Axenic cultivation of Entamoeba dispar in newly designed yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium

Yeast extract-iron-gluconic acid-dihydroxyacetone-serum medium that allows axenic cultivation of Entamoeba dispar was designed based on casein-free yeast extract-iron-serum (YI-S) medium, and the usefulness of the medium was assessed. The main differences from YI-S medium are replacement of glucose by gluconic acid, addition of dihydroxyacetone and D-galacturonic acid monohydrate, and sterilization by filtration. This medium promoted the axenic growth of 5 strains of E. dispar (2 strains of nonhuman primate isolates and 3 strains of human isolates). In addition, to clarify the biological basis for the growth of E. dispar in this medium, analyses of relevant enzymes on the glycolytic pathway of the amoebae as well as of the protozoans that are the best culture supplement for amoebae are being performed.     

Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with delta12-desaturase gene expression

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Delta12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in M. alpina 1S-4, we silenced the Delta12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Delta12-desaturation-defective mutants previously identified. Thus, RNAi occurs in M. alpina and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.     

A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair

In DNA damage responses, the Fanconi anemia (FA) protein, FancD2, is targeted to chromatin and forms nuclear foci following its monoubiquitination, a process likely catalyzed by the FA core complex. Here, we show that a chicken FancD2-ubiquitin fusion protein, carrying a Lys-Arg substitution removing the natural monoubiquitination site (D2KR-Ub), could reverse cisplatin hypersensitivity and localize to chromatin in FANCD2-deficient DT40 cells. Importantly, the chromatin targeting was dependent on three core complex components as well as the hydrophobic surface of ubiquitin that may direct protein-protein interactions. Furthermore, a constitutively chromatin bound fusion of D2KR-histone H2B could complement cisplatin sensitivity in FANCD2- but not FANCC-, FANCG-, or FANCL-deficient cells. Thus these core complex components have an additional function in the DNA repair, which is independent of the monoubiquitination and chromatin targeting of FancD2. These results define functional consequences of FancD2 monoubiquitination and reveal previously hidden functions for the FA protein core complex.     

The comparison between cool and warm-hot environments on lipolytic response during prolonged exercise

The purpose of this study was to investigate the effect of cool exposure on lipolytic response during prolonged intermediate-intensity exercise in humans. Eight male subjects participated in this study; they performed 120-min cycle ergometer exercise at 60% of maximal oxygen uptake (VO2max) in a climatic chamber at 10 degrees C (C) and 30 degrees C (WH). There were no significant differences in oxygen uptake and respiratory exchange ratio between the two conditions during the prolonged exercise. Significant influences of cool exposure were observed in the changes in both heart rate and rectal temperature (p<0.01). Although cool exposure had no significant effects on plasma triglyceride, free fatty acid, and glycerol levels, changes in adrenaline and noradrenaline levels at C were significantly lower than WH during the prolonged exercise (p<0.01). Changes in the ratio of glycerol to noradrenaline (Gly/Nad), as an index of lipolytic efficiency, were significantly high at C as compared with WH (p<0.01). These results suggest that cool exposure has an influence on lipid metabolism during prolonged intermediate-intensity exercise, from the viewpoint of efficiency in lipolysis.     

Structure-based functional identification of a novel heme-binding protein from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8

The TT1485 gene from Thermus thermophilus HB8 encodes a hypothetical protein of unknown function with about 20 sequence homologs of bacterial or archaeal origin. Together they form a family of uncharacterized proteins, the cluster of orthologous group COG3253. Using a combination of amino acid sequence analysis, three-dimensional structural studies and biochemical assays, we identified TT1485 as a novel heme-binding protein. The crystal structure reveals that this protein is a pentamer and each monomer exhibits a β-barrel fold. TT1485 is structurally similar to muconolactone isomerase, but this provided no functional clues. Amino acid sequence analysis revealed remote homology to a heme enzyme, chlorite dismutase. Strikingly, amino acid residues that are highly conserved in the homologous hypothetical proteins and chlorite dismutase cluster around a deep cavity on the surface of each monomer. Molecular modeling shows that the cavity can accommodate a heme group with a strictly conserved His as a heme ligand. TT1485 reconstituted with iron protoporphyrin IX chloride gave a low chlorite dismutase activity, indicating that TT1485 catalyzes a reaction other than chlorite degradation. The presence of a possible Fe–His–Asp triad in the heme proximal site suggests that TT1485 functions as a novel heme peroxidase to detoxify hydrogen peroxide within the cell.     

Inference of S-system models of genetic networks using a cooperative coevolutionary algorithm

MOTIVATION: To resolve the high-dimensionality of the genetic network inference problem in the S-system model, a problem decomposition strategy has been proposed. While this strategy certainly shows promise, it cannot provide a model readily applicable to the computational simulation of the genetic network when the given time-series data contain measurement noise. This is a significant limitation of the problem decomposition, given that our analysis and understanding of the genetic network depend on the computational simulation. RESULTS: We propose a new method for inferring S-system models of large-scale genetic networks. The proposed method is based on the problem decomposition strategy and a cooperative coevolutionary algorithm. As the subproblems divided by the problem decomposition strategy are solved simultaneously using the cooperative coevolutionary algorithm, the proposed method can be used to infer any S-system model ready for computational simulation. To verify the effectiveness of the proposed method, we apply it to two artificial genetic network inference problems. Finally, the proposed method is used to analyze the actual DNA microarray data.     

Food habits of sika deer in the Shiranuka Hills,eastern Hokkaido: a northern example from the north–south variations in food habits in sika deer

Seasonal changes in the composition of the diet of the sika deer population in the Shiranuka Hills, eastern Hokkaido, in 1998 were determined by fecal analyses. The deer were dependent on Sasa nipponica, a dwarf bamboo, throughout the year, particularly in winter when it accounted for as much as 77.7% of the diet. It accounted for 33.1% and 45.6% in spring and summer, respectively, and this decreased to 12.2% in autumn. Besides S. nipponica, all the graminoid categories accounted for large amounts (66–96.7%), while dicotyledonous plants accounted for little (3–8%) except in autumn when they accounted for 31%. The strong dependence of the Shiranuka population on graminoids was different from other Hokkaido deer populations, for example the population from Ashoro/Onbetsu and the extremely high density population on Nakanoshima Island. In spite of these differences, food for all Hokkaido sika deer was poor in winter. Along the north–south geographical cline in the food composition of sika deer along the Japanese archipelago, the Shiranuka population was positioned as a grazer type, in contrast to the southern populations. However, it is important to note that variations are great among local populations in Hokkaido.     

Four-cell stage mouse blastomeres have different developmental properties

Blastomeres of the early mouse embryo are thought to be equivalent in their developmental properties at least until the eight-cell stage. However, the experiments that have led to this conclusion could not have taken into account either the spatial origin of individual blastomeres or the spatial allocation and fate of their progeny. We have therefore readdressed this issue having defined cell lineages in mouse embryos undergoing different patterns of cleavage in their second division cycle. This has enabled us to identify a major group of embryos in which we can predict not only the spatial origin of each given four-cell blastomeres, but also which region of the blastocyst is most likely to be occupied by its progeny. We show that a pattern of second cleavage divisions in which a meridional division is followed by one that is equatorial or oblique allows us to identify blastomeres that differ in their fate and in their developmental properties both from each other and from their cousins. We find that one of these four-cell stage blastomeres that inherits some vegetal membrane marked in the previous cleavage cycle tends to contribute to mural trophectoderm. The progeny of its sister tend to donate cells to part of the ICM lining the blastocyst cavity and its associated trophectoderm. Chimaeras made entirely of these equatorially or obliquely derived blastomeres show developmental abnormalities in both late preimplantation and early postimplantation development. By contrast, chimaeras made from four-cell stage blastomeres from early meridional divisions develop normally. The developmental defects of chimaeras made from the most vegetal blastomeres that result from later second cleavages are the most severe and following transplantation into foster mothers they fail to develop to term. However, when such individual four-cell blastomeres are surrounded by blastomeres from random positions, they are able to contribute to all embryonic lineages. In conclusion, this study shows that while all four-cell blastomeres can have full developmental potential, they differ in their individual developmental properties according to their origin in the embryo from as early as the four-cell stage.     

Polymorphisms of TNFalpha, IL1beta, and IL1RN genes in chronic obstructive pulmonary disease

It is recognized that genetic factors play a role in the susceptibility to COPD. COPD is characterized by airflow limitation. Chronic inflammation causes small airway disease and parenchymal destruction, leading to the airflow limitation. Polymorphisms in pro-inflammatory cytokine genes may confer a risk for the development of COPD. A case-control association study was performed in Japanese population (88 COPD patients and 61 controls) and Egyptian population (106 patients and 72 controls). Genotype and allele frequencies of the TNFalpha -308 G/A and +489 G/A polymorphisms, the IL1beta -511 C/T, -31 T/C, and +3954 C/T polymorphisms, and a VNTR polymorphism in intron 2 of the IL1RN gene were investigated. In addition, pairwise haplotype frequencies were analyzed. When studied independently, none of the polymorphisms were associated with the development of COPD in both populations. However, in the Egyptian population, the distributions of the haplotype (IL1beta -31 T/C : IL1beta +3954 C/T) were significantly different between the COPD patients and the controls (p(corr)=0.0037). Our findings suggest that this haplotype within the IL1beta gene may be involved in the pathogenesis of COPD and that the genetic factors of COPD susceptibility might be different between different populations.