Changes in aerobic and anaerobic ATP-synthesizing activities in hypoxic mouse brain

The changes in cerebral metabolism in mice in severe hypoxia were investigated by analyses of changes in the levels of energy metabolites and near-infrared spectrophotometric assessment of the states of hemoglobin and cytochrome oxidase. Under 4.4% O2, the contribution of anaerobic ATP production was at most about 20% of the demand. However, the cerebral ATP level was kept at the control level until about 1 min before death. Pentobarbital anesthesia, which reduced the cerebral rate of metabolism, prolonged the survival time, although anaerobic ATP production still did not support ATP demand. Under these conditions, deoxygenation of hemoglobin and reduction of cytochrome oxidase proceeded rapidly within 1 min. Hemoglobin reached the maximum state of deoxygenation in the middle phase of hypoxia, with no further change until death. However, cytochrome oxidase was reduced slowly with one phase of partial reoxidation due to increase of cerebral blood volume, and reached the completely reduced state at death. From these results it was concluded that the aerobic ATP synthesis, which supplied more than 80% of the cerebral demand, decreased gradually because of limitation of oxygen supply, and that the failure of oxidative phosphorylation to meet demand triggered the decrease in the cellular ATP level that led to death.     

Oxidative stress is associated with aluminum toxicity recovery in apex of pea root

Aims

Although many studies on the mechanism of Al toxicity and tolerance have been conducted independently, events occurring during the recovery process from Al injury is limited. This study was to investigate Al toxicity recovery mechanism focusing in morphological and physiological aspect.

Methods

We investigated the mechanisms underlying Al toxicity recovery in terms of oxidative stress using the pea root apex as a model system.

Results

The accumulation of reactive oxygen species was remarkably high in the root under continued Al treatment but decreased in the recovering root. The superoxide anion exuded in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) showed a similar tendency with respect to the accumulation of reactive oxygen species. A similar pattern of lignin content and superoxide dismutase activity was observed among the treatments, while the increased peroxidation in the root under continued Al treatment did not decline with recovery treatment. A longitudinal section of the root under continued Al treatment showed the accumulation of superoxide anion, lignin and peroxide (H₂O₂) at the epidermal and outer cortex region where the Al induced injuries, including ruptures, are detected.

Conclusions

Oxidative stress is associated with the mechanism of Al toxicity recovery. The recovery process might include the elongation of the central cylinder as a consequence of the oxidative stress-induced formation of the zonal region (ZR). The results further suggest a plausible role for the ZR in the programmed cell death-like function involved in Al toxicity recovery.     

Deficiency of inducible NO synthase reduces advanced but not early atherosclerosis in apolipoprotein E-deficient mice

The inducible nitric oxide synthase (iNOS) is abundantly expressed by smooth muscle cells and macrophages in atherosclerotic lesions. Apolipoprotein E-deficient (apoE(-/-)) mice develop early and advanced atherosclerotic lesions. The role of iNOS in both early and advanced atherosclerotic formation was determined in apoE(-/-) mice. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 12 weeks of age on chow diet, iNOS(-/-)/apoE(-/-) mice developed comparable sizes of early atherosclerotic lesions in the aortic root as did iNOS(+/+)/apoE(-/-) mice (30,993+/-4746 vs. 26,648+/-6815 microm(2)/section; P=0.608). After being fed the Western diet for 12 weeks, iNOS(-/-)/apoE(-/-) mice developed significantly smaller advanced lesions than iNOS(+/+)/apoE(-/-) mice (458,734+/-14,942 vs. 519,570+/-22,098 microm(2)/section; P=0.029). This reduction in lesion formation could not be explained by differences in plasma lipid levels. To examine whether iNOS contributed to LDL oxidation, smooth muscle cells were isolated from the aorta, activated with TNF-alpha, and then incubated with native LDL in the absence or presence of N-Omega-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by smooth muscle cells from iNOS(+/+)/apoE(-/-) mice (P=0.048), but it had no effect on LDL oxidation by cells from iNOS(-/-)/apoE(-/-) mice. iNOS(-/-)/apoE(-/-) mice had a significantly lower plasma lipoperoxide level on the Western diet (2.74+/-0.23 vs. 3.89+/-0.41 microM MDA; P=0.021) but not on chow diet (1.02+/-0.07 vs. 1.51+/-0.29 microM MDA; P=0.11). Thus, the absence of iNOS-mediated LDL oxidation may contribute to the reduction in advanced lesion formation of iNOS(-/-)/apoE(-/-) mice.     

24β-Ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)-dehydrolophenol in seeds of three cucurbitaceae species

The structures of two 4α-methylsterols is isolated from Cucumis sativus(Cucurbitaceae) seeds were determined based mainly on their ¹³CNMR spectra as 24β-ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)- dehydrolophenol, respectively, of which the former is a new sterol from natural sources. These two 4α-methylsterols were identified in the seeds of two other Cucurbitaceae species, Lagenaria leucantha var. Gourda and Citrullus battich. The probable biogenetic significance of the two 4α-methylsterols is discussed. Other 4α-methylsterols identified in the seeds of the three Cucurbitaceae species were obtusifoliol, cycloeucalenol and gramisterol.     

Glycolipid composition of ascitic fluids from patients with cancer

The glycolipid composition of ascitic fluids from nine patients with cancer and one pleural effusion from a hepatoma patient was studied. Glucosylceramide, lactosylceramide, globotriaosylceramide, and globotetraosylceramide were found in all samples and also in normal human serum. These glycolipids accounted for more than 90% of the neutral glycolipid fraction and the composition in ascitic fluids was similar to that in normal human serum. From ascitic fluids, several minor glycolipids, which could not be detected in normal human serum, were isolated and characterized by exoglycosidase treatment. Lactoneotetraosylceramide was found in eight samples of ascitic fluids, and globopentaosylceramide was detected in two samples from hepatoma and one from pancreatic cancer. A fucolipid which was converted to lactoneotetraosylceramide by alpha-L-fucosidase treatment was recognized in two samples from hepatoma patients. In the ganglioside fraction, GM3 was the predominant component both in normal human serum and in ascitic fluid. The GM2 content in ascitic fluids was much higher than that in normal human serum. From these results, lactoneotetraosylceramide and GM2 are possible candidates as cancer markers, because they seemed to be derived from cancer tissues by shedding.     

Heterologous production of horseradish peroxidase C1a by the basidiomycete yeast Cryptococcus sp. S-2 using codon and signal optimizations

In the present study, we attempted to improve the production of recombinant horseradish peroxidase C1a (HRP-C1a; a heme-binding protein) by Cryptococcus sp. S-2. Both native and codon-optimized HRP-C1a genes were expressed under the control of a high-level expression promoter. When the HRP-C1a gene with native codons was expressed, poly(A) tails tended to be added within the coding region, producing truncated messenger RNAs (mRNAs) that lacked the 3′ ends. Codon optimization prevented polyadenylation within the coding region and increased both the mRNA and protein levels of active HRP-C1a. To improve secretion of the recombinant protein, we tested five types of N-terminal signal peptide (NTP). These included the native HRP-C1a NTP (C1a-NTP), short and long xylanase secretion signals (X1-NTP and X2-NTP), cutinase signal (C-NTP), and amylase signal (A-NTP), with and without a C-terminal propeptide (CTP). X2-NTP without CTP resulted in the highest HRP-C1a secretion into the culture medium. HRP-C1a secretion was further increased by using xylose fed-batch fermentation. The production of HRP-C1a in this study was 2.7 and 15 times higher than the production reported in previous studies that used insect cell and Pichia expression systems, respectively.     

The micronucleus assay with peripheral blood reticulocytes by acridine orange supravital staining with 1-beta-D-arabinofuranosylcytosine.

Dose-dependent induction of micronuclei with 1-beta-D-arabinofuranosylcytosine (ara-C) was clearly shown in CD-1 mouse peripheral blood reticulocytes (RETs) using an acridine orange (AO) supravital staining method, as well as in the conventional bone marrow assay. The maximum frequencies of micronucleated RETs (MNRETs) in peripheral blood and of micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow were comparable, as shown in two laboratories independently. The maximum frequencies of MNRETs in peripheral blood lagged about 24 and 12 h behind those of MNPCEs in bone marrow in experiments with 24- and 12-h sampling intervals, respectively. The proportion of each type of RET was examined periodically after treatment with ara-C at doses ranging from 6.25 to 50.0 mg/kg. The proportion of type I RETs among total RETs decreased 24 or 48 h after treatment according to the dose level. This suggest that this ratio could be a good indicator of the bone marrow cell toxicity of test chemicals.     

Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain

We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.