Long-Chain Aldehyde Dehydrogenase That Participates in n-Alkane Utilization and Wax Ester Synthesis in Acinetobacter sp. Strain M-1

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C₄ to C₁₄), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Δ) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Δ strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.     

Cross‐species functional complementation of cellulose synthase during the development of cellular slime molds

Cellulose is a major and important component of the extracellular matrix during the development of Dictyostelium discoideum. Upon starvation, solitary amoebae of D. discoideum gather and form fruiting bodies in which cells differentiate into stalk cells and spores. The stalk tubes and walls of spores and stalk cells are made of cellulose. In the genus Acytostelium, however, all cells are destined to become spores and the stalks comprise only a cellulose tube, suggesting species‐specific regulation of cellulose synthesis. In this study, we cloned a putative cellulose synthase gene (cesA) of Acytostelium subglobosum and performed comparative analyses with the D. discoideum cellulose synthase gene (dcsA). Although the deduced amino acid sequences were highly conserved between cesA and dcsA, the numbers of transmembrane spans preceding the catalytic domain were dissimilar; 2 and 3, respectively. Since ectopic expression of cesA in dcsA^?null cells failed to restore the developmental defects of the mutant, we constructed a series of chimerical genes for complementation analyses and found that the catalytic domain of cesA was functional in D. discoideum cells if the preceding transmembrane region was swapped with dcsA. The non‐functional products that contained the cesA‐derived transmembrane region were localized to lysosomes. These results indicate that the transmembrane region of cellulose synthase is essential for proper accumulation of cellulose during the development of D. discoideum and that its differential localization in A. subglobosum may be related to the characteristic morphogenesis in this species.     

Oscillator decomposition of infant fNIRS data

The functional near-infrared spectroscopy (fNIRS) can detect hemodynamic responses in the brain and the data consist of bivariate time series of oxygenated hemoglobin (oxy-Hb) and deoxygenated hemoglobin (deoxy-Hb) on each channel. In this study, we investigate oscillatory changes in infant fNIRS signals by using the oscillator decompisition method (OSC-DECOMP), which is a statistical method for extracting oscillators from time series data based on Gaussian linear state space models. OSC-DECOMP provides a natural decomposition of fNIRS data into oscillation components in a data-driven manner and does not require the arbitrary selection of band-pass filters. We analyzed 18-ch fNIRS data (3 minutes) acquired from 21 sleeping 3-month-old infants. Five to seven oscillators were extracted on most channels, and their frequency distribution had three peaks in the vicinity of 0.01-0.1 Hz, 1.6-2.4 Hz and 3.6-4.4 Hz. The first peak was considered to reflect hemodynamic changes in response to the brain activity, and the phase difference between oxy-Hb and deoxy-Hb for the associated oscillators was at approximately 230 degrees. The second peak was attributed to cardiac pulse waves and mirroring noise. Although these oscillators have close frequencies, OSC-DECOMP can separate them through estimating their different projection patterns on oxy-Hb and deoxy-Hb. The third peak was regarded as the harmonic of the second peak. By comparing the Akaike Information Criterion (AIC) of two state space models, we determined that the time series of oxy-Hb and deoxy-Hb on each channel originate from common oscillatory activity. We also utilized the result of OSC-DECOMP to investigate the frequency-specific functional connectivity. Whereas the brain oscillator exhibited functional connectivity, the pulse waves and mirroring noise oscillators showed spatially homogeneous and independent changes. OSC-DECOMP is a promising tool for data-driven extraction of oscillation components from biological time series data.     

NAD+-dependent xylitol dehydrogenase (xdhA) and l-arabitol-4-dehydrogenase (ladA) deletion mutants of Aspergillus oryzae for improved xylitol production

Xylitol dehydrogenase (XDHA) and l-arabitol dehydrogenase (LADA) are two key enzymes in xylan metabolism catalyzing the oxidation of xylitol to d-xylulose and arabitol to l-xylulose, respectively. In Aspergillus oryzae, XDHA and LADA are encoded by xdhA and ladA. We deleted xdhA and ladA and xdhA–ladA to generate mutants with decreased dehydrogenase activities and increased xylitol production. The mutants were constructed by homologous transformation into A. oryzae P4 (?pyrG) using pyrG as a selectable marker. The xylitol productivity of the mutants was measured using d-xylose as the sole carbohydrate source. xdhA, ladA, and the double-deletion mutant produced, respectively, 12.4 g xylitol/l with a yield of 0.24 g/g d-xylose, 12.4 g/l with a yield of 0.33 g/g d-xylose, and 8.6 g/l at a yield of 0.26 g/g d-xylose.     

Genetic analysis of the Neurospora crassa RAD14 homolog mus-43 and the RAD10 homolog mus-44 reveals that they belong to the mus-38 pathway of two nucleotide excision repair systems

Saccharomyces cerevisiae Rad14 and Rad10 proteins are essential for nucleotide excision repair (NER). Rad14 is a UV-damaged DNA binding protein and Rad10 is a structure-specific endonuclease that functions in a complex with Rad1. In this study, we identified and characterized the RAD14 and RAD10 homolog genes in Neurospora crassa, which we named mus-43 and mus-44, respectively. Disruption of mus-43 and mus-44 conferred sensitivity to UV and 4-nitroquinoline 1-oxide, but not to methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, camptothecin, hydroxyurea, or bleomycin. The mus-44 mutant was more sensitive to UV than the mus-43 mutant. Genetic analysis indicated that mus-43 and mus-44 are epistatic to mus-38 which is a homolog of the S. cerevisiae RAD1, but not to mus-18 which belongs to a second excision repair pathway. Immunological assays demonstrated that both mus-43 and mus-44 retained the ability to excise UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts, but that excision ability was completely abolished in the mus-43 mus-18 and mus-44 mus-18 double mutants. These double mutants exhibited extremely high sensitivity to UV. In mus-43 and mus-44 mutants, the UV-induced mutation frequency increased compared to that of the wild-type. The mus-44 mutants also exhibited a partial photoreactivation defect phenotype similar to mus-38. These results suggest that both mus-43 and mus-44 function in the mus-38 NER pathway, but not in the mus-18 excision repair pathway.     

Leptin Activates Hepatic 5′-AMP-activated Protein Kinase through Sympathetic Nervous System and α1-Adrenergic Receptor: A POTENTIAL MECHANISM FOR IMPROVEMENT OF FATTY LIVER IN LIPODYSTROPHY BY LEPTIN*

Leptin is an adipocyte-derived hormone that regulates energy homeostasis. Leptin treatment strikingly ameliorates metabolic disorders of lipodystrophy, which exhibits ectopic fat accumulation and severe insulin-resistant diabetes due to a paucity of adipose tissue. Although leptin is shown to activate 5′-AMP-activated protein kinase (AMPK) in the skeletal muscle, the effect of leptin in the liver is still unclear. We investigated the effect of leptin on hepatic AMPK and its pathophysiological relevance in A-ZIP/F-1 mice, a model of generalized lipodystrophy. Here, we demonstrated that leptin activates hepatic AMPK through the central nervous system and α-adrenergic sympathetic nerves. AMPK activities were decreased in the fatty liver of A-ZIP/F-1 mice, and leptin administration increased AMPK activities in the liver as well as in skeletal muscle with significant reduction in triglyceride content. Activation of hepatic AMPK with A769662 also led to a decrease in hepatic triglyceride content and blood glucose levels in A-ZIP/F-1 mice. These results indicate that the down-regulation of hepatic AMPK activities plays a pathophysiological role in the metabolic disturbances of lipodystrophy, and the hepatic AMPK activation is involved in the therapeutic effects of leptin.     

Novel germline hMSH2 genomic deletion and somatic hMSH2 mutations in a hereditary nonpolyposis colorectal cancer family

Germline and somatic mutations of the hMSH2 gene were determined in a Japanese hereditary nonpolyposis colorectal cancer (HNPCC) family fulfilling the Amsterdam criteria. PCR-SSCP-sequencing of genomic DNA detected a somatic hMSH2 mutation of an A deletion at codon 227-229 in a duodenal carcinoma and a somatic hMSH2 mutation of an A insertion at codon 21 in a gastric carcinoma from affected family members, both carcinomas exhibiting high microsatellite instability. However, no germline hMSH2 mutation was detected by the PCR-SSCP-sequencing method. Genomic DNA was then analyzed by Southern blot hybridization using three hMSH2 cDNA probes (probe A involving exons 1-5, probe B involving exons 4-11 and probe C involving exons 9-16) after digestion by restriction enzymes, EcoRI, HindIII and NsiI. The NsiI digest of DNA from normal tissues of affected members exhibited an aberrant 8.6 kb restriction fragment, in addition to the normal 10.6 kb fragment, when hybridized to probes A and B. This suggested the presence of a heterozygous 2kb genomic deletion encompassing exon 4, 5 or 6. RT-PCR-sequencing revealed that the deleted region encompassed exon 5. This novel genomic deletion of the hMSH2 gene was confirmed to be pathogenic, and the Southern hybridization pattern was applied to the pre-symptomatic diagnosis.     

Lysophosphatidic Acid Sensitises Ca Influx through Mechanosensitive Ion Channels in Cultured Lens Epithelial Cells

We investigated the effect of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) to mechanical stress in cultured bovine lens epithelial cells. Spritzing of bath solution onto cells as mechanical stress caused marked increase in [Ca²⁺]_i in the presence of LPA and this increase was concentration-dependent (1–10 μM), whereas neither addition of LPA alone nor the mechanical stress in the absence of LPA affected [Ca²⁺]_i. The mechanical stress-induced increase in [Ca²⁺]_i in the presence of LPA was inhibited by removing extracellular Ca²⁺ or by addition of Gd³⁺, a blocker of mechanosensitive cation channels, but not by nicardipine, thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump, or U73122, a phospholipase C inhibitor. These results show that LPA sensitises Ca²⁺ influx through cation-selective mechanosensitive channels, but does not sensitise Ca²⁺ release from intracellular stores, triggered by changes in mechanical stress. On the other hand, phosphatidic acid had less of a sensitising effect than LPA, and neither lysophosphatidylcholine nor chlorpromazine had any effect. Also Ca²⁺ mobilising agonists, ATP, histamine and carbachol, did not sensitise Ca²⁺ response to the mechanical stress. These results show that LPA sensitises mechanoreceptor-linked response in lens epithelial cells, suggesting that it plays a role in the development of cataracts due to increases in [Ca²⁺]_i induced by mechanical stress.