Direct evidence for two distinct forms of the flavoprotein subunit of human mitochondrial complex II (succinate-ubiquinone reductase)

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in both the tricarboxylic acid cycle and aerobic respiration. A recent study showed that defects in human complex II are associated with cancers as well as mitochondrial diseases. Mutations in the four subunits of human complex II are associated with a wide spectrum of clinical presentations. Such tissue-specific clinical symptoms suggest the presence of multiple isoforms of the subunits, but subunit isoforms have not been previously reported. In the present study, we identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual, and demonstrated expression of these two isoforms in skeletal muscle, liver, brain, heart and kidney. Interestingly, one of the Fp isoforms was encoded as an intronless gene.     

Copper and zinc fractions affecting microorganisms in long-term sludge-amended soils

The influences of Zn and Cu on soil enzyme activities (acid phosphatase, alkaline phosphatase, arylsulfatase, cellulase, dehydrogenase, protease (z-FLase), urease, beta-D-glucosidase and beta-D-fructofuranosidase (invertase)) and microbial biomass carbon were investigated in agricultural soils amended with municipal sewage sludge or compost since 1978. The trace metals in the soils were fractionated using a sequential extraction method. Long-term application of the sewage sludge and composts caused accumulations of Cu and Zn in the soils, ranging from 140 to 144 and from 216 to 292 mg kg(-1), respectively. The percentage of Cu was highest in the NaOH- and HNO3-extractable fractions (44-51% and 38-46%, respectively), while the percentage of Zn was highest in the HNO3- and EDTA-extractable fractions (65-83% and 11-32%, respectively). Although the percentage of the bioavailable fractions (sum of KNO3 + H2O-, NaOH-, and EDTA-extractable amounts) of Cu (53-64%) was higher than that of Zn (15-37%), the percentage of the most labile fractions (KNO3 + H2O) of Zn (2.1-5.9%) was larger than that of Cu (1.1-2.4%). The size of the microbial biomass carbon increased with the application of sewage sludge or compost. For some enzymes, however, the ratio of the enzyme activity to microbial biomass was lower in the soils amended with sewage sludge or compost than that in the control soil. The soil enzyme activities were more adversely affected by Zn than by Cu. From a multiple regression analysis, it was found that dehydrogenase, urease, and beta-D-glucosidase activities were reduced by the KNO3 + H2O-extractable fraction of Zn in the soils. These microbial activities seem to be sensitive to Zn stress, indicating the possibility that they might be useful bioindicators for evaluation of the toxic effects of Zn on microorganisms in the soils.     

Phylogenetic utility of mitochondrial COI and nuclear Gpdh genes in Drosophila

Phylogenetic utility of the mitochondrial COI (cytochrome oxidase subunit I) and nuclear Gpdh (glycerol-3-phosphate dehydrogenase) genes was studied in the Drosophila melanogaster species group. The rate of substitution was higher in the COI gene than in the Gpdh gene. In addition, multiple substitutions, not only for transitional but also for transversional substitutions, occurred faster in the COI gene. None of the trees obtained using the COI gene supported the well-established monophyly of the ananassae subgroup. In addition, the incongruence length difference test, Templeton test, and partitioned Bremer support revealed that the trees based on the COI data are considerably different from those based on the Gpdh and the combined data set. Thus, the COI gene did not show good phylogenetic performance in the melanogaster group. The present analyses based on the Gpdh gene and the combined data set revealed that the ananassae subgroup branched off first in the melanogaster group followed by the montium subgroup and further by the melanogaster subgroup in contrast to the most recent phylogenetic hypothesis based on Amy multigenes.     

Control of life, death, and differentiation in cultured midgut cells of the lepidopteran, Heliothis virescens

Summary Differentiated cells in the insect midgut depend on stem cells for renewal. We have immunologically identified Integrin β₁, a promotor of cell-cell adhesion that also induces signals mediating proliferation, differentiation, and apoptosis on the surfaces of culturedHeliothis virescens midgut cells; clusters of immunostained integrin β₁-like material, indicative of activated integrin, were detected on aggregating midgut columnar cells. Growth factor-like peptides (midgut differentiation factors 1 and 2 [MDF1 and MDF2]), isolated from conditioned medium containingManduca sexta midgut cells, may be representative of endogenous midgut signaling molecules. Exposing the cultured midgut cells toBacillus thuringiensis (Bt) toxin caused large numbers of mature differentiated cells to die, but the massive cell death simultaneously induced a 150–200% increase in the numbers of midgut stem and differentiating cells. However, after the toxin was washed out, the proportions of cell types returned to near-control levels within 2 d, indicating endogenous control of cell-population dynamics. MDF1 was detected immunologically in larger numbers of Bt-treated columnar cells than controls, confirming its role in inducing the differentiation of rapidly produced stem cells. However, other insect midgut factors regulating increased proliferation, differentiation, as well as inhibition of proliferation and adjustment of the ratio of cell types, remain to be discovered. Products mentioned in this article are not endorsed by the U.S. Department of Agriculture.     

Anti-inflammatory activity of a novel selective cyclooxygenase-2 inhibitor, FR140423, on type II collagen-induced arthritis in Lewis rats

The mechanism of action of FR140423 (3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)-phenyl]pyrazole), a novel and selective cyclooxygenase (COX)-2 inhibitor, in rat type II collagen-induced arthritis was investigated and compared with that of indomethacin. We tested the inhibitory effects of FR140423 on paw edema and the formation of arachidonic acid metabolites in inflamed paws immunized with type II collagen. Oral administration of FR 140423 showed a dose-dependent anti-inflammatory effect and was two-fold more potent than indomethacin. The increase of prostaglandin (PG) E2 and thromboxane (TX) B2 but not leukotriene B4 in inflamed paws was associated with the development of paw edema. FR140423 and indomethacin dose-dependently suppressed the levels of PGE2 and TXB2 in arthritic rat paws. Unlike indomethacin, FR140423 did not induce gastric lesions in arthritic rats. These results suggest that FR140423 shows a potent anti-inflammatory effect mediated by inhibition of prostanoids produced by COX-2 in inflamed tissues immunized with type II collagen, with a greatly improved safety profile compared to indomethacin.     

A novel gene that is up-regulated during recovery from cold shock in Drosophila melanogaster

Gene expression during recovery at 25°C (rearing temperature) after cold shock (0°C) was studied in Drosophila melanogaster using a subtraction technique. A novel gene (Frost, abbreviated as Fst) was considerably up-regulated during recovery after cold shock. In addition, a prolongation of cold shock was more effective for induction. In contrast to cold shock, Fst gene did not respond to heat shock. This gene is apparently the same as the unidentified gene, CG9434. Fst has high internal repeats not only in nucleotide but also in amino acid sequences. In addition, FST protein has a proline-rich region. The deduced amino acid sequence revealed a modular structure; i.e., a signal peptide in the N-terminal region followed by a long hydrophilic region. Therefore, this protein is likely to be directed into ER and secreted into extracellular space.     

Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity

Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.     

Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.     

Effects of recombinant cholera toxin B subunit on IL-1beta production by macrophages in vitro

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.