Diversity of substrate utilization and growth characteristics of sulfate-reducing bacteria isolated from estuarine sediment in Japan

Two different isolation methods, the dilution colony-counting method (colony-isolation) and enrichment culture, were used to isolate sulfate-reducing bacteria (SRBs) from estuarine sediment in Japan. Lactate was used as an electron donor for colony-isolation, and lactate or propionate was used for enrichment culture. All isolates were classified into six different phylogenetic groups according to the 16S rRNA gene-based analysis. The closest relatives of the colony-isolates (12 strains) were species in the genera of Desulfobacterium, Desulfofrigus, Desulfovibrio and Desulfomicrobium. The closest known relative of the lactate-enrichment isolates was Desulfovibrio acrylicus and that of the propionate-enrichment isolates was Desulfobulbus mediterraneus. All isolates were incompletely-oxidizing SRBs. Overall patterns of utilization of electron donors and acceptors, as well as fermentative substrates, differed depending on the affiliation of the strain. Furthermore, even if several strains used the same substrate, the growth rates were often significantly different depending on the strain. It was strongly suggested that various species of SRBs could coexist in the sediment by competing for common substrates as well as taking priority in favorable or specific substrates for each species and the community of SRBs should be able to oxidize almost all major intermediates of anaerobic decomposition of organic matter such as lower fatty acids, alcohols and H2 as well as amino acids. Thus, it was indicated by the phylogenetic and physiological analyses of the isolates that the SRB community composed of diverse lineages of bacteria living in anoxic estuarine sediment should be able to play an extensive role in the carbon cycle as well as the sulfur cycle of the earth.     

Lamprey TLRs with properties distinct from those of the variable lymphocyte receptors

Fish express mammalian-type (M-type) TLRs consisting of leucine-rich repeats (LRRs) and Toll-IL-1R (TIR) homology domain for immunity, whereas invertebrates in deuterostomes appear to have no orthologs of M-type TLRs. Lampetra japonica (lamprey) belongs to the lowest class of vertebrates with little information about its TLRs. We have identified two cDNA sequences of putative TLRs in the lamprey (laTLRs) that contain LRRs and TIR domains. The two laTLRs were 56% homologous to each other, and their TIRs were similar to those of members of the human TLR2 subfamily, most likely orthologs of fish TLR14. We named them laTLR14a and laTLR14b. We raised a rabbit polyclonal Ab against laTLR14b and identified a 85-kDa protein in a human HEK293 transfectant by immunoblotting using the Ab. FACS, histochemical, and confocal analyses showed that laTLR14b is expressed intracellularly in lamprey gill cells and that the overexpressed protein resides in the endoplasmic reticulum of human and fish (medaka) cell lines. Because natural agonists of TLR14 remained unidentified, we made a chimera construct of extracellular CD4 and the cytoplasmic domain of laTLR14. The chimera molecule of laTLR14b, when expressed in HEK293 cells, elicited activation of NF-kappaB and, consequently, weak activation of the IFN-beta promoter. laTLR14b mRNA was observed in various organs and leukocytes. This lamprey species expressed a variable lymphocyte receptor structurally independent of laTLR14 in leukocytes. Thus, the jawless vertebrate lamprey possesses two LRR-based recognition systems, the variable lymphocyte receptor and TLR, and the M-type TLRs are conserved across humans, fish, and lampreys.     

Modulation of larval nutrition affects midgut neutral lipid storage and temporal pattern of transcription factor expression during mosquito metamorphosis

During holometabolous insect development the critical weight marks a physiological transition after which juvenile hormone (JH) concentration decreases to such a level that a subsequent increase in ecdysone titer will initiate metamorphosis. Starvation experiments indicate that the Aedes aegypti critical weight is achieved by 24 h after the last larval-larval molt. When grown at 24 degrees C with excess food, the time between the critical weight and maximum weight (interval to cessation of growth) is about 24 h and pupation occurs about 24 h after the maximum weight is achieved. Oil Red O staining of 3rd and early 4th instars indicates that the midgut is a neutral lipid storage organ during this period. Coincident with the attainment of the critical weight is the depletion of stored midgut neutral lipid. Application of methoprene to 24 h post-molt 4th instars results in renewed midgut storage of neutral lipid suggesting that midgut neutral lipid storage is a JH dependent process. Starvation of 4th instars during the 24 h post-molt period suspends development in a fraction of the larvae, and with the resumption of feeding, development resumes. A regimen of starvation and resumption of feeding of 4th instars suggests that JH concentration decreased over a 40 h period after the resumption of feeding and maximum weight is attained about 48 h after the resumption of feeding. We hypothesize that this results in a shortening of the interval to cessation of growth. Real-time PCR experiments indicate that shortening the interval to cessation of growth compresses the time period during which increases in AHR3, AaEcR-B and AaUSP-a expression occur.     

The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin gamma chains in integrin binding by laminins

Laminins are the major cell-adhesive proteins in the basement membrane, consisting of three subunits termed alpha, beta, and gamma. The putative binding site for integrins has been mapped to the G domain of the alpha chain, although trimerization with beta and gamma chains is necessary for the G domain to exert its integrin binding activity. The mechanism underlying the requirement of beta and gamma chains in integrin binding by laminins remains poorly understood. Here, we show that the C-terminal region of the gamma chain is involved in modulation of the integrin binding activity of laminins. We found that deletion of the C-terminal three but not two amino acids within the gamma1 chain completely abrogated the integrin binding activity of laminin-511. Furthermore, substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the gamma1 chain, also abolished the integrin binding activity, underscoring the role of Glu-1607 in integrin binding by the laminin. We also found that the conserved Glu residue of the gamma2 chain is necessary for integrin binding by laminin-332, suggesting that the same mechanism operates in the modulation of the integrin binding activity of laminins containing either gamma1 or gamma2 chains. However, the peptide segment modeled after the C-terminal region of gamma1 chain was incapable of either binding to integrin or inhibiting integrin binding by laminin-511, making it unlikely that the Glu residue is directly recognized by integrin. These results, together, indicate a novel mechanism operating in ligand recognition by laminin binding integrins.     

Neurons and astrocytes exhibit lower activities of global genome nucleotide excision repair than do fibroblasts

Nucleotide excision repair (NER) is a DNA repair pathway, which eliminates various types of helix-distorting DNA damage including some forms of oxidative damage and UV-induced photoproducts. To understand why patients with NER-defective disorders develop progressive neurological abnormalities, we investigated NER capabilities in neural cells. Primary cultured neurons and astrocytes derived from rat embryonic brains were prepared in mixed-cell cultures, and fibroblasts from the same embryos were cultured for comparison. Neurons in culture were unable to proliferate, while cultured astrocytes maintained that capacity. Determination of (6-4) photoproducts in situ using antibodies against those DNA lesions was used to measure NER capabilities in individual neural cells, which were identified by staining with specific cell markers. The results demonstrate that both neurons and astrocytes have significantly lower NER capabilities than fibroblasts. That result was consistent with the finding that levels of an NER-related protein (proliferating cell nuclear antigen, PCNA) recruited at the localized UV-damage sites were lower in neurons and in astrocytes than in fibroblasts. Interestingly, the degrees of NER deterioration in those neural cells were almost equivalent to those found in NER-defective human fibroblasts (TTD2VI) that show an increased sensitivity to UV. Thus, the present study suggests that an attenuated NER capacity is not specific to post-mitotic neurons, but may be common to neural cells constituting the central nervous system regardless of their residual proliferative capacity. Although the reduced but substantial NER capability of neural cells is indispensable to preventing progressive neurological abnormalities, that low NER capability might have implications for brain ageing.     

A developed determination of midazolam and 1'-hydroxymidazolam in plasma by liquid chromatography-mass spectrometry: application of human pharmacokinetic study for measurement of CYP3A activity

This paper describes sensitive and reliable determination of midazolam (MDZ) and its major metabolite 1'-hydroxymidazolam (1-OHMDZ) in human plasma by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. MDZ, 1-OHMDZ and diazepam as an internal standard were extracted from 1ml of alkalinized plasma using n-hexane-chloroform (70:30, v/v). The extract was injected into an analytical column (YMC-Pak Pro C(18), 50mmx2.0mmi.d.). The mobile phase for separation consisted of 10mM ammonium acetate and methanol (50:50, v/v) and was delivered at a flow-rate of 0.2ml/min. The drift voltage was 100V. The sampling aperture was heated at 120 degrees C and the shield temperature was 260 degrees C. The total time for chromatographic separation was less than 16min. The validated concentration ranges of this method were 0.25-50ng/ml for both MDZ and 1-OHMDZ. Mean recoveries were 93.6% for MDZ and 86.6% for 1-OHMDZ. Intra- and inter-day coefficient variations were less than 6.5 and 5.5% for MDZ, and 6.1 and 5.7% for 1-OHMDZ at 0.3, 4, 20 and 40ng/ml. The limits of quantification were 0.25ng/ml for both MDZ and 1-OHMDZ. This method was sensitive and reliable enough for pharmacokinetic studies on healthy volunteers, and was applied for the measurement of CYP3A activity in humans after an intravenous (1mg) and a single-oral administration (2mg) of subtherapeutic MDZ dose.     

Gonad development and size‐at‐maturity of silver therapon Leiopotherapon plumbeus (Kner 1864; Teleostei:Terapontidae) in tropical volcanic lakes in south Luzon,Philippines

Gonad development of the silver therapon Leiopotherapon plumbeus in two volcanic crater  lake  habitats (Sampaloc Lake, Taal Lake) in south Luzon, Philippines was examined during the annual reproductive cycle. The minimum body size‐at‐maturity of fish in these two lake habitats was also compared. Four gonad development stages were characterized as basis for the classification of ovarian (immature, maturing, mature, spawned) and testicular maturation (immature, maturing, mature) phases. The occurrence of all development stages in individual gonads suggest an asynchronous development whereby advanced stages are recruited continuously from a pool of younger stage germ cells to result in elevated female and male GSI throughout the annual cycle due to active gonadogenesis. Together with the increasing occurrence of advanced stage oocytes and spermatozoa from March until October, the elevated GSI of fish may indicate peak gonadal growth during the onset of the dry season (December–January) for eventual spawning from the beginning (May–June) until the end of the wet season (October–November). In both lake habitats, male fish were smaller than females but, regardless of sex, the minimum size‐at‐maturity of fish in Sampaloc Lake was significantly smaller than fish in Taal Lake. Overall, asynchronous development during oogenesis and spermatogenesis allows for year‐round reproduction of silver therapon, with elevated gonad growth in the dry season in preparation for spawning during the wet season. Compared with fish in Taal Lake, a smaller size‐at‐maturity of fish in Sampaloc Lake may be a response of the wild fishery stock to long‐term high fishing mortality and degradation of the lake habitat.     

Soy sauce increased the oxidative stress tolerance of nematode via p38 MAPK pathway

Soy sauce – a fermented food made from soybeans and wheat – is considered a healthy seasoning, but little scientific evidence is available to support this. In this study, physiological effects of soy sauce were analyzed using Caenorhabditis elegans. When soy sauce was fed to C. elegans together with Escherichia coli OP50, fat accumulation decreased, and resistance to oxidative stress by H₂O₂ was greatly increased in the nematodes. qRT-PCR revealed that mRNA expression of oxidative stress tolerance genes, including sod, ctl, and gpx, was markedly increased in soy sauce-fed nematodes. Worms ingesting soy sauce showed high mitochondrial membrane potential and reactive oxygen species (ROS) and low intracellular ROS, suggesting that soy sauce induced mitohormesis and decreased cytoplasmic ROS. Therefore, soy sauce ingestion affects the mitochondria and may alter the fat metabolism in C. elegans. Furthermore, the increase in oxidative stress tolerance is mediated through p38 MAPK pathway.     

Comparative analysis of the reactive oxygen species‐producing enzymatic activity of Arabidopsis NADPH oxidases

Reactive oxygen species (ROS) produced by NADPH oxidases, called respiratory burst oxidase homologs (Rbohs), play crucial roles in development as well as biotic and abiotic stress responses in plants. Arabidopsis has 10 Rboh genes, AtRbohA to AtRbohJ. Five AtRbohs (AtRbohC, ‐D, ‐F, ‐H and ‐J) are synergistically activated by Ca²⁺‐binding and protein phosphorylation to produce ROS that play various roles in planta, although the activities of the other Rbohs remain unknown. With a heterologous expression system, we found a range of ROS‐producing activity among the AtRbohs with differences up to 100 times, indicating that the required amounts of ROS are different in each situation where AtRbohs act. To specify the functions of AtRbohs involved in cell growth, we focused on AtRbohC, ‐H and ‐J, which are involved in tip growth of root hairs or pollen tubes. Ectopic expression of the root hair factor AtRbohC/ROOT HAIR DEFECTIVE 2 (RHD2) in pollen tubes restored the atrbohH atrbohJ defects in tip growth of pollen tubes. However, expression of AtRbohH or ‐J in root hairs did not complement the tip growth defect in the atrbohC/rhd2 mutant. Our data indicate that Rbohs possess different ranges of enzymatic activity, and that some Rbohs have evolved to carry specific functions in cell growth.