Purification and characterization of aquacobalamin reductase (NADPH) from Euglena gracilis

Euglena aquacobalamin reductase (NADPH: EC 1.6.99.-) was purified, and its subcellular distribution was studied to elucidate the mechanism of the conversion of hydroxocobalamin to 5'-deoxyadenosylcobalamin. The enzyme was found in the mitochondria. It was purified about 150-fold over the Euglena mitochondrial extract in a yield of 38%. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis. Spectra of the purified enzyme showed that it was a flavoprotein. The molecular weight of the enzyme was calculated to be 66,000 by Sephadex G-100 gel filtration and 65,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific to NADPH with an apparent Km of 43 microM and to hydroxocobalamin with an apparent Km of 55 microM. The enzyme did not require FAD or FMN as a cofactor. The optimum pH and temperature were 7.0 and 40 degrees C, respectively.     

Change of incidence of antinuclear antibodies in serum of NOD mouse with aging

Extractable nuclear antigens (ENA) were prepared from liver of C57BL/6J mouse and analyzed by SDS PAGE Western-immunoblotting techniques. Some protein components of the ENA, with molecular weights of 94 K, 65 K, 32 K, and 26 K, reacted with antinuclear antibodies in the sera of NOD mice. Incidence of antinuclear antibodies in the sera of NOD mice with aging were measured by ELISA method using the ENA as antigen. The antinuclear antibodies were not detected in young NOD mice (10 weeks old). However, the incidence increased with aging and reached 100% in the female NOD mice of 40 weeks. In the male NOD mice, the incidence of antinuclear antibodies was delayed and low in comparison with that in female.     

Detection of nuclear protein antigens to antinuclear antibodies in serum of NOD mouse

Nuclear protein antigens to the antinuclear antibodies in serum of non-obese diabetic (NOD) mice were investigated. In the serum of diabetic NOD female mice (20 weeks old), the antinuclear antibodies were detected by indirect immunofluorescence assay using frozen sections of liver of C 57 BL/6 J or NOD mice as antigen. Nuclei were separated from the liver of C 57 BL/6 J mice and solubilized. Solubilized nuclear antigens were analyzed by SDS PAGE-Western immunoblotting techniques. Nuclear protein antigens with molecular weights of 26,000, 32,000 and 65,000 showed strongly positive reactions with the antinuclear antibodies in the serum of the NOD mouse.     

Measurement of lactose concentrations in milk of Microtus montebelli with methylamine reaction

Concentrations of lactose in milk of Microtus montebelli were measured with the method using with the methylamine reaction. In this method, a small amount of sample (0.5 g) was sufficient for assay, and reproducibility and sensitivity were excellent. This method was very useful for measuring of lactose concentrations in experimental small animals. The average lactose concentration in milk of Microtus montebelli was 1.57g/100g, considerably low in comparison with the value of ICR mice (2.70g/100g). The low concentration of lactose in milk was considered to be one of metabolic characteristics of Microtus montebelli as a herbivorous animal.     

Site-directed insertion mutagenesis with cloned fragments in Escherichia coli by P1 phage transduction

Summary A cloned gene with an insertion, which was made by introducing cat, was ligated to the cloning site of the phage gt11. P1 phage grown on cells lysogenized with the recombinant phage could transduce the mutant gene into the original site on the Escherichia coli chromosome.     

Distribution of Glycinebetaine in Old and Young Leaf Blades of Salt-Stressed Barley Plants

In barley plants exposed to stepwise salt-stress (up to 200mM NaCl), sodium and chloride ions accumulated preferentiallyin old rather than in young leaf blades. Furthermore, the levelof glycinebetaine in young leaf blades was approximately threetimes that in old leaf blades. ³Present address: Department of Biochemistry, Faculty of Science,Kasesart University, Bangkok, 10900 Thailand     

Cloning of cDNA coding for dihydroflavonol-4-reductase (DFR) and characterization of dfr expression in the corollas of Gerbera hybrida var. Regina (Compositae)

We are approaching corolla differentiation in Compositae by studying the regulation of flavonoid pathway genes during inflorescence development in gerbera. We have cloned a dfr cDNA from a ray floret corolla cDNA library of Gerbera hybrida var. Regina by a PCR technique based on homologies found in genes isolated from other plant species. The functionality of the clone was tested in vivo by complementing the dihydrokaempferol accumulating petunia mutant line RL01. By Southern blot analysis, G. hybrida var. Regina was shown to harbour a small family of dfr genes, one member of which was deduced to be mainly responsible for the DFR activity in corolla. Dfr expression in corolla correlates with the anthocyanin accumulation pattern: it is basipetally induced, epidermally specific and restricted to the ligular part of corolla. By comparing the dfr expression in different floret types during inflorescence development, we could see that dfr expression reflects developmental schemes of the outermost ray and trans florets, contrasted with that of the disc florets.     

Carbon uptake in Eurasian boreal forests dominates the high-latitude net ecosystem carbon budget

Arctic-boreal landscapes are experiencing profound warming, along with changes in ecosystem moisture status and disturbance from fire. This region is of global importance in terms of carbon feedbacks to climate, yet the sign (sink or source) and magnitude of the Arctic-boreal carbon budget within recent years remains highly uncertain. Here, we provide new estimates of recent (2003–2015) vegetation gross primary productivity (GPP), ecosystem respiration (R_eco), net ecosystem CO₂ exchange (NEE; R_eco − GPP), and terrestrial methane (CH₄) emissions for the Arctic-boreal zone using a satellite data-driven process-model for northern ecosystems (TCFM-Arctic), calibrated and evaluated using measurements from >60 tower eddy covariance (EC) sites. We used TCFM-Arctic to obtain daily 1-km² flux estimates and annual carbon budgets for the pan-Arctic-boreal region. Across the domain, the model indicated an overall average NEE sink of −850 Tg CO₂-C year⁻¹. Eurasian boreal zones, especially those in Siberia, contributed to a majority of the net sink. In contrast, the tundra biome was relatively carbon neutral (ranging from small sink to source). Regional CH₄ emissions from tundra and boreal wetlands (not accounting for aquatic CH₄) were estimated at 35 Tg CH₄-C year⁻¹. Accounting for additional emissions from open water aquatic bodies and from fire, using available estimates from the literature, reduced the total regional NEE sink by 21% and shifted many far northern tundra landscapes, and some boreal forests, to a net carbon source. This assessment, based on in situ observations and models, improves our understanding of the high-latitude carbon status and also indicates a continued need for integrated site-to-regional assessments to monitor the vulnerability of these ecosystems to climate change.     

Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog.

Capsiate is a nonpungent capsaicin analog, a recently identified principle of the nonpungent red pepper cultivar CH-19 Sweet. In the present study, we report that 2-wk treatment of capsiate increased metabolic rate and promoted fat oxidation at rest, suggesting that capsiate may prevent obesity. To explain these effects, at least in part, we examined uncoupling proteins (UCPs) and thyroid hormones. UCPs and thyroid hormones play important roles in energy expenditure, the maintenance of body weight, and thermoregulation. Two-week treatment of capsiate increased the levels of UCP1 protein and mRNA in brown adipose tissue and UCP2 mRNA in white adipose tissue. This dose of capsiate did not change serum triiodothyronine or thyroxine levels. A single dose of capsiate temporarily raised both UCP1 mRNA in brown adipose tissue and UCP3 mRNA in skeletal muscle. These results suggest that UCP1 and UCP2 may contribute to the promotion of energy metabolism by capsiate, but that thyroid hormones do not.     

Affinity immunosensor for milk progesterone: identification of critical parameters

The effects of several experimental parameters on the performance characteristics of a competitive-type immunochromatographic assay of milk progesterone were studied. Increasing the size of the colloidal gold particles used as a label increased both maximal signal obtained and sensitivity of the assay measured as slope of the progesterone standard curve. The concentration of the antibody used to prepare the detection zone was found to be a critical factor, in that low concentrations of antibody resulted in a poor sensitivity. The compatibilities of various buffer systems with the assay were studied. The assay worked well with buffers having a broad pH range of 4·5–8·5.