Amperometric estimation of BOD by using living immobilized yeasts

Summary A microbial electrode consisting of immobilized living whole cells of yeasts, porous membrane and an oxygen electrode was prepared for continuous estimation of biochemical oxygen demand (BOD). Immobilized Trichosporon cutaneum was employed for the microbial electrode sensor for BOD. When a sample solution containing the equivalent amount of glucose and glutamic acid was injected into the sensor system, the current of the electrode decreased markedly with time until steady state was reached. The response time was within 18 min. A linear relationship was observed between the current decrease and the concentration below 41 mg l ^– of glucose and 41 mg l ^– glutamic acid (5-day BOD 60 mg l ^–). The current decrease was reproducible within ± 6% of the relative error when a sample solution containing 27 mg l ^– of glucose and 27 mg l ^– of glutamic acid (5-day BOD 40 mg l ^–) was employed. The microbial electrode sensor was applied to untreated waste waters from a fermentation factory. Good comparative results were obtained between BOD estimated by the microbial electrode and that determined by the conventional 5-day method (regression coefficient was 1.2). Furthermore, the effect of various compounds on BOD estimation was also examined. The current output of the microbial electrode sensor was almost constant for 17 d and 400 tests.     

HLA antigens in Japanese populations.

HLA antigens in 841 healthy, unrelated Japanese from nine widely separated geographic localities were studied. The five most common antigens observed in order of decreasing frequency were for the HLA-A locus: HLA-A9, A2, A10, AW32 and A11; and for the HLA-B locus: HLA-'B5' (= HLA-B5+B17), BW40, B12, B14 and B8. The allelic frequency of undetected antigens of the HLA-A locus was .14-.37, and that of the HLA-B locus, .32-.67, indicating that there were serological difficulties in typing for Japanese antigens using antisera from Caucasians. Marked gene frequency clines were observed for HLA-A9 and HLA-A2 from south (Okinawa) to north (Nagoya). Two haplotypes, HLA-A9, B5 and HLA-A10, BW40 were shown to be in linkage disequilibrium in four of the nine subpopulations.     

Excitotoxic Insult Results in a Long-Lasting Activation of CaMKIIα and Mitochondrial Damage in Living Hippocampal Neurons

Over-activation of excitatory NMDA receptors and the resulting Ca²⁺ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1–3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca²⁺ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca²⁺-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults.     

Genetic structure of Pacific crown-of-thorns starfish (Acanthaster cf. solaris) in southern Japan based on genome-wide RADseq analysis

Coral Reefs - Pacific crown-of-thorns starfish (Acanthaster cf. solaris) are known to be widely distributed, and previous studies (showing limited genetic structure) suggest that there are high...     

Cortisol promotes stress tolerance via DAF-16 in Caenorhabditis elegans

In this study, we studied the effects of cortisol and cortisone on the age-related decrease in locomotion in the nematode Caenorhabditis elegans and on the tolerance to heat stress at 35 °C and to oxidative stress induced by the exposure to 0.1% H₂O₂. Changes in mRNA expression levels of C. elegans genes related to stress tolerance were also analyzed. Cortisol treatment restored nematode movement following heat stress and increased viability under oxidative stress, but also shortened worm lifespan. Cortisone, a cortisol precursor, also restored movement after heat stress. Additionally, cortisol treatment increased mRNA expression of the hsp-12.6 and sod-3 genes. Furthermore, cortisol treatment failed to restore movement of daf-16-deficient mutants after heat stress, whereas cortisone failed to restore the movement of dhs-30-deficient mutants after heat stress. In conclusion, the results suggested that cortisol promoted stress tolerance via DAF-16 but shortened the lifespan, whereas cortisone promoted stress tolerance via DHS-30.     

Simultaneous Determination of Purine and Pyrimidine Metabolites in Hprt-Deficient Cell Lines

Genetic mutations in the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT), are known to cause Lesch–Nyhan syndrome and Kelley–Seegmiller syndrome. In patients, purine metabolism is different from that of normal persons. We have previously developed a method for simultaneously determining the concentration of purine and pyrimidine nucleosides and nucleotides. This system was applied to determine the concentrations of nucleosides and nucleotides in HPRT-deficient cell lines. The amount of inosine 5′-monophosphate (IMP) was different in Lesch–Nyhan syndrome, Kelley–Seegmiller syndrome, and control cell lines. The difference in the amount of IMP confirmed the mutation of the enzyme.     

A heat‐inducible CRE/LOXP gene induction system in medaka

We established three lines of transgenic medaka, a heat‐shock element (HSE) monitor line (hse‐GFP line), heat‐inducible driver lines (hse‐cre lines), and effector lines (gapdh‐loxP[DsRed]‐GFP lines). We employed these to comprehensively analyze gene induction at different time points in various tissues. These analyses demonstrate a good response of synthetic HSEs by heat treatment during embryogenesis and the mosaic gene induction by cre/loxP‐mediated recombination, thus providing practical information regarding the feasibility of a heat‐inducible cre/loxP‐mediated system in medaka. We also activated recombination by local heat‐treatment using a metal probe and an infrared laser. Our results collectively indicate that these lines allow us to perform lineage tracing and mosaic analysis and provide the platform to investigate gene functions at later developmental stage and adult. genesis 51:59–67, 2013. © 2012 Wiley Periodicals, Inc.     

Ovarian Cancer Stem Cells Are Enriched in Side Population and Aldehyde Dehydrogenase Bright Overlapping Population

Cancer stem-like cells (CSCs)/cancer-initiaiting cells (CICs) are defined as a small population of cancer cells that have self-renewal capacity, differentiation potential and high tumor-initiating ability. CSCs/CICs of ovarian cancer have been isolated by side population (SP) analysis, ALDEFLUOR assay and using cell surface markers. However, these approaches are not definitive markers for CSCs/CICs, and it is necessary to refine recent methods for identifying more highly purified CSCs/CICs. In this study, we analyzed SP cells and aldehyde dehydrogenese bright (ALDH^Br) cells from ovarian cancer cells. Both SP cells and ALDH^Br cells exhibited higher tumor-initiating ability and higher expression level of a stem cell marker, sex determining region Y-box 2 (SOX2), than those of main population (MP) cells and ALDH^Low cells, respectively. We analyzed an SP and ALDH^Br overlapping population (SP/ALDH^Br), and the SP/ALDH^Br population exhibited higher tumor-initiating ability than that of SP cells or ALDH^Br cells, enabling initiation of tumor with as few as 10² cells. Furthermore, SP/ADLH^Br population showed higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDH^Low, MP/ALDH^Br and MP/ALDH^Low cells. Gene knockdown of SOX2 suppressed the tumor-initiation of ovarian cancer cells. An SP/ALDH^Br population was detected in several gynecological cancer cells with ratios of 0.1% for HEC—1 endometrioid adenocarcinoma cells to 1% for MCAS ovary mucinous adenocarcinoma cells. Taken together, use of the SP and ALDH^Br overlapping population is a promising approach to isolate highly purified CSCs/CICs and SOX2 might be a novel functional marker for ovarian CSCs/CICs.     

Structural Basis of α-Catenin Recognition by EspB from Enterohaemorrhagic E. coli Based on Hybrid Strategy Using Low-Resolution Structural and Protein Dissection

Enterohaemorrhagic E. coli (EHEC) induces actin reorganization of host cells by injecting various effectors into host cytosol through type III secretion systems. EspB is the natively partially folded EHEC effector which binds to host α-catenin to promote the actin bundling. However, its structural basis is poorly understood. Here, we characterize the overall structural properties of EspB based on low-resolution structural data in conjunction with protein dissection strategy. EspB showed a unique thermal response involving cold denaturation in the presence of denaturant according to far-UV circular dichroism (CD). Small angle X-ray scattering revealed the formation of a highly extended structure of EspB comparable to the ideal random coil. Various disorder predictions as well as CD spectra of EspB fragments identified the presence of α-helical structures around G41 to Q70. The fragment corresponding to this region indicated the thermal response similar to EspB. Moreover, this fragment showed a high affinity to C-terminal vinculin homology domain of α-catenin. The results clarified the importance of preformed α-helix of EspB for recognition of α-catenin.