Purification and molecular cloning of an intracellular 3-hydroxybutyrate-oligomer hydrolase from Paucimonas lemoignei

An intracellular 3-hydroxybutyrate-oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Paucimonas lemoignei. It hydrolyzed the 3-hydroxybutyrate dimer with the highest specific activity of any of the enzymes reported so far. The gene was cloned and sequenced. The deduced amino acid sequence showed that the enzyme is a homolog of the PhaZc of Ralstonia eutropha H16.     

Cloning of a gene for D-sorbitol dehydrogenase from Gluconobacter oxydans G624 and expression of the gene in Pseudomonas putida IFO3738

We have cloned a novel gene for d-sorbitol dehydrogenase (SLDH), which efficiently converted D-sorbitol to L-sorbose, from Gluconobacter oxydans G624 (FERM BP-4415). A cosmid library of the genomic DNA was screened by assaying SLDH activity. The inserted DNA from a positive clone was downsized by subcloning into charomid and pUCP plasmid, successively. Sequencing analysis of the DNA responsible for SLDH activity revealed an open reading frame of 1455 bp coding for 485 amino acid residues with a calculated molecular mass of 53,642 Da. The amino acid sequence showed 42.2% identity with a NAD+-dependent mannitol dehydrogenase (MDH), which catalyzed conversion of d-sorbitol to d-fructose, from Pseudomonas fluorescens DSM50106. Since the intact SLDH was found to be very unstable during isolation and purification, this SLDH fused to 6 x His-tag was expressed in Pseudomonas putida IFO3738 and purified by immobilized metal affinity chromatography using cobalt-based resins. The 6 x His-tag SLDH catalyzed the oxidation of D-sorbitol to L-sorbose and exhibited 15 times higher activity in the presence of NADP+ than that of NAD+. These results indicate that the SLDH is a novel kind of dehydrogenase distinct from MDH previously reported.     

Survey of histamine content in seafood on the market

Food poisoning involving histamine has occurred almost every year for 20 years in Tokyo, and is usually due to ingestion of fish with lean meat, such as sardine, mackerel, horse mackerel and so on. Therefore, we were investigated the levels of histamine and 4 non-volatile amines (tyramine, putrescine, cadaverine, spermidine) in 637 samples on the market. The water activity of samples in which histamine was detected at 5 mg/100 g and over was examined. Histamine, tyramine, putrescine, cadaverine and spermidine were detected in 66, 43, 26, 64 and 5 samples, and the detection ranges were 5-340, 5-51, 5-42, 5-180 and 5-8 mg/100 g, respectively. Most of the samples in which histamine was detected were semi-dried round and split sardine. Water activity of 24 samples of semi-dried round and split sardine in which histamine was detected was in the range of 0.68-0.96.     

Effect of fermented milk prepared with two probiotic strains on Japanese cedar pollinosis in a double-blind placebo-controlled clinical study

There has been much interest in the potential of using probiotic bacteria for treating allergic diseases. A double-blind, placebo-controlled study was conducted to examine the effectiveness of Lactobacillus GG (LGG) and L. gasseri TMC0356 (TMC0356) in alleviating Japanese cedar pollinosis (JCP), a seasonal allergic rhinitis caused by Japanese cedar pollen. Fermented milk prepared with the tested bacteria or placebo yoghurt was administered to 40 subjects with a clinical history of JCP for 10 weeks. Subjective symptoms, self-care measures and blood samples were compared between the two groups. Peripheral blood mononuclear cells (PBMCs) were collected from seven patients with JCP and in vitro cytokine production by the isolated PBMCs was analysed in the presence of heat-killed lactic acid bacteria. Consumption of the fermented milk significantly decreased the mean symptom score for nasal blockage after 9 weeks (P<0.05) and mean symptom-medication scores after 9 and 10 weeks when compared with the placebo group (P<0.01 and P<0.05 respectively). The tested strains of lactic acid bacteria affected cytokine production by isolated PBMCs in vitro in a strain-dependent manner. LGG significantly inhibited IL-4 and IL-5 production by PBMCs stimulated by both Cry j 1 and PHA. TMC0356 only suppressed IL-5 production stimulated by PHA. The fermented milk prepared with LGG and TMC0356 might be beneficial in JCP because of its effect on nasal blockage. The effects of LGG and TMC0356 might arise at least partly from their specific down-regulation of the human Th2 immune response.     

Low ergosterol content in yeast adh1 mutant enhances chitin maldistribution and sensitivity to paraquat-induced oxidative stress

Alcohol dehydrogenases catalyse the reversible oxidation of alcohols to aldehydes or ketones, with concomitant reduction of NAD(+) or NADP(+) . Adh1p is responsible for the reduction of acetaldehyde to ethanol, while Adh2p catalyses the reverse reaction, the oxidation of ethanol to acetaldehyde. Lack of Adh1p shifts the cellular redox balance towards excess NADH/NADPH and acetaldehyde, while absence of Adh2p does the opposite. Yeast mutant adh1Δ had a slow growth rate, whereas adh2Δ grew like the isogenic wild-type (WT) during prediauxic shift fermentative metabolism. After 48 h WT and mutants reached the same number of viable cells. When exponentially growing (LOG) cells were exposed to calcofluor white, only mutant adh1Δ displayed an irregular deposition of chitin. Quantitative analyses of both LOG and stationary-phase cells showed that adh1Δ mutant contained significantly less ergosterol than cells of WT and adh2Δ mutant, whereas the erg3Δ mutant contained extremely low ergosterol pools. Both adh1Δ and adh2Δ mutants showed higher-than-WT resistance to heat shock and to H(2) O(2) but had WT resistance when exposed to ultraviolet (UV) light and the DNA cross-linking agent diepoxyoctane, indicating normal DNA repair capacity. Mutant adh1Δ was specifically sensitive to acetaldehyde and to membrane peroxidizing paraquat. Our results link the pleiotropic phenotype of adh1Δ mutants to low pools of ergosterol and to reductive stress, and introduce the two new phenotypes, resistance to heat shock and to H(2) O(2) , for the adh2Δ mutant, most probably related to increased ROS production in mitochondria, which leads to the induction of oxidative stress protection.     

Simultaneous induction of calcium transients in embryoid bodies using microfabricated electrode substrates

Precise control of differentiation processes of pluripotent stem cells is a key component for the further development of regenerative medicine. For this purpose, combining a cell-aggregate-size treatment for regulating intercellular signal transmissions and an electrical stimulation technique for inducing cellular responses is a promising approach. In the present study, we developed microfabricated electrode substrates that allow simultaneous stimulation of embryoid bodies (EBs) of P19 cells. Mouse embryonal carcinoma P19 cells can be induced to differentiate into three germ layers and serve as a promising stem cell model. Microcavity–array patterns were fabricated onto indium–tin–oxide (ITO) substrates using a standard photo-lithography technique, and uniform-sized EBs of P19 cells were inserted into each microcavity. Electrical stimulation was applied to the EBs through substrate electrodes and stimulus-induced intracellular calcium transients were monitored. We confirmed that the developed electrode device could simultaneously stimulate smaller (200 μm diameter) and larger (500 μm diameter) EBs inserted in the microcavities and induce specific spatio-temporal patterns of intracellular calcium transients in the EBs with fine reproducibility. We concluded that the developed microcavity array with embedded electrodes could simultaneously and effectively stimulate uniform-sized EBs inserted in it. Therefore, it is a promising experimental tool for precisely controlling cell differentiation processes.     

Multiresidue method for pesticides and veterinary drugs in bovine milk using GC/MS and LC/MS/MS

A simple, sensitive and selective method with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed to detect 342 pesticides and veterinary drugs contaminating bovine milk at the maximum residue limits (MRLs) defined in the "positive list system". Sample preparation was performed by extracting the analytes with acetonitrile, followed by salting-out with sodium chloride. For some pesticides, the extract was further cleaned up by n-hexane partitioning and PSA cartridge column chromatography. GC/MS-EI or -NCI was used to determine pesticide residues, while LC/MS/MS-ESI was applicable to the determination of pesticide and veterinary drug residues. The variation of the recoveries of these drugs at MRL was relatively wide; however the relative standard deviations of the recovery of each drug were within 28%, suggesting that the present method is good enough for use as a screening test for contaminants at the MRLs. These results show that this method is useful for multiresidue analysis of numerous pesticides and veterinary drugs in bovine milk.     

Determination of captafol,cyhexatin, 1-naphthylacetic acid and quintozene in apple,Japanese pear and melon by simultaneous extraction

A simple and rapid method is described for the determination of the non-registered pesticides, captafol, quintozene (PCNB), cyhexatin and 1-naphthylacetic acid (NAA), in fruits. These pesticides were extracted with acidified acetone, then captafol and PCNB were purified with a Florisil mini column and analyzed by GC-ECD. Cyhexatin was ethylated with ethylmagnesium bromide, and the ethyl derivative was analyzed by GC-FPD (Sn filter). NAA was purified with liquid-liquid extraction and determined by HPLC equipped with a fluorescence detector. These analytes were identified with GC/MS or LC/MS. The minimum identified concentration of the pesticides was below 0.2 ng per injection, which corresponds to a detection limit of below 0.02 microgram/g in the original samples. Recoveries of the pesticides spiked at 0.1 microgram/g into apple, Japanese pear and melon were greater than 61%.     

Size-dependent volatility of diesel nanoparticles: chassis dynamometer experiments

We analyzed the size-dependent volatility of nanoparticles in a diameter range of 30-70 nm in diesel exhaust emissions. The test system included a medium-duty diesel truck on a chassis dynamometer, a single-stage dilution tunnel, a tandem differential mobility analyzer (TDMA) equipped with an electric furnace, and a condensation particle counter. The size shifts of monodispersed diesel nanoparticles under changing furnace temperatures were measured by TDMA in the gas phase. Together with the reduction of average particle size and volume, we observed the development of bimodal size distributions resulting from the separation between semivolatile and nonvolatile species as the furnace temperature was increased. While 91-98% of the particles were found to be semivolatile species by total volume during the idling engine condition, only 6-9% were semivolatile during the one-half engine load condition. We also found that smaller particles contained a larger fraction of semivolatile species.     

Perfluorinated surfactants in surface, subsurface water and microlayer from Dalian Coastal waters in China

Two predominant perfluorinated compounds (PFCs), Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA), in surface water (SW, 0-20 cm), subsurface water (SSW, > 30 cm depth), and sea surface microlayer (SML, 50 microm thickness) were measured from Dalian Coastal waters in China. The SML samples were collected using glass-plate dipping method. Analysis of the PFCs was conducted through solid-phase extraction, followed by LC/MS-SIM. The PFC's concentrations in SW samples were consistent with previously reported data in this region. Significantly higher concentrations of PFCs were found in SML samples than corresponding SSW samples. The enrichment factors (EF = C(SML)/C(SSW)) for PFOS were as high as 24-109 atthree near-shore sites. The concentration in SW was also generally higher than corresponding SSW samples, giving C(SW)/C(SSW) mean ratios of 1.5 and 1.4 for PFOS and PFOA, respectively. This apparent enrichment of PFCs in surface water, especially in the microlayer, has implications for designing measurement techniques, understanding their distributions, and sea spray-mediated transport in the environment.