A new 4016-marker radiation hybrid map for porcine-human genome analysis

We constructed a 5000-rad comprehensive radiation hybrid (RH) map of the porcine (Sus scrofa) genome and compared the results with the human genome. Of 4475 typed markers, 4016 (89.7%) had LOD >5 compared with the markers used in our previous RH map by means of two-point analysis and were grouped onto the 19 porcine chromosomes (SSCs). All mapped markers had LOD >3 as determined by RHMAPPER analysis. The current map comprised 430 microsatellite (MS) framework markers, 914 other MS markers, and 2672 expressed sequence tags (ESTs). The whole-genome map was 8822.1 cR in length, giving an average marker density of 0.342 Mb/cR. The average retention frequency was 35.8%. Using BLAST searches of porcine ESTs against the RefSeq human nucleotide and amino acid sequences (release 22), we constructed high-resolution comparative maps of each SSC and each human chromosome (HSA). The average distance between ESTs in the human genome was 1.38 Mb. SSC contained 50 human chromosomal syntenic groups, and SSC11, SSC12, and SSC16 were only derived from the HSA13q, HSA17, and HSA5 regions, respectively. Among 38 porcine terminal regions, we found that at least 20 regions have been conserved between the porcine and human genomes; we also found four paralogous regions for the major histocompatibility complex (MHC) on SSC7, SSC2, SSC4, and SSC1. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

2-Aminoethyl diphenylborinate analogues: selective inhibition for store-operated Ca2+ entry

Capacitative calcium entry (CCE), the mechanism that replenishes the internal Ca2+ stores with Ca2+ from the extracellular milieu in response to depletion of the store, is mediated by Ca2+ channels in the plasma membrane generally referred to as store-operated channels (SOCs). However, the roles of SOCs in the more physiological context have been fully elucidated. 2-Aminoethyl diphenylborinate (2-APB) strongly inhibits SOCs, as well as inositol-1,4,5 trisphosphate (IP3) receptors. In the present study, we screened a library of 166 2-APB analogues for effects on CCE and IP3-induced Ca2+ release in order to discover specific SOC inhibitors, and found that some blocked both store-operated and receptor-operated Ca2+ influx more strongly and selectively than 2-APB. Indeed, these new compounds ceased the prolonged intracellular Ca2+ oscillations induced by a low concentration of ATP in CHO-K1 cells. These novel SOC inhibitors will be valuable pharmacological and biochemical tools for elucidating the physiological roles.     

Genomic regions affecting backfat thickness and cannon bone circumference identified by genome‐wide association study in a Duroc pig population

We performed a genome‐wide association study using the porcine 60K SNP array to detect QTL regions for nine traits in a three‐generational Duroc samples (n = 651), viz. generations 1, 2 and 3 from a population selected over five generations using a closed nucleus breeding scheme. We applied a linear mixed model for association mapping to detect SNP effects, adjusting for fixed effects (sex and season) and random polygenic effects (reflecting genetic relatedness), and derived a likelihood ratio statistic for each SNP using the efficient mixed‐model association method. We detected a region on SSC6 for backfat thickness (BFT) and on SSC7 for cannon bone circumference (CANNON), with a genome‐wide significance of P < 0.01 after Bonferroni correction. These regions had been detected previously in other pig populations. Six genes are located in the BFT‐associated region, while the CANNON‐associated region includes 66 genes. In the future, significantly associated SNPs, derived by sequencing the coding regions of the six genes in the BFT region, can be used in marker‐assisted selection of BFT, whereas haplotypes constructed from the SSC7 region with strong LD can be used to select for the CANNON trait in our resource family.     

The Relationships between Metabolic Disorders (Hypertension,Dyslipidemia, and Impaired Glucose Tolerance) and Computed Tomography-Based Indices of Hepatic Steatosis or Visceral Fat Accumulation in Middle-Aged Japanese Men

Background

Most studies on the relationships between metabolic disorders (hypertension, dyslipidemia, and impaired glucose tolerance) and hepatic steatosis (HS) or visceral fat accumulation (VFA) have been cross-sectional, and thus, these relationships remain unclear. We conducted a retrospective cohort study to clarify the relationships between components of metabolic disorders and HS/VFA.

Methods

The participants were 615 middle-aged men who were free from serious liver disorders, diabetes, and HS/VFA and underwent multiple general health check-ups at our institution between 2009 and 2013. The data from the initial and final check-ups were used. HS and VFA were assessed by computed tomography. HS was defined as a liver to spleen attenuation ratio of ≤1.0. VFA was defined as a visceral fat cross-sectional area of ≥100 cm² at the level of the navel. Metabolic disorders were defined using Japan’s metabolic syndrome diagnostic criteria. The participants were divided into four groups based on the presence (+) or absence (-) of HS/VFA. The onset rates of each metabolic disorder were compared among the four groups.

Results

Among the participants, 521, 55, 24, and 15 were classified as HS(-)/VFA(-), HS(-)/VFA(+), HS(+)/VFA(-), and HS(+)/VFA(+), respectively, at the end of the study. Impaired glucose tolerance was more common among the participants that exhibited HS or VFA (p = 0.05). On the other hand, dyslipidemia was more common among the participants that displayed VFA (p = 0.01).

Conclusions

It is likely that VFA is associated with impaired glucose tolerance and dyslipidemia, while HS might be associated with impaired glucose tolerance. Unfortunately, our study failed to detect associations between HS/VFA and metabolic disorders due to the low number of subjects that exhibited fat accumulation. Although our observational study had major limitations, we consider that it obtained some interesting results. HS and VFA might affect different metabolic disorders. Further large-scale longitudinal studies are needed to reveal the relationships between the components of metabolic disorders and HS/VFA.     

Angiotensin II stimulates DNA synthesis of rat pancreatic stellate cells by activating ERK through EGF receptor transactivation

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [3H]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation-ERK activation pathway.     

Quinate oxidation in Gluconobacter oxydans IFO3244: purification and characterization of quinoprotein quinate dehydrogenase

Quinoprotein quinate dehydrogenase (QDH) is a membrane-bound enzyme containing pyrroloquinoline quinone (PQQ) as the prosthetic group. QDH in Gluconobacter oxydans IFO3244 was found to be inducible by quinate and it is not constitutively expressed in the absence of quinate. The purification of holo-form of QDH to nearly homogeneity was achieved. The purified QDH appears to have two subunits of approximately 65 and 21 kDa, which could be the result of proteolysis of single polypeptide. Kinetic analysis indicated that the purified enzyme is much more specific to quinate than QDH from Acinetobacter calcoaceticus. The efficiency of the artificial electron acceptor was also determined.     

Single-molecule detection of phosphorylation-induced plasticity changes during ezrin activation

Ezrin-radixin-moesin protein family provides a regulated link between the cortical actin cytoskeleton and the plasma membrane. Phosphorylation of ezrin has been functionally linked to membrane dynamics and plasticity. Our recent study demonstrated that phosphorylation of the conserved T567 residue of ezrin alters the physiology of gastric parietal cells. However, the molecular mechanism of phosphorylation-induced ezrin activation has remained elusive. Here we use atomic force microscopy (AFM) to probe phosphorylation-mediated activation of ezrin in single molecules. The phospho-mimicking and non-phosphorylatable mutant ezrin proteins were generated and purified to homogeneity. Comparative analyses of two ezrin mutants by AFM demonstrate the unfolding of the N- and C-terminal domains upon the phospho-activation. To measure the physical force underlying the inter-domain contact during mechanical unfolding, we probed the defined region of ezrin using the N-terminal ezrin coated onto the AFM tip. Comparative force measurements indicate that T567 phosphorylation-induced unfolding of ezrin favors the inter-molecular association. Taken together, these results provide molecular illustration of phosphorylation elicited functional activation of ERM proteins and indicate that stimulus-induced protein conformational change can be used as a signaling mechanism orchestrating cellular dynamics.     

Impaired clustered protocadherin‐α leads to aggregated retinogeniculate terminals and impaired visual acuity in mice

Clustered protocadherins (cPcdhs) comprising cPcdh‐α, ‐β, and ‐γ, encode a large family of cadherin‐like cell‐adhesion molecules specific to neurons. Impairment of cPcdh‐α results in abnormal neuronal projection patterns in specific brain areas. To elucidate the role of cPcdh‐α in retinogeniculate projections, we investigated the morphological patterns of retinogeniculate terminals in the lateral geniculate (LG) nucleus of mice with impaired cPcdh‐α. We found huge aggregated retinogeniculate terminals in the dorsal LG nucleus, whereas no such aggregated terminals derived from the retina were observed in the olivary pretectal nucleus and the ventral LG nucleus. These aggregated terminals appeared between P10 and P14, just before eye opening and at the beginning of the refinement stage of the retinogeniculate projections. Reduced visual acuity was observed in adult mice with impaired cPcdh‐α, whereas the orientation selectivity and direction selectivity of neurons in the primary visual cortex were apparently normal. These findings suggest that cPcdh‐α is required for adequate spacing of retinogeniculate projections, which may be essential for normal development of visual acuity.

     

Allele-specific primer polymerase chain reaction for a single nucleotide polymorphism (C1205T) of swine toll-like receptor 5 and comparison of the allelic frequency among several pig breeds in Japan and the Czech Republic

In the present study, an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 5 (TLR5) (C1205T; P402L) that is related to the impaired recognition of Salmonella enterica serovar Choleraesuis (SC) was developed. The allele frequencies in several pig breeds in Japan and the Czech Republic were also compared. The swine TLR5 C1205T mutation was successfully determined by ASP-PCR using genomic DNA samples in Japan that had previously been genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from blood obtained from 110 pigs from seven different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR completely matched the results from the sequencing method. The allele frequency of the swine TLR5 C1205T mutation was 27.5% in the Landrace breed of the Czech Republic compared with 50.0% in Japanese Landrace. In Japan, the C1205T mutation was found only in the Landrace breed, whereas in the Czech Republic it was found in both the Landrace and Piétrain breeds. These results indicate the usefulness of ASP-PCR for detecting a specific SNP for swine TLR5 affecting ligand recognition. They also suggest the possibility of genetically improving pigs to enhance their resistance against SC infection by eliminating or selecting this specific SNP of swine TLR5.     

Biased distribution of single nucleotide polymorphisms (SNPs) in porcine Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR5, and TLR6 genes

Toll-like receptors (TLRs) recognize various microbial components and induce immune responses. Polymorphisms in TLRs may influence their recognition of pathogen-derived molecules; swine TLRs are predicted to be associated with responses to infectious diseases such as pneumonia. In this study, we searched for single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR1, TLR2, TLR4, TLR5, and TLR6 genes in 96 pigs from 11 breeds and elucidated 21, 11, 7, 13, and 11 SNPs, respectively, which caused amino acid substitutions in the respective TLRs. Distribution of these nonsynonymous SNPs was biased; many were located in the leucine-rich repeats, particularly in TLR1. These data demonstrated that the heterogeneity of TLR genes was preserved in various porcine breeds despite intensive breeding that was carried out for livestock improvement. It suggests that the heterogeneity in TLR genes is advantageous in increasing the possibility of survival in porcine populations.Electronic SupplementaryMaterial Supplementary material is available for this article at