Changes in gamma-aminobutyric acid content during beni-koji making

The changes in the gamma-aminobutyric acid (GABA) content during the making of beni-koji prepared with Monascus pilosus IFO 4520 vs. the difference in the rate of tomo koji (10%, 30%, and 50%) were examined. The increased proportion of tomo koji would increase the GABA production and the productions of GABA peaked on the fifth day and thereafter declined. The glutamate decarboxylase activity during beni-koji making with 50% tomo koji steadily increased after the start of the koji making, reaching its peak on the fifth day. The succinic acid content increased after the sixth day. The mycelial growth was in the stationary phase after the sixth day. Therefore, the GABA content increases with an increase in the proportion of tomo koji. It is presumed that the maximum amount of GABA reached on the fifth day was the cause of the increasing amount of conversion of GABA into succinic acid, in addition to the decline in the GAD activity after the fifth day of koji making.     

Histological change after interferon therapy in chronic hepatitis C in view of iron deposition in the liver

We examined the efficacy of interferon (IFN) therapy for chronic hepatitis C (CHC) in view of the change of liver histology and iron staining before and after IFN therapy. Enrolled in this study were 109 patients with CHC who completed IFN treatment and were followed for at least 1 yr after the end of IFN therapy. Serum iron, unsaturated-iron-binding capacity (UIBC), and total-iron-binding capacity (TIBC) were assessed before IFN therapy. Knodell’s histological activity index (HAI) score and iron staining were examined in 55 patients in whom liver biopsy was performed at two points: before and 1 yr after IFN therapy. Serum iron levels before IFN therapy did not correlate with the response to IFN. The HAI score significantly decreased after IFN therapy in complete responders (p<0.01) and biochemical responders (p<0.01). Three factors in the HAI, periportal necrosis, intralobular necrosis, and portal inflammation, but not fibrosis, were significantly decreased in complete responders (p<0.01) and biochemical responders (p<0.01). Of 55 patients, 23 (41.8%) were positive for iron staining before IFN therapy and 14 of 55 (25.5%) after IFN therapy. The positive rate for iron staining tended to decrease after IFN therapy, not correlating to the response to IFN, but the change was not statistically significant. In conclusion, the histological improvement by IFN therapy was mostly seen in necroinflammatory changes but not in fibrosis at least 1 yr after IFN, and iron staining tended to decrease after IFN therapy.     

Convection of waters in Lakes Maninjau and Singkarak,tropical oligomictic lakes

The ecology of a lake is mainly controlled by mixing processes; particularly, in tropical oligomictic lakes, the occurrence frequency and magnitude of convection govern the vertical mixing of chemicals and organisms. In this study, vertical profiles of water temperature, dissolved oxygen, electric conductivity, turbidity, and chlorophyll a were measured in 2014, 2015, 2017, and 2018 in two Sumatran deep lakes, Lakes Maninjau and Singkarak. In Lake Maninjau, intensive surveys on the profiles were also conducted in three different seasons in 2018. The comparison of the profiles between 2015 and 2017 indicated the events of large convection down to the lake bottoms happened in both of the lakes. Similarly, small convection down to around 30 m depth was found in the period between May and Jul, 2018. Air temperature drops up to five degrees centigrade were observed in these periods, confirmed by the changes in lake surface temperature estimated by MODIS imagery for the lakes. The magnitudes of the convective events were discussed with the observed amounts of heat loss and the estimated heat transfer through lake surface. Furthermore, the influences of such events on anoxic hypolimnetic waters were evaluated and considered from the view of climate change.

     

Cutting edge: a critical role of nitric [corrected] oxide in preventing inflammation upon apoptotic cell clearance

Apoptotic cells are removed by phagocytes without causing inflammation. It remains largely unresolved whether anti-inflammatory mediators prevent neutrophil infiltration upon apoptotic cell clearance in vivo. In this study, we showed that, upon induction of apoptosis in the thymus by x-ray, inducible NO synthase knockout (KO) mice exhibited higher levels of neutrophil infiltration and production of MIP-2 and keratinocyte-derived chemokine (KC) in the thymus than wild-type (WT) mice. Furthermore, administration of NG-nitro-L-arginine methyl ester, an inhibitor of NO synthase, to x-irradiated WT mice increased the level of neutrophil infiltration to that of KO mice by the augmentation of MIP-2 and KC production. Additionally, thymic macrophages isolated from x-irradiated KO mice produced more MIP-2 and KC than those from WT mice. Thus, although apoptosis is believed to be noninflammatory, this is actually achieved by the production of immunosuppressive signals such as NO that counteract proinflammatory chemokines such as MIP-2 and KC.     

Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation

Many G-protein-coupled receptors can activate more than one G-protein subfamily member. Leukotriene B4 receptor type 1 (BLT1) is a high affinity G-protein-coupled receptors for leukotriene B4 functioning in host defense, inflammation, and immunity. Previous studies have shown that BLT1 utilizes different G-proteins (the Gi family and G16 G-proteins) in mediating diverse cellular events and that truncation of the cytoplasmic tail of BLT1 does not impair activation of Gi and G16 proteins. To determine responsive regions of BLT1 for G-protein coupling, we performed an extensive mutagenesis study of its intracellular loops. Three intracellular loops (i1, i2, and i3) of BLT1 were found to be important for both Gi and G16 coupling, as judged by Gi-dependent guanosine 5'-(gamma-thio) triphosphate (GTPgammaS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPgammaS binding but intact inositol phosphate accumulation triggered by leukotriene B4 stimulation. These results suggest that the i3-1 region is required only for Gi activation. Moreover, in the i3-1 mutant, the deficiency in Gi activation was accompanied by a loss of the high affinity leukotriene B4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of Gi proteins.     

DNA recombination protein-dependent mechanism of homoplasmy and its proposed functions

Homoplasmy is a basic genetic state of mitochondria, in which all of the hundreds to thousands of mitochondrial (mt)DNA copies within a cell or an individual have the same nucleotide-sequence. It was recently found that "vegetative segregation" to generate homoplasmic cells is an active process under genetic control. In the yeast Saccharomyces cerevisiae, the Mhr1 protein which catalyzes a key reaction in mtDNA homologous recombination, plays a pivotal role in vegetative segregation. Conversely, within the nuclear genome, homologous DNA recombination causes genetic diversity. Considering these contradictory roles of this key reaction in DNA recombination, possible functions of homoplasmy are discussed.     

Genetic structure of three Oryza AA genome species (O. rufipogon, O. nivara and O. sativa) as assessed by SSR analysis on the Vientiane Plain of Laos

Microsatellite (SSR) markers can reveal a high level of polymorphic loci, and are increasingly being used in population genetic structure studies. On the Vientiane plain of Laos all components of the rice crop complex exist, wild annual (O. nivara), wild perennial (O. rufipogon) and weedy relatives of rice as well as rice itself. To understand gene flow in the rice complex, the genetic structures of O. rufipogon (10 populations), O. nivara (10 populations) and O. sativa (24 samples) from across the Vientiane Plain, Laos, were compared. Higher genetic differentiation was detected among O. nivara populations (G _ST = 0.77, R _ST = 0.71) than O. rufipogon populations (G _ST = 0.29, R _ST = 0.28), whereas genetic diversity for all populations of these two wild species showed similar values (H _T = 0.77 and 0.64 in O. rufipogon and O. nivara, respectively). Based on neighbor-joining tree constructed on the basis of genetic distance (D _A), three genetic clusters were detected, corresponding to (1) O. sativa samples, (2) O. nivara populations and (3) O. rufipogon populations. Pairwise tests confirmed the genetic differentiation of the three species. Although none of the wild rice individuals used in this study had any cultivated-specific phenotypic traits, genetic admixture analysis detected more than 10% O. sativa membership in three O. rufipogon and one O. nivara populations, indicating that O. sativa alleles may cryptically persist in natural populations of O. rufipogon and O. nivara on the Vientiane Plain.     

Nonrandom RNAseq gene expression associated with RNAlater and flash freezing storage methods

RNA sequencing is a popular next‐generation sequencing technique for assaying genome‐wide gene expression profiles. Nonetheless, it is susceptible to biases that are introduced by sample handling prior gene expression measurements. Two of the most common methods for preserving samples in both field‐based and laboratory conditions are submersion in RNAlater and flash freezing in liquid nitrogen. Flash freezing in liquid nitrogen can be impractical, particularly for field collections. RNAlater is a solution for stabilizing tissue for longer‐term storage as it rapidly permeates tissue to protect cellular RNA. In this study, we assessed genome‐wide expression patterns in 30‐day‐old fry collected from the same brood at the same time point that were flash‐frozen in liquid nitrogen and stored at ?80°C or submerged and stored in RNAlater at room temperature, simulating conditions of fieldwork. We show that sample storage is a significant factor influencing observed differential gene expression. In particular, genes with elevated GC content exhibit higher observed expression levels in liquid nitrogen flash‐freezing relative to RNAlater storage. Further, genes with higher expression in RNAlater relative to liquid nitrogen experience disproportionate enrichment for functional categories, many of which are involved in RNA processing. This suggests that RNAlater may elicit a physiological response that has the potential to bias biological interpretations of expression studies. The biases introduced to observed gene expression arising from mimicking many field‐based studies are substantial and should not be ignored.     

Nonlinear force-length relationship in the ADP-induced contraction of skeletal myofibrils

The regulatory mechanism of sarcomeric activity has not been fully clarified yet because of its complex and cooperative nature, which involves both Ca²⁺ and cross-bridge binding to the thin filament. To reveal the mechanism of regulation mediated by the cross-bridges, separately from the effect of Ca²⁺, we investigated the force-sarcomere length (SL) relationship in rabbit skeletal myofibrils (a single myofibril or a thin bundle) at SL > 2.2 μm in the absence of Ca²⁺ at various levels of activation by exogenous MgADP (4-20 mM) in the presence of 1 mM MgATP. The individual SLs were measured by phase-contrast microscopy to confirm the homogeneity of the striation pattern of sarcomeres during activation. We found that at partial activation with 4-8 mM MgADP, the developed force nonlinearly depended on the length of overlap between the thick and the thin filaments; that is, contrary to the maximal activation, the maximal active force was generated at shorter overlap. Besides, the active force became larger, whereas this nonlinearity tended to weaken, with either an increase in [MgADP] or the lateral osmotic compression of the myofilament lattice induced by the addition of a macromolecular compound, dextran T-500. The model analysis, which takes into account the [MgADP]-and the lattice-spacing-dependent probability of cross-bridge formation, was successfully applied to account for the force-SL relationship observed at partial activation. These results strongly suggest that the cross-bridge works as a cooperative activator, the function of which is highly sensitive to as little as ≤1 nm changes in the lattice spacing.