Rat mutations cvd and hob with cerebellar malformations map to chromosome 2.

In this paper, we executed genome mapping and comparative mapping analyses for cvd and hob, autosomal recessive mutations with cerebellar vermis defect and cerebellar dysplasia in the rat. For the linkage analysis, we produced three sets of backcross progeny, (ACI x CVD)F(1) and (F344 x CVD)F(1) females crossed to a cvd homozygous male rat, and (HOB x WKY)F(1) males crossed to hob homozygous female rats. Analysis of the segregation patterns of simple sequence length polymorphism (SSLP) markers scanning the whole rat genome allowed the mapping of these autosomal recessive mutations to rat Chromosome (Chr) 2. The most likely gene order is D2Mgh12 - D2Rat86 - D2Mit15 - D2Rat185 - cvd - D2Rat66 - D2Mgh13, and D2Mit18 - Fga -D2Mit14 - D2Rat16 - hob - D2Mgh13. Crossing test between a proven cvd heterozygous and a hob heterozygous rats demonstrated their allelism. Furthermore, comparative mapping indicated the cvd locus corresponds to mouse chromosome 3 and a strong candidate gene Unc5h3, a causative gene for the rostral cerebellar malformation mouse, was implicated.     

Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function

Thyroid-stimulating hormone (TSH) controls thyroid growth and hormone secretion through binding to its G protein-coupled receptor (TSHR) and production of cyclic AMP (cAMP). Serum TSH is a sensitive indicator of thyroid function, and overt abnormalities in thyroid function lead to common endocrine disorders affecting approximately 10% of individuals over a life span. By genotyping 362,129 SNPs in 4,300 Sardinians, we identified a strong association (p = 1.3 x 10(-11)) between alleles of rs4704397 and circulating TSH levels; each additional copy of the minor A allele was associated with an increase of 0.13 muIU/ml in TSH. The single-nucleotide polymorphism (SNP) is located in intron 1 of PDE8B, encoding a high-affinity cAMP-specific phosphodiesterase. The association was replicated in 4,158 individuals, including additional Sardinians and two genetically distant cohorts from Tuscany and the Old Order Amish (overall p value = 1.9 x 10(-20)). In addition to association of TSH levels with SNPs in PDE8B, our genome scan provided evidence for association with PDE10A and several biologically interesting candidates in a focused analysis of 24 genes. In particular, we found evidence for association of TSH levels with SNPs in the THRB (rs1505287, p = 7.3 x 10(-5)), GNAQ (rs10512065, p = 2.0 x 10(-4)), TG (rs2252696, p = 2.2 x 10(-3)), POU1F1 (rs1976324, p = 3.9 x 10(-3)), PDE4D (rs27178, p = 8.3 x 10(-3)), and TSHR (rs4903957, p = 8.6 x 10(-3)) loci. Overall, the results suggest a primary effect of PDE8B variants on cAMP levels in the thyroid. This would affect production of T4 and T3 and feedback to alter TSH release by the pituitary. PDE8B may thus provide a candidate target for the treatment of thyroid dysfunction.     

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.     

Impaired autophagy by soluble endoglin,under physiological hypoxia in early pregnant period,is involved in poor placentation in preeclampsia

In early pregnancy, trophoblasts and the fetus experience hypoxic and low-nutrient conditions; nevertheless, trophoblasts invade the uterine myometrium up to one third of its depth and migrate along the lumina of spiral arterioles, replacing the maternal endothelial lining. Here, we showed that autophagy, an intracellular bulk degradation system, occurred in extravillous trophoblast (EVT) cells under hypoxia in vitro and in vivo. An enhancement of autophagy was observed in EVTs in early placental tissues, which suffer from physiological hypoxia. The invasion and vascular remodeling under hypoxia were significantly reduced in autophagy-deficient EVT cells compared with wild-type EVT cells. Interestingly, soluble endoglin (sENG), which increased in sera in preeclamptic cases, suppressed EVT invasion by inhibiting autophagy. The sENG-inhibited EVT invasion was recovered by TGFB1 treatment in a dose-dependent manner. A high dose of sENG inhibited the vascular construction by EVT cells and human umbilical vein endothelial cells (HUVECs), meanwhile a low dose of sENG inhibited the replacement of HUVECs by EVT cells. A protein selectively degraded by autophagy, SQSTM1, accumulated in EVT cells in preeclamptic placental biopsy samples showing impaired autophagy. This is the first report showing that impaired autophagy in EVT contributes to the pathophysiology of preeclampsia.     

Identifying sequence determinants of reduction potentials of metalloproteins

The reduction potential of an electron transfer protein is one of its most important functional characteristics. Although the type of redox site and the protein fold are the major determinants of the reduction potential of a redox-active protein, its amino acid sequence may tune the reduction potential as well. Thus, homologous proteins can often be divided into different classes, with each class characterized by a biological function and a reduction potential. Site-specific mutagenesis of the sequence determinants of the differences in the reduction potential between classes should change the reduction potential of a protein in one class to that of the other class. Here, a procedure is presented that combines energetic and bioinformatic analysis of homologous proteins to identify sequence determinants that are also good candidates for site-specific mutations, using the [4Fe–4S] ferredoxins and the [4Fe–4S] high-potential iron–sulfur proteins as examples. This procedure is designed to guide site-specific mutations or more computationally expensive studies, such as molecular dynamics simulations. To make the procedure more accessible to the general scientific community, it is being implemented into CHARMMing, a Web-based portal, with a library of density functional theory results for the redox site that are used in the setting up of Poisson–Boltzmann continuum electrostatics calculations for the protein energetics.     

Quantitation using a stable isotope dilution assay (SIDA) and thresholds of taste-active pyroglutamyl decapeptide ethyl esters (PGDPEs) in sake

A stable isotope dilution assay (SIDA) for two taste-active pyroglutamyl decapeptide ethyl esters (PGDPE1; (pGlu)LFGPNVNPWCOOC₂H₅, PGDPE2; (pGlu)LFNPSTNPWCOOC₂H₅) in sake was developed using deuterated isotopes and high-resolution mass spectrometry. Recognition thresholds of PGDPEs in sake were estimated as 3.8 μg/L for PGDPE1 and 8.1 μg/L for PGDPE2, evaluated using 11 student panelists aged in their twenties. Quantitated concentrations in 18 commercial sake samples ranged from 0 to 27 μg/L for PGDPE1 and from 0 to 202 μg/L for PGDPE2. The maximum levels of PGDPE1 and PGDPE2 in the sake samples were approximately 8 and 25 times higher than the estimated recognition thresholds, respectively. The results indicated that PGDPEs may play significant sensory roles in the sake. The level of PGDPEs in unpasteurized sake samples decreased during storage for 50 days at 6 °C, suggesting PGDPEs may be enzymatically decomposed.     

A familial case of visceral toxocariasis due to consumption of raw bovine liver

We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in their livers and lungs. Serological examinations using the larval excretory–secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.     

Multiple mechanisms for accumulation of myosin II filaments at the equator during cytokinesis

Total internal reflection fluorescence microscopy revealed how individual bipolar myosin II filaments accumulate at the equatorial region in dividing Dictyostelium cells. Direct observation of individual filaments in live cells provided us with much convincing information. Myosin II filaments accumulated at the equatorial region by at least two independent mechanisms: (i) cortical flow, which is driven by myosin II motor activities and (ii) de novo association to the equatorial cortex. These two mechanisms were mutually redundant. At the same time, myosin II filaments underwent rapid turnover, repeating their association and dissociation with the actin cortex. Examination of the lifetime of mutant myosin filaments in the cortex revealed that the turnover mainly depended on heavy chain phosphorylation and that myosin motor activity accelerated the turnover. Double mutant myosin II deficient in both motor and phosphorylation still accumulated at the equatorial region, although they displayed no cortical flow and considerably slow turnover. Under this condition, the filaments stayed for a significantly longer time at the equatorial region than at the polar regions, indicating that there are still other mechanisms for myosin II accumulation such as binding partners or stabilizing activity of filaments in the equatorial cortex.     

Structure,interaction and real‐time monitoring of the enzymatic reaction of wild‐type APOBEC3G

Human APOBEC3G exhibits anti‐human immunodeficiency virus‐1 (HIV‐1) activity by deaminating cytidines of the minus strand of HIV‐1. Here, we report a solution structure of the C‐terminal deaminase domain of wild‐type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real‐time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3′ → 5′ order. Virus infectivity factor (Vif) of HIV‐1 counteracts the anti‐HIV‐1 activity of APOBEC3G. The structure of the N‐terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species‐specific sensitivity of APOBEC3G to Vif action.     

Laboratory appraisal of optimal gaseous conditions for growth of zoonotic Helicobacter felis ATCC 49179

An attempt was made to assess the hitherto undescribed optimal gaseous conditions for growth of zoonotic Helicobacter felis, focusing on the ratio of spiral-forms amongst the whole cells examined. The largest mean colony diameter was obtained under the gaseous condition of O₂ 12% and CO₂ 10%. In analyzing the five day old colonies, the highest percentage of spiral forms (85.5%) was observed under the condition of O₂ 18% and CO₂ 5%. In contrast, the lowest percentage of spiral forms (2.3%) was demonstrated under the condition of O₂ 1% and CO₂ 10%. The condition of O₂ 12% and CO₂ 10% was concluded to be optimal for obtaining cells with the largest colony sizes, although colonies proliferated under such conditions definitely contain many more coccoid cells than spiral forms. In culturing H. felis strains, optimal gaseous conditions should be employed according to the purposes or preferences of study designs.