A temperature-dependent timing mechanism is involved in the circadian system that drives locomotor rhythms in the fruit fly Drosophila melanogaster

The circadian clock of Drosophila melanogaster is thought to include rhythmic expression of period gene. Recent studies suggested, however, that a per-less oscillation is also involved in the regulation of circadian locomotor rhythms. In the present study, we examined the existence and the property of the possible per-less oscillation using arrhythmic clock mutant flies carrying per (01), tim(01), dClk(Jrk) or cyc(01), which lack rhythmic per expression. When temperature cycles consisting of 25 degrees C and 30 degrees C with various periods (T=8-32 hr) were given, wild-type (Canton-S) flies showed locomotor rhythms entrained to temperature cycles over a wide range of period (T=8-32 hr) in constant light (LL) while only to T=24 hr in constant darkness (DD). The mutant flies showed rhythms synchronizing with the given cycle both under LL and DD. In per(01) and tim(01) flies, the phase of a major peak slightly changed dependent on Ts in DD, while it did not in dClk(Jrk) and cyc(01) flies. When they were transferred from a constant temperature to a temperature cycle under DD, several cycles were necessary to establish a clear temperature entrainment in per(01) and tim (01) flies. These results suggest that per(01) and tim(01) flies have a temperature-entrainable weak oscillatory mechanism and that the per-less oscillatory mechanism may require dClk and cyc. In addition, per (01) and tim(01) flies changed from thermoactive in DD to cryoactive in LL, while dClk(Jrk) and cyc(01) flies did not. It is thus suggested that dClk and cyc are also involved in determining the light-associated temperature preference in per(01) and tim(01) flies.     

Characteristic microbial community of a dry thermophilic methanogenic digester: its long-term stability and change with feeding

Thermophilic dry anaerobic digestion of sludge for cellulose methanization was acclimated at 53 °C for nearly 5 years using a waste paper-based medium. The stability of the microbial community structure and the microbial community responsible for the cellulose methanization were studied by 16S rRNA gene-based clone library analysis. The microbial community structure remained stable during the long-term acclimation period. Hydrogenotrophic methanogens dominated in methanogens and Methanothermobacter, Methanobacterium, Methanoculleus, and Methanosarcina were responsible for the methane production. Bacteria showed relatively high diversity and distributed mainly in the phyla Firmicutes, Bacteroidetes, and Synergistetes. Ninety percent of operational taxonomic units (OTUs) were affiliated with the phylum Firmicutes, indicating the crucial roles of this phylum in the digestion. Relatives of Clostridium stercorarium, Clostridium thermocellum, and Halocella cellulosilytica were dominant cellulose degraders. The acclimated stable sludge was used to treat garbage stillage discharged from a fuel ethanol production process, and the shift of microbial communities with the change of feed was analyzed. Both archaeal and bacterial communities had obviously changed: Methanoculleus spp. and Methanothermobacter spp. and the protein- and fatty acid-degrading bacteria became dominant. Accumulation of ammonia as well as volatile fatty acids led to the inhibition of microbial activity and finally resulted in the deterioration of methane fermentation of the garbage stillage.     

Production of D-lactic acid from defatted rice bran by simultaneous saccharification and fermentation

Production of d-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueckii IFO 3202 produced 28 kgm(-3) lactic acid from 100 kgm(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kgm(-3) from 100 kgm(-3) of the bran) was 78%. The optical purity of produced d-lactic acid was 95% e.e.     

Structural basis for target protein recognition by the protein disulfide reductase thioredoxin

Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares a mechanism with glutaredoxin and glutathione transferase for correctly positioning substrate cysteine residues at the catalytic groups but possesses a unique structural element that allows recognition of protein disulfides.     

(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent against HL-60 cells

(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-1-ol were prepared using the Wittig olefination of Garner's aldehyde (N-Boc-N,O-isopropylidene-L- or D-serinal) from L- or D-serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60 cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with N-acetyl-D-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order D-e-C2-Cer approximately L-e-C2-Cer approximately (2R,3Z)- approximately (2S,3Z)->(2R,3E)- approximately (2S,3E)- approximately (2R)- approximately (2S)-derivative. Qualitative analysis of DNA fragmentation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and (2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly synthesized compounds, (3Z)-2-Acetylamino-3-octadecen-1-ol, have similar characteristics and apoptosis-inducing activities against HL-60 cells with C2-Cer.     

Physiological role of metastin/kisspeptin in regulating gonadotropin-releasing hormone (GnRH) secretion in female rats

Various studies have attempted to unravel the physiological role of metastin/kisspeptin in the control of gonadotropin-releasing hormone (GnRH) release. A number of evidences suggested that the population of metastin/kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) is involved in generating a GnRH surge to induce ovulation in rodents, and thus the target of estrogen positive feedback. Females have an obvious metastin/kisspeptin neuronal population in the AVPV, but males have only a few cell bodies in the nucleus, suggesting that the absence of the surge-generating mechanism or positive feedback action in males is due to the limited AVPV metastin/kisspeptin neuronal population. On the other hand, the arcuate nucleus (ARC) metastin/kisspeptin neuronal population is considered to be involved in the regulation of tonic GnRH release. The ARC metastin/kisspeptin neurons show no sex difference in their expression, which is suppressed by gonadal steroids in both sexes. Thus, the ARC population of metastin/kisspeptin neurons is a target of estrogen negative feedback action on tonic GnRH release. The lactating rat model provided further evidence indicating that ARC metastin/kisspeptin neurons are involved in GnRH pulse generation, because pulsatile release of luteinizing hormone (LH) is profoundly suppressed by suckling stimulus and the LH pulse suppression is well associated with the suppression of ARC metastin/kisspeptin and KiSS-1 gene expression in lactating rats.     

Localization of ghrelin-producing cells in the stomach of the rainbow trout (Oncorhynchus mykiss)

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), was isolated from the rat stomach and determined to be n-octanoylated 28-amino-acid peptide. In this study, we studied the distribution of ghrelin-producing cells (ghrelin cells) in the gastrointestinal tract of male and female rainbow trout (Oncorhynchus mykiss) by immunohistochemistry using N-terminal region-recognizing antibody and also by in situ hybridization using a trout ghrelin-specific cRNA probe. Ghrelin cells were found in the mucosal layer of the stomach but not in the myenteric plexus, and no ghrelin cells were observed in other regions of the gastrointestinal tract. Ghrelin cells could be classified into two types: closed- and opened-type cells. The density of ghrelin cells increased gradually in the direction from the cardiac to pyloric portions of the stomach in both sexes. The number of ghrelin cells per unit area seemed to be higher in females than in males. In conclusion, trout ghrelin cells exist in the stomach and are classified into two types of cells, closed- and opened-type cells.     

Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

Environment-mediated accumulation of diacyl lipoproteins over their triacyl counterparts in Staphylococcus aureus

Bacterial lipoproteins are believed to exist in only one specific lipid-modified structure, such as the diacyl form or the triacyl form, in each bacterium. In the case of Staphylococcus aureus, recent extensive matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis revealed that S. aureus lipoproteins exist in the α-aminoacylated triacyl form. Here, we discovered conditions that induce the accumulation of diacyl lipoproteins that lack α-aminoacylation in S. aureus. The accumulation of diacyl lipoproteins required a combination of conditions, including acidic pH and a post-logarithmic-growth phase. High temperatures and high salt concentrations additively accelerated the accumulation of the diacyl lipoprotein form. Following a post-logarithmic-growth phase where S. aureus MW2 cells were grown at pH 6, SitC lipoprotein was found almost exclusively in its diacyl structure rather than in its triacyl structure. This is the first report showing that the environment mediates lipid-modified structural alterations of bacterial lipoproteins.     

Activation of macrophage-stimulating protein by human airway trypsin-like protease

Macrophage-stimulating protein (MSP) circulates as a proform protein and requires proteolytic processing for activation. Respiratory ciliated cells express the MSP receptor, recepteur d'origine nantais (RON), at the apical surface, which reportedly has an important role in ciliary function. Like RON, human airway trypsin-like protease (HAT) is also expressed at the apical surface of ciliated cells. Here we show that HAT cleaves proMSP at the physiological activation site, Arg483-Val484. MSP processed by HAT could induce chemotactic responses and morphological changes of peritoneal macrophages. In human respiratory epithelial cells, knock down of HAT expression reduced proMSP processing and RON autophosphorylation. We suggest that HAT is important for MSP-RON signaling in the respiratory tract.