses to hypoxia in Presbyterian mice. We conclude that chronically enhanced O2 delivery to peripheral tissues can reduce ventilation during acute hypercapnic and hypoxic exposures.     

Are there two forms of beta 2-N-acetylglucosaminyltransferase I in rat testicular and epididymal fluids?

The activity of alpha 3-D-mannoside-beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101), which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins, was detected in rat testicular and cauda epididymal fluids. The GnT I activity of testicular fluid had a pH optimum of 6.0, whereas that of the cauda epididymal fluid was optimal at pH 7.0. The enzyme in testicular fluid had an absolute requirement for either Co2+, or Mn2+, Mg2+ and Ca2+, the activity being stimulated by these cations in the above order, whereas that of cauda epididymal fluid had an absolute requirement for Mn2+ or Ca2+, with Co2+ and Mg2+ being ineffective. The specific activity of GnT I in cauda epididymal fluid was somewhat higher than in testicular fluid. The apparent Km value for alpha 1-3 alpha 1-6mannopentaose of GnT I in the testicular and epididymal fluids was 0.57 and 0.38 mM, respectively. The substrate specificity for both GnT I activities decreased in the following order: alpha1-3 alpha 1-6mannopentaose>alpha1-3 alpha 1-6mannotriose>alpha 1-3mannobiose>alpha 1-6mannobiose. These data suggest that two forms of GnT I exist in the testicular and epididymal fluids.     

Structure and physicochemical properties of starches from kidney bean seeds at immature,premature and mature stages of development

Starches from kidney bean (Phaseolus vulgaris L. cv. Toramame) seeds at the immature, premature, mature stages of development were examined. The starch content increased from 94, 219 to 265 mg per seed. Starches showed the C(a)-crystalline type composed of small (<5 micrometer) and large (10-35 micrometer) granules, with the large granules largely increasing with maturity. The amylose content increased from 21, 26 to 27%, and rapid viscograms and DSC thermograms suggested that the mature-stage starch was gelatinized with ease. The amylose increased in size from DPn 820, 1000 to 1080 and a number of chains per molecule (NC) from 3.3, 4.2 to 4.5. The branched amylose was a minor component (11-18% by mole) with NC 20-22. The amylopectin was similar in CL (23), beta-amylolysis limit (59%), and chain-length distribution, but reduced in size (DPn 17,100-5270) and increased in content of phosphorus (114-174 ppm) with an increase in the amount of phosphorus linked to C-6 of the glucose residue (8-66%).     

T cell hyporesponsiveness induced by oral administration of ovalbumin is associated with impaired NFAT nuclear translocation and p27kip1 degradation

Oral tolerance is an important physiological component of the immune system whereby the organism avoids dangerous reactions such as hypersensitivity to ingested food proteins and other luminal Ags which may cause tissue damage and inflammation. In addition, it has been shown in animal models and in humans that oral tolerance can be applied to controlling undesired immune responses, including autoimmune diseases, allergies, and organ transplant rejections. However, the molecular mechanisms of oral tolerance have been poorly defined. In this study, we investigated the molecular basis underlying the hyporesponsiveness of orally tolerant CD4 T cells using a TCR transgenic mouse system in which oral tolerance was induced by long-term feeding with high dose Ag. We demonstrate that the hyporesponsive state of the CD4 T cells was maintained by a selective impairment in the TCR-induced calcium/NFAT signaling pathway and in the IL-2R-induced degradation of p27(kip1) and cell cycle progression. Thus, physiological mucosal tolerance is revealed to be associated with a unique type of T cell hyporesponsiveness which differs from previously described anergic T cells.     

Ras activation in T cells determines the development of antigen-induced airway hyperresponsiveness and eosinophilic inflammation

The central role for Th2 cells in the development of Ag-induced airway hyperresponsiveness and eosinophilic inflammation is well documented. We have reported a crucial role for TCR-induced activation of the Ras/extracellular signal-regulated kinase mitogen-activated protein kinase cascade in Th2 cell differentiation. Here, we show that the development of both OVA-induced airway hyperresponsiveness and eosinophilic airway inflammation in a mouse asthma model are attenuated in transgenic mice by the overexpression of enzymatically inactive Ras molecules in T cells. In addition, reduced levels of IL-5 production and eosinophilic inflammation induced by nematode infection (Nippostrongylus brasiliensis or Heligmosomoides polygyrus) were detected. Thus, the level of Ras activation in T cells appears to determine Th2-dependent eosinophilic inflammation and Ag-induced airway hyperresponsiveness.