BRYOPSIS MAXIMA (CHLOROPHYTA) GLUTAMATE DEHYDROGENASE: MULTIPLE GENES AND ISOZYMES

Subcellular localization of glutamate dehydrogenase (GDH) was investigated in the green alga Bryopsis maxima. Both intact and pure chloroplasts and mitochondria were isolated by two methods: successive centrifugation and continuous Percoll density gradient centrifugation. The NADP-dependent GDH activities of the chloroplastic, mitochondrial, and cytosolic portions were estimated as 64.3, 9.8, and 25.9%, respectively, and NAD-dependent GDH activity was observed only in the chloroplasts. Three organelle-specific isozymes—chloroplastic NADP-GDH1, cytosolic/mitochondrial NADP-GDH2, and cytosolic/mitochondrial NADP-GDH3—were purified. The molecular masses of these isozymes were estimated to be the same (280 kDa). K_m values of NADP-GDH1, NADP-GDH2, and NADP-GDH3 for NADPH in the amination reaction were 30, 110, and 34 μM, respectively, and those for NADH were 185, 1490, and 974 μM, respectively, showing different cofactor affinities. Several NADP-GDHs and one NAD-GDH were induced in the chloroplasts during incubation of the collected thalli in either continuous light or darkness in aerated seawater for 0 to 5 days, whereas the cytosolic and mitochondrial NADP-GDHs decreased to an almost undetectable level in 5 days. Two distinct DNA fragments (BmF-1 and BmF-2) encoding B. maxima Okamura GDH were identified and sequenced. They showed 90% homology in their deduced amino acid sequences, whereas synonymous nucleotide substitution was observed in the third position of 52% of the codons. Genomic Southern analysis suggested that the two genes are located at two different loci on the B. maxima chromosome. Thus, B. maxima GDH has been confirmed to be multiple in terms of both protein and gene. The localization of other nitrogen-assimilating enzymes was also determined. Glutamine synthetase was located in the chloroplasts and the cytosol, glutamate synthase was located in the chloroplasts, and nitrate reductase was located in the cytosol.     

Male-killing Wolbachia in the butterfly Hypolimnas bolina

Some lines of the butterfly Hypolimnas bolina L. (Lepidoptera: Nymphalidae) are characterized by their female‐biased sex ratio. In these lines, most males die before reaching the middle larval stage. However, the cause of the bias remains unclear. We detected the proteobacterium Wolbachia in all individuals in the female‐biased butterfly lines and in some of the lines with a normal sex ratio. Tetracycline treatment of adult females of a female‐biased line led to a significant increase in both the hatch rate of their eggs (F1) and the male‐to‐female ratio of F1 pupae. In addition, certain assays of tetracycline treatment on mother butterflies significantly increased the male to female ratio of F1 adults. Known bacterial sex ratio distorters other than Wolbachia were not detected by diagnostic PCR assay, nor by the sequencing of 16S rDNA amplified using general prokaryotic 16S rDNA primers. These results strongly suggest that the distortion of the sex ratio is due to the killing of males by the inherited Wolbachia. Sequences of the 16S rDNA amplified using Wolbachia‐specific primers, the cell division protein gene (ftsZ), the molecular chaperone groE genes (groE operon), and the Wolbachia surface protein gene (wsp) from Wolbachia in lines belonging to three subspecies of the butterfly (bolina, jacintha, and philippensis) revealed no variation among lines nor between female‐biased lines and a normal one.     

Elimination of Pasteurella pneumotropica from a contaminated mouse colony by oral administration of Enrofloxacin.

Enrofloxacin, a fluoroquinolone bactericidal antibiotic, was administered in an attempt to eradicate Pasteurella pneumotropica (P. pneumotropica) from a contaminated mouse colony. Contaminated mice, maintained within 4 animal rooms, were administered Enrofloxacin in drinking water at a daily dosage of 25.5 mg/kg for 2 weeks. Following one week of Enrofloxacin treatment, mice were selected randomly from each room and examined for P. pneumotropica. This procedure was repeated two or three times until all mice examined tested negative for the Pasteurella strain. With the exception of one room, treated mice consistently tested negative for P. pneumotropica for up to 45 weeks following completion of Enrofloxacin treatment. Thus, oral administration of Enrofloxacin significantly eliminated P. pneumotropica from a contaminated mouse colony.     

Differential effects of glycosphingolipids on protein kinase C activity in PC12D pheochromocytoma cells

Previous studies have shown that certain glycosphingolipids may function as modulators of protein kinase C (PKC) activity. To study the structure-activity relationship, we examined the effects of 17 gangliosides, 10 neutral glycolipids, as well as sulfatide, psychosine and ceramide on PKC activity in PC12D cells. Using an in vitro assay system, we found that all but one (GQ1b) ganglioside inhibited PKC activity at concentrations between 25 and 100 µM, and the potency was proportional to the number of sialic acid residues. However, at lower concentrations several gangliosides, including GM1 and LM1 behaved as mild activators of PKC activity. GQ1b had no effect within the range 0.1–10 µM, but acted as a mild activator of PKC activity at 25 µM. On the other hand, fucosyl-GM1 and GM1 containing blood group B determinant, which are abundant in PC12 cells, were potent inhibitors of PKC activity. Among the neutral glycosphingolipids tested, LacCer, Gb3, GalGb3, and GA1, all of which have a terminal galactose residue, were found to be ineffective or acted as mild activators of PKC activity. In contrast, GA2, Gb4 and Gb5 which have a terminal N-acetylgalactosamine residue, were potent inhibitors of the PKC activity. Thus, the terminal sugar residue may play a pivotal role in determining the effect of glycosphingolipids in modulating PKC activity. In addition, we also found that GalCer containing normal fatty acids acted as potent activators of PKC activity. Ceramide and GlcCer appeared to be ineffective in modulating PKC activity, whereas psychosine and sulfatides appeared to be inhibitory. We conclude that the carbohydrate head groups and the hydrophobic groups of gangliosides and neutral glycolipids may modulate the PKC system in unique manners, which may in turn affect various biological processes in the cell.     

Effect of lung-protective ventilation on severe Pseudomonas aeruginosa pneumonia and sepsis in rats

Pneumonia caused by Pseudomonas aeruginosa carries a high rate of morbidity and mortality. A lung-protective strategy using low tidal volume (V(T)) ventilation for acute lung injury improves patient outcomes. The goal of this study was to determine whether low V(T) ventilation has similar utility in severe P. aeruginosa infection. A cytotoxic P. aeruginosa strain, PA103, was instilled into the left lung of rats anesthetized with pentobarbital. The lung-protective effect of low V(T) (6 ml/kg) with or without high positive end-expiratory pressure (PEEP, 10 or 3 cmH(2)O) was then compared with high V(T) with low PEEP ventilation (V(T) 12 ml/kg, PEEP 3 cmH(2)O). Severe lung injury and septic shock was induced. Although ventilatory mode had little effect on the involved lung or septic physiology, injury to noninvolved regions was attenuated by low V(T) ventilation as indicated by the wet-to-dry weight ratio (W/D; 6.13 +/- 0.78 vs. 3.78 +/- 0.26, respectively) and confirmed by histopathological examinations. High PEEP did not yield a significant protective effect (W/D, 4.03 +/- 0.32) but, rather, caused overdistension of noninvolved lungs. Bronchoalveolar lavage revealed higher concentrations of TNF-alpha in the fluid of noninvolved lung undergoing high V(T) ventilation compared with those animals receiving low V(T). We conclude that low V(T) ventilation is protective in noninvolved regions and that the application of high PEEP attenuated the beneficial effects of low V(T) ventilation, at least short term. Furthermore, low V(T) ventilation cannot protect the involved lung, and high PEEP did not significantly alter lung injury over a short time course.     

EPR studies of 5-bromouracil crystal after irradiation with X rays in the bromine K-edge region

Radicals induced in a single crystal of 5-bromouracil (BrUra) by synchrotron soft X rays in the bromine K-edge region (13.461-13.482 keV) were investigated using the X-band EPR method. The crystal was irradiated at three peak energies of the absorption spectrum at room temperature or at 80 K. A hydrogen abstraction radical derived from N1 of the pyrimidine ring was commonly observed for all of the energies used, though with some variation in quantity. Similar characteristics were also observed in the EPR signal for the off-K-edge low-energy (13.42 keV) and (60)Co gamma rays used for comparison. When irradiated at 80 K, a much larger exposure (roughly 10 times) of soft X rays was needed to obtain the same signal intensity as that observed at room temperature. EPR signals were not detectable with gamma irradiation at liquid nitrogen temperature.     

Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-gamma. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1-/- MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1-/- MRL/lpr mice displayed significantly reduced IFN-gamma production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.     

Distinct E2F-mediated transcriptional program regulates p14ARF gene expression

The tumor suppressor p14(ARF) gene is induced by ectopically expressed E2F, a positive regulator of the cell cycle. The gene is expressed at low levels in normally growing cells in contrast to high levels in varieties of tumors. How p14(ARF) gene is regulated by E2F in normally growing cells and tumor cells remains obscure. Here we show that regulation of p14(ARF) gene by E2F is distinct from that of classical E2F targets. It is directly mediated by E2F through a novel E2F-responsive element that varies from the typical E2F site. The element responds to E2F activity resulting from ectopic E2F1 expression, inactivation of pRb by adenovirus E1a or shRNA, but not to phosphorylation of pRb by serum stimulation or ectopic cyclin D1/cyclin-dependent kinase-4 expression in normal human fibroblasts. The element has activity in various tumor cells with defective pRb, but not in normally growing cells. These results indicate that the distinct regulation constitutes the basis of p14(ARF) function as a tumor suppressor, discriminating abnormal growth signals caused by defects in pRb function from normal growth signals.