Semilunar postlarval settlement periodicity ensuing from semilunar larval release cycle in the Nihonotrypaea harmandi (Crustacea,Decapoda, Axiidea,Callianassidae) population on an intertidal sandflat in an open marine coastal embayment

Many decapod crustaceans in marine intertidal habitats release larvae toward coastal oceans, from which postlarvae (decapodids: settling-stage larvae) return home. Decapodid settlement processes are poorly understood. Previous studies showed that in Kyushu, Japan, the callianassid shrimp population on an intertidal sandflat of an open bay joining the coastal ocean near a large estuary released eight batches of larvae basically in a semilunar cycle from June through October and that decapodids performed diel vertical migration, occurring in the water column nocturnally. We conducted (a) frequent sampling for population density and size-composition on the sandflat through one reproductive season, (b) planktonic and benthic sampling for decapodids around the bay mouth, and (c) current meter deployment at three points across the bay mouth for tidal harmonic analysis. On the sandflat, six batches of newly-settled decapodids (settlers) occurred in a semilunar periodicity until October, with peaks occurring 0–3 days before syzygy dates except for the first one. For larval Batches 1–4, buoyancy-driven shoreward subsurface currents during July to mid-October would transport some pre-decapodid-stage larvae (zoeae) toward the bay. The absence of expected settler Batches 7–8 would be due to the converse subsurface currents caused by water-column mixing and seasonal winds after mid-October, carrying zoeae offshore. Once in the bay, phasing of night and nighttime-averaged shoreward tidal current explained the settlement pattern for Batches 1–4. For Batches 5–6 occurring in mid-September to mid-October, water currents generated by seasonal wind and tidal forcings may have caused peak settlement after the time expected from tidally-driven decapodid transport.     

Intra-strain biological and epidemiological characterization of plum pox virus

Plum pox virus (PPV) is one of the most important plant viruses causing serious economic losses. Thus far, strain typing based on the definition of 10 monophyletic strains with partially differentiable biological properties has been the sole approach used for epidemiological characterization of PPV. However, elucidating the genetic determinants underlying intra-strain biological variation among populations or isolates remains a relevant but unexamined aspect of the epidemiology of the virus. In this study, based on complete nucleotide sequence information of 210 Japanese and 47 non-Japanese isolates of the PPV-Dideron (D) strain, we identified five positively selected sites in the PPV-D genome. Among them, molecular studies showed that amino acid substitutions at position 2,635 in viral replicase correlate with viral titre and competitiveness at the systemic level, suggesting that amino acid position 2,635 is involved in aphid transmission efficiency and symptom severity. Estimation of ancestral genome sequences indicated that substitutions at amino acid position 2,635 were reversible and peculiar to one of two genetically distinct PPV-D populations in Japan. The reversible amino acid evolution probably contributes to the dissemination of the virus population. This study provides the first genomic insight into the evolutionary epidemiology of PPV based on intra-strain biological variation ascribed to positive selection.     

The role of protein kinase C in the increased generation in isolated rat hepatocytes of the hydroxyl radical by puromycin aminonucleoside

Puromycin aminonucleoside (PAN) has been known to induce proteinuria. The increased generation of reactive oxygen species (ROS) has been implicated in this toxicity of PAN. We have reported that PAN increases the synthesis of methylguanidine (MG) and creatol which are the products of the reaction of creatinine and the hydroxyl radical in isolated rat hepatocytes. However, the mechanism for the increased ROS induced by PAN is still unclear. In this paper, we investigate the role of protein kinase C (PKC) on the PAN induced reactive oxygen generation in isolated rat hepatocytes. Isolated hepatocytes were incubated in Krebs-Henseleit bicarbonate buffer containing 3% BSA, 16.6 mM creatinine and tested reagents. MG and creatol were determined by high-performance liquid chromatography using 9,10-phenanthrenequinone for the post-labeling. PAN increased MG and creatol synthesis in isolated rat hepatocytes by 60%. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, at 10 and 100 μM significantly inhibited MG and creatol synthesis with or without PAN. The inhibition rate is dose dependent from 10 to 100 μM. H1004, a reagent used as control for H-7, did not affect (at 10 μM) or increased little (at 100 μM) the synthesis of MG and creatol. Ro31-8425, a potent PKC inhibitor, significantly inhibited (at 10 μM) MG synthesis in the presence of PAN. PKC in the membrane fraction, a marker of PKC activation, increased over the initial concentration by a factor of 1.65-fold at 60 min incubation and 2.16-fold at 120 min with PAN, while it changed little without PAN. These results indicate that PAN activates PKC resulting in increased hydroxyl radical generation in isolated rat hepatocytes.     

Homogeneous Fluorescence Assays for RNA Diagnosis by Pyrene-Conjugated 2′- O -Methyloligoribonucleotides

We developed a bispyrene-conjugated 2 ′-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply.     

Substrate specificity,plasma membrane localization,and lipid modification of the aldehyde dehydrogenase ALDH3B1

The accumulation of reactive aldehydes is implicated in the development of several disorders. Aldehyde dehydrogenases (ALDHs) detoxify aldehydes by oxidizing them to the corresponding carboxylic acids. Among the 19 human ALDHs, ALDH3A2 is the only known ALDH that catalyzes the oxidation of long-chain fatty aldehydes including C16 aldehydes (hexadecanal and trans-2-hexadecenal) generated through sphingolipid metabolism. In the present study, we have identified that ALDH3B1 is also active in vitro toward C16 aldehydes and demonstrated that overexpression of ALDH3B1 restores the sphingolipid metabolism in the ALDH3A2-deficient cells. In addition, we have determined that ALDH3B1 is localized in the plasma membrane through its C-terminal dual lipidation (palmitoylation and prenylation) and shown that the prenylation is required particularly for the activity toward hexadecanal. Since knockdown of ALDH3B1 does not cause further impairment of the sphingolipid metabolism in the ALDH3A2-deficient cells, the likely physiological function of ALDH3B1 is to oxidize lipid-derived aldehydes generated in the plasma membrane and not to be involved in the sphingolipid metabolism in the endoplasmic reticulum.     

Development of simple sequence repeat markers and construction of a high-density linkage map of Capsicum annuum

To facilitate marker-assisted breeding and genetic analyses of pepper (Capsicum annuum), we developed non-redundant 2- or 3-base simple sequence repeat (SSR) markers from enriched C. annuum genomic libraries and from C. annuum cDNA sequences in public databases. The SSR-enriched libraries were constructed using combinations of three restriction enzymes (AluI, HaeIII, and RsaI) and two biotinylated oligonucleotides [b(GA)₁₅ and b(CA)₁₅]. Ultimately, we obtained 1,736 genomic SSR markers and 1,344 cDNA-derived SSR markers from 6,528 clones and 13,003 sequences, respectively. We mapped 597 markers, including 265 of the newly developed SSR markers, onto a linkage map by using doubled-haploid (DH) lines derived from an intraspecific cross of two pure lines of C. annuum (K9-11 × MZC-180). The map, designated as the KL-DH map, consisted of 12 linkage groups. The map covered a genetic distance of 2,028 cM, and the average distance between markers was less than 4 cM. The frame structure of the KL-DH map was compared with the published standard conserved ortholog set II (COSII) map, which was derived from an interspecific F₂ population (C. frutescens × C. annuum), by using tomato (Solanum lycopersicum) chromosomal sequences to bridge the two maps. The intraspecific KL-DH map constructed in this study and the interspecific COSII map were similar in map length and marker distribution, suggesting that the KL-DH map covers nearly the whole genome of C. annuum.     

Farsenyl pyrophosphate synthase is a potential molecular drug target of risedronate in Babesia bovis

A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011 bp with predicted 336 amino acids and molecular mass of 38 kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the K_m value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophosphate was 2.494 ± 1.536 μM. Risedronate inhibited the activity of BbFPPS yielding IC₅₀ value of 8.4 ± 1.2 nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar concentration of risedronate (IC₅₀ = 4.02 ± 0.91 μM). No regrowth of B. bovis was observed at 10 μM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis.     

Identification and Characterization of a Novel aac(6′)-Iag Associated with the bla IMP-1–Integron in a Multidrug-Resistant Pseudomonas aeruginosa

In a continuing study from Dec 2006 to Apr 2008, we characterized nine multi-drug resistant Pseudomonas aeruginosa strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis of SpeI-digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-β-lactamase. PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated as In124, that carries an array of four gene cassettes: a novel aminoglycoside (AG) resistance gene, aac(6′)-Iag, bla _IMP-1, a truncated form of bla _IMP-1, and a truncated form of aac(6′)-Iag. The aac(6′)-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6′)-Iz. Recombinant AAC(6′)-Iag protein showed aminoglycoside 6′-N-acetyltransferase activity using thin-layer chromatography (TLC) and MS spectrometric analysis. Escherichia coli carrying aac(6′)-Iag showed resistance to amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not to gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for bla _IMP-1 and aac(6′)-Ig strongly suggested In124 is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually all AGs, suggesting that the In124 conjugal plasmid also possesses a gene conferring resistance to gentamicin.     

Comparison of Barium and Arsenic Concentrations in Well Drinking Water and in Human Body Samples and a Novel Remediation System for These Elements in Well Drinking Water

Health risk for well drinking water is a worldwide problem. Our recent studies showed increased toxicity by exposure to barium alone (≤700 µg/L) and coexposure to barium (137 µg/L) and arsenic (225 µg/L). The present edition of WHO health-based guidelines for drinking water revised in 2011 has maintained the values of arsenic (10 µg/L) and barium (700 µg/L), but not elements such as manganese, iron and zinc. Nevertheless, there have been very few studies on barium in drinking water and human samples. This study showed significant correlations between levels of arsenic and barium, but not its homologous elements (magnesium, calcium and strontium), in urine, toenail and hair samples obtained from residents of Jessore, Bangladesh. Significant correlation between levels of arsenic and barium in well drinking water and levels in human urine, toenail and hair samples were also observed. Based on these results, a high-performance and low-cost adsorbent composed of a hydrotalcite-like compound for barium and arsenic was developed. The adsorbent reduced levels of barium and arsenic from well water in Bangladesh and Vietnam to <7 µg/L within 1 min. Thus, we have showed levels of arsenic and barium in humans and propose a novel remediation system.