Association test algorithm between a qualitative phenotype and a haplotype or haplotype set using simultaneous estimation of haplotype frequencies, diplotype configurations and diplotype-based penetrances

Analysis of the association between haplotypes and phenotypes is becoming increasingly important. We have devised an expectation-maximization (EM)-based algorithm to test the association between a phenotype and a haplotype or a haplotype set and to estimate diplotype-based penetrance using individual genotype and phenotype data from cohort studies and clinical trials. The algorithm estimates, in addition to haplotype frequencies, penetrances for subjects with a given haplotype and those without it (dominant mode). Relative risk can thus also be estimated. In the dominant mode, the maximum likelihood under the assumption of no association between the phenotype and presence of the haplotype (L(0max)) and the maximum likelihood under the assumption of association (L(max)) were calculated. The statistic -2 log(L(0max)/L(max)) was used to test the association. The present algorithm along with the analyses in recessive and genotype modes was implemented in the computer program PENHAPLO. Results of analysis of simulated data indicated that the test had considerable power under certain conditions. Analyses of two real data sets from cohort studies, one concerning the MTHFR gene and the other the NAT2 gene, revealed significant associations between the presence of haplotypes and occurrence of side effects. Our algorithm may be especially useful for analyzing data concerning the association between genetic information and individual responses to drugs.     

A fluorescent probe-labeled Escherichia coli aspartate transcarbamoylase that monitors the allosteric conformational state

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.     

Regulatory mechanisms and function of ERK MAP kinases

Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is a key factor for determining the specificity of cellular responses including cell proliferation, cell differentiation and cell survival. The fidelity of this signaling is regulated by docking interactions as well as scaffolding. Subcellular localization of ERK is controlled by cytoplasmic ERK anchoring proteins that have a nuclear export signal (NES), such as MEK. In quiescent cells, ERK and MEK localize to the cytoplasm. In response to stimulation, dissociation of the MEK-ERK complex is induced and activated ERK translocates to the nucleus. Recently, several negative regulators for Ras/ERK signaling have been identified and their detailed molecular mechanisms have been analyzed. Among them, Sprouty and Sef act as a temporal and a spatial regulator, respectively, for Ras/ERK signaling. Thus, multiple factors are involved in control of Ras/ERK signaling.     

Shp2, an SH2-containing protein-tyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty

Src homology 2-containing phosphotyrosine phosphatase (Shp2) functions as a positive effector in receptor tyrosine kinase (RTK) signaling immediately proximal to activated receptors. However, neither its physiological substrate(s) nor its mechanism of action in RTK signaling has been defined. In this study, we demonstrate that Sprouty (Spry) is a possible target of Shp2. Spry acts as a conserved inhibitor of RTK signaling, and tyrosine phosphorylation of Spry is indispensable for its inhibitory activity. Shp2 was able to dephosphorylate fibroblast growth factor receptor-induced phosphotyrosines on Spry both in vivo and in vitro. Shp2-mediated dephosphorylation of Spry resulted in dissociation of Spry from Grb2. Furthermore, Shp2 could reverse the inhibitory effect of Spry on FGF-induced neurite outgrowth and MAP kinase activation. These findings suggest that Shp2 acts as a positive regulator in RTK signaling by dephosphorylating and inactivating Spry.     

Improved catalytic and stereoselective glycosylation with glycosyl N-trichloroacetylcarbamate: application to various 1-hydroxy sugars

Efficient catalytic and stereoselective glycosylation was achieved by activating a glycosyl N-trichloroacetylcarbamate with a catalytic amount of Lewis acid in the presence of a glycosyl acceptor and 5 ? molecular sieves. Catalytic one-pot dehydrative glycosylation of a 1-hydroxy carbohydrate was achieved stereoselectively by reaction with trichloroacetyl isocyanate, followed by activation with a catalytic amount of activators.     

Isolation of porphyran-degrading marine microorganisms from the surface of red alga, Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.     

Spatial changes in carbon and nitrogen stable isotopes of the plankton food web in a saline lake ecosystem

We investigated spatial changes in the isotope ratios of the plankton food web in Lake Chany, Siberia, Russia, especially at an estuarine transition zone of the lake. The δ¹³C values of particulate organic matter (POM) varied among the sampling sites, and increased with increasing pH of the lake water. This may reflect a shift by phytoplankton from using CO₂ to using bicarbonate for photosynthesis with increasing pH. The δ¹³C values of zooplankton community also changed at each site along with those of the POM. This was indicative of carbon isotope changes of plankton food webs between the stations along an environmental gradient.     

Chlamydospores of Rhizopus microsporus var. rhizopodiformis in Tissue of Pulmonary Mucormycosis

Hyphae are usually the only fungal elements found in tissue of mucormycosis, and other fungal elements are quite rarely encountered. We found chlamydospores in bronchial lumina in autopsied tissue of pulmonary mucormycosis of a diabetic patient. Chlamydospores are thick-walled, asexually produced spores arising from the modification of a hyphal segment. This is the first histologic demonstration of chlamydospores in mucormycosis in which the causative fungus is culturally identified to species level. Rhizopus microsporus var. rhizopodiformis was isolated from the present autopsied pulmonary tissue. A literature review of human infection by this fungus found 27 cases with histopathologic evidence.     

Aging modulates susceptibility to mouse liver Mallory-Denk body formation

Mallory-Denk bodies (MDBs) are hepatocyte cytoplasmic inclusions found in several liver diseases and consist primarily of the cytoskeletal proteins, keratins 8 and 18 (K8/K18). Recent evidence indicates that the extent of stress-induced protein misfolding, a K8>K18 overexpression state, and transglutaminase-2 activation promote MDB formation. In addition, the genetic background and gender play an important role in mouse MDB formation, but the effect of aging on this process is unknown. Given that oxidative stress increases with aging, the authors hypothesized that aging predisposes to MDB formation. They used an established mouse MDB model-namely, feeding non-transgenic male FVB/N mice (1, 3, and 8 months old) with 3,5 diethoxycarbonyl-1,4-dihydrocollidine for 2 months. MDB formation was assessed using immunofluorescence staining and biochemically by demonstrating keratin and ubiquitin-containing crosslinks generated by transglutaminase-2. Immunofluorescence staining showed that old mice had a significant increase in MDB formation compared with young mice. MDB formation paralleled the generation of high molecular weight ubiquitinated keratin-containing complexes and induction of p62. Old mouse livers had increased oxidative stress. In addition, 20S proteasome activity and autophagy were decreased, and endoplasmic reticulum stress was increased in older livers. Therefore, aging predisposes to experimental MDB formation, possibly by decreased activity of protein degradation machinery.