Molecular characteristics of extended-spectrum beta-lactamases and qnr determinants in Enterobacter species from Japan

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing bla(CTX-M), bla(SHV), or bla(TEM) were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan.     

Human α/β hydrolase domain containing 10 (ABHD10) is responsible enzyme for deglucuronidation of mycophenolic acid acyl-glucuronide in liver

Mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), is primarily metabolized by glucuronidation to a phenolic glucuronide (MPAG) and an acyl glucuronide (AcMPAG). It is known that AcMPAG, which may be an immunotoxic metabolite, is deglucuronidated in human liver. However, it has been reported that recombinant β-glucuronidase does not catalyze this reaction. AcMPAG deglucuronidation activity was detected in both human liver cytosol (HLC) and microsomes (HLM). In this study, the enzyme responsible for AcMPAG deglucuronidation was identified by purification from HLC with column chromatographic purification steps. The purified enzyme was identified as α/β hydrolase domain containing 10 (ABHD10) by amino acid sequence analysis. Recombinant ABHD10 expressed in Sf9 cells efficiently deglucuronidated AcMPAG with a K(m) value of 100.7 ± 10.2 μM, which was similar to those in HLM, HLC, and human liver homogenates (HLH). Immunoblot analysis revealed ABHD10 protein expression in both HLC and HLM. The AcMPAG deglucuronidation by recombinant ABHD10, HLC, and HLH were potently inhibited by AgNO(3), CdCl(2), CuCl(2), PMSF, bis-p-nitrophenylphosphate, and DTNB. The CL(int) value of AcMPAG formation from MPA, which was catalyzed by human UGT2B7, in HLH was increased by 1.8-fold in the presence of PMSF. Thus, human ABHD10 would affect the formation of AcMPAG, the immunotoxic metabolite.     

TNFR1 promotes tumor necrosis factor-mediated mouse colon epithelial cell survival through RAF activation of NF-kappaB

Tumor necrosis factor (TNF) is a therapeutic target in the treatment of inflammatory bowel disease; however, the exact role of TNF signaling in the colon epithelium remains unclear. We demonstrate that TNF activation of TNF receptor (R)1 stimulates both pro- and anti-apoptotic signaling pathways in the colon epithelium; however, TNFR1 protects against colon epithelial cell apoptosis following TNF exposure. To investigate anti-apoptotic signaling pathways downstream of TNFR1, we generated an intestinal epithelium-specific Raf knock-out mouse and identified Raf kinase as a key regulator of colon epithelial cell survival in response to TNF. Surprisingly, Raf promotes NF-kappaB p65 phosphorylation, independent of MEK signaling, to support cell survival. Taken together, these data demonstrate a novel pathway in which Raf promotes colon epithelial cell survival through NF-kappaB downstream of TNFR1 activation. Thus, further understanding of colon epithelial cell-specific TNFR signaling may result in the identification of new targets for inflammatory bowel disease treatment and define novel mediators of colitis-associated cancer.     

Real-Time Polymerase Chain Reaction Assays for Rapid Detection and Quantification of <Emphasis Type="Italic">Noctiluca scintillans</Emphasis> Zoospore

Application and availability of real-time polymerase chain reaction (PCR) assay to detect and quantify the Noctiluca scintillans zoospore were investigated seasonally. Specific primer set for N. scintillans 18S rDNA was designed and applied to real-time PCR assay using the serial dilutions of N. scintillans zoospores. The real-time PCR assays with Ns63F and Ns260R primers were applied to sea water samples collected weekly in Manazuru Port of Sagami Bay, Japan from April 2005 to June 2006. We developed effective DNA preparation steps for collecting the template DNA of N. scintillans zoospore: size fraction and filter concentration of the water samples, fixation with Lugol solution, cell lysis, and purification. This method is useful for the monitoring of the zoospores of N. scintillans, and can also be used for other small and physiologically fragile planktonic cell. Variation in the density of zoospore was successfully detected in the field samples. The peak density of N. scintillans zoospore was observed to occur just before or at the same time as the peak of the vegetative cells. Moreover, zoospores were detected in seawater even when the vegetative cells were not observed. The presence of zoospore was found all year round in the present study. In this regards, this information is essential for the study of the life cycle and seasonal variation of N. scintillans in the coastal waters.     

Nutritional diagnosis of phytoplankton in Lake Baikal

To diagnose the nutritional status of phytoplankton in Lake Baikal, surveys for the determination of concentrations of particulate carbon (PC), nitrogen (PN) and phosphorus (PP) and their ratios were conducted at six stations in March, June, August and October 1999. The concentrations of PC and PN were lower than, and those of PP were similar to, those in another mesotrophic lake except at the station near the mouth of the largest input river, Selenga River, of Lake Baikal. The PC : PN : PP ratio was 102 : 13 : 1, considerably close to the Redfield ratio. The ratio was constant against spatiotemporal changes. These indicate that phytoplankton in Lake Baikal were exposed to no deficiency in nitrogen nor phosphorus. From a viewpoint of the nutritional status of phytoplankton, Lake Baikal might be viewed as an ocean rather than as a lake.     

Life history response and age-specific tolerance to starvation in Brachionus plicatilis O.F. Müller (Rotifera)

To examine the life history response and age-specific tolerance to starvation in the rotifer Brachionus plicatilis O.F. Müller, we carried out two series of individual culture experiments. In the first experiment, rotifers were fed until each of the ages of 1-4 days, and were then starved during the rest of their lifetimes. The control group was fed throughout their lifespans. Rotifers stopped active reproduction just after the onset of food deprivation, and showed shorter subsequent survival times when they were starved at older ages. The finding that the larger the number of offspring produced before food deprivation, the shorter the subsequent lifetime under starvation, appeared to reflect a trade-off with the cost of reproduction. In the second experiment, newborns were starved until each of the ages of 1-5 days, and were fed thereafter. The lifespans of the rotifers starved up to the age of 3 days were not statistically different from those that were not starved. Although the starved rotifers began to reproduce once fed again, their lifetime fecundity decreased significantly from that of the non-starved group. Based on these results, it was suggested that the reproductive suppression caused by starvation would cause rotifers to have a longer lifespan to allow for future reproduction.     

Variability in Toxicity of the Dinoflagellate Alexandrium Tamarense Isolated from Hiroshima Bay, Western Japan, as a Reflection of Changing Environmental Conditions

The variability of cellular toxin content in the dinoflagellateAlexandrium tamarense isolated from Hiroshima Bay was analyzedunder a variety of culture conditions. Growth and toxicity wererepresented as a function of light (80, 90, 110, 160 and 350µmol m^–2 s ^–1), temperature (12, 17 and 22°C),salinity (13, 16.5, 19.5, 25, 29, 33, 36.5 and 38 PSU) and ammoniumconcentration (0.11, 0.22 and 0.44 mM). Toxicity was measuredby the tissue culture bioassay using mouse neuroblastoma cells,and expressed as saxitoxin concentration equivalents. Cellulartoxicity increased with decreasing salinity. At temperaturesof 17 and 22°C, maximum toxin content was observed at thelowest light intensity and growth rate. At the lowest temperatureof 12°C, maximum toxin content was observed at intermediatelight intensities and growth rates. A drastic increase in toxincontent with an increase in ammonium concentration from 0.11to 0.22 mM supported the idea that ammonium utilization fortoxin production directly brings about a high toxin contentinA. tamarense. Our results ecologically imply that the cellsbecome highly toxic in environments with low salinity and highammonium concentration, and successive cloudy days. Such environmentalconditions may lead to increasing risk of shellfish toxification.     

Why do rotifer populations present a typical sigmoid growth curve?

To determine the underlying processes to population growth in the rotifer Brachionus plicatilis, we conducted an experiment using 1.5 ml cultures for 70 days. All individuals were transferred daily to culture media containing algae, and the number of individuals, clutch sizes and number of deaths were counted. The population dynamics showed a typical sigmoid curve. The population density increased exponentially from 10 to 682 individuals during the first 7 days (exponential growth phase), and gradually up to about 1500 individuals during the next 30 days (post-exponential growth phase). The population density then remained at a constant level with small fluctuations during the rest of the experimental period (stationary phase). Mortalities appeared from the post-exponential growth phase and were almost constant at about 2% throughout the experimental period. The clutch size decreased from 5 to 1 during the first 5 days, and afterwards females laid only one egg each. The proportion of non-reproductive females increased from 30% (exponential growth phase) to 80% (post-exponential growth phase) to 90% (stationary phase). These results suggest that the exponential growth phase resulted from the imbalance between a high birth rate and a low death rate, while the stationary phase was maintained by the compensation between low birth and death rates.     

Molecular cloning of endo-beta-D-1,4-glucanase genes,rce1, rce2, and rce3, from Rhizopus oryzae

Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision ZYGOMYCOTA: All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45. The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one. rce1, rce2, and rce3 had various lengths of linker sequences.