Genetic profile of the SMXA recombinant inbred mouse strains revealed with restriction landmark genomic scanning

We have applied the restriction landmark genomic scanning (RLGS) method to the SMXA recombinant inbred (RI) mouse strain set to reveal its detailed genetic profile. A total of 663 polymorphic RLGS spot loci were identified, 576 of which were assigned to chromosomes. Strain distribution patterns (SDPs) at 55 microsatellite marker loci were also obtained. As a result, the total number of loci with distinct SDPs on chromosomes increased to 400. These loci were dispersed on all chromosomes, except for the Chromosome (Chr) Y, and effectively covered the genome with an average spacing of 4 cM. The SMXA RI strain set, hereby, would be of value for genetic study. Received: 20 February 1998 / Accepted: 19 May 1998     

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.     

Estimating hominid life history: the critical interbirth interval

Unlike any great apes, humans have expanded into a wide variety of habitats during the course of evolution, beginning with the transition by australopithecines from forest to savanna habitation. Novel environments are likely to have imposed hominids a demographic challenge due to such factors as higher predation risk and scarcer food resources. In fact, recent studies have found a paucity of older relative to younger adults in hominid fossil remains, indicating considerably high adult mortality in australopithecines, early Homo, and Neanderthals. It is not clear to date why only human ancestors among all hominoid species could survive in these harsh environments. In this paper, we explore the possibility that hominids had shorter interbirth intervals to enhance fertility than the extant apes. To infer interbirth intervals in fossil hominids, we introduce the notion of the critical interbirth interval, or the threshold length of birth spacing above which a population is expected to go to extinction. We develop a new method to obtain the critical interbirth intervals of hominids based on the observed ratios of older adults to all adults in fossil samples. Our analysis suggests that the critical interbirth intervals of australopithecines, early Homo, and Neanderthals are significantly shorter than the observed interbirth intervals of extant great apes. We also discuss possible factors that may have caused the evolutionary divergence of hominid life history traits from those of great apes.     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.     

Studies on the relationship between paracoccidioidomycosis in ddY mice and their estrous cycle

The relationship between paracoccidioidomycosis in ddY mouse and its estrous cycle was studied. Adult ddY mice of both sexes were used as experimental animals. Estrous cycle of female mice was examined before inoculation of Paracoccidioides brasiliensis yeast cells and mice were divided into 5 groups such as proestrus, estrus, metestrus-I, metestrus-II and diestrus. Each mouse was inoculated intravenously with 10⁶ P. brasiliensis yeast cell units and sacrificed on day 28 after inoculation. Their internal organs were cultured, and in addition, their histopathologies were studied. As a result, there was no difference in the organ cultures among the male and the female mice of 5 groups. However, histopathologically, the female groups at estrus, metestrus-I and metestrus-II were affected more severely than the male group, and the susceptibility of the female mice to the fungus was closely related to their estrous cycles.Abbreviations BHI-D brain heart infusion agar supplemented with 1.0% of anhydrous dextrose - PAS periodic acid-Schiff techniques - PBS phosphate buffered saline solution - SD standard deviation     

Possible role of ILK-affixin complex in integrin-cytoskeleton linkage during platelet aggregation

Integrin-mediated adhesion induces the formation of focal adhesions that link the extracellular matrix and intracellular actin cytoskeletal networks. We previously showed that integrin-linked kinase (ILK), which can interact with beta1 and beta3 integrins, and its interacting protein, affixin, play an essential role in the initial assembly of focal adhesion structures and actin stress fibers. Although the relevant structures are also observed in integrin alphaIIbbeta3 in platelets, the precise underlying molecular mechanism remains unclarified. Here, we found that ILK stably forms a complex with ss-affixin in platelets. Thrombin stimulation induces their association with integrin beta3, which is followed by their incorporation into the Triton-insoluble membrane-cytoskeletal fraction. During the course of thrombin-induced platelet aggregation, ILK activity was enhanced within 90s to 2.1-fold of the basal level, independent of phosphatidylinositol 3-kinase. Taken together with the observation that the treatment with an anti-integrin beta3 antibody stimulates ILK activity without inducing platelet aggregation, these results suggest that the outside-in signaling induced by fibrinogen binding to integrin enhances ILK activity and results in the initial phase to reorganize the actin cytoskeleton.     

Clasping structure in the double cone of the retina of the turtle (Gyoclemys reevesii)

Summary The double cone of the turtle retina was reconstructed three-dimensionally from electron micrographs of 400 serial sections. Cytoplasm of the accessory cone extends bilaterally to surround the principal cone incompletely. The cytoplasmic extensions are 0.1 m in width and 2 m in length at their longest portions. They arise at approximately the median portion of the ellipsoid (consisting of mitochondria) and terminate at the level of the outer limiting membrane. The possible function of these extensions as a clasping structure is discussed.     

Genetic Diversity and Distribution of the Ciguatera-Causing Dinoflagellate Gambierdiscus spp. (Dinophyceae) in Coastal Areas of Japan

Background

The marine epiphytic dinoflagellate genus Gambierdiscus produce toxins that cause ciguatera fish poisoning (CFP): one of the most significant seafood-borne illnesses associated with fish consumption worldwide. So far, occurrences of CFP incidents in Japan have been mainly reported in subtropical areas. A previous phylogeographic study of Japanese Gambierdiscus revealed the existence of two distinct phylotypes: Gambierdiscus sp. type 1 from subtropical and Gambierdiscus sp. type 2 from temperate areas. However, details of the genetic diversity and distribution for Japanese Gambierdiscus are still unclear, because a comprehensive investigation has not been conducted yet.

Methods/Principal Finding

A total of 248 strains were examined from samples mainly collected from western and southern coastal areas of Japan during 2006–2011. The SSU rDNA, the LSU rDNA D8–D10 and the ITS region were selected as genetic markers and phylogenetic analyses were conducted. The genetic diversity of Japanese Gambierdiscus was high since five species/phylotypes were detected: including two reported phylotypes (Gambierdiscus sp. type 1 and Gambierdiscus sp. type 2), two species of Gambierdiscus (G. australes and G. cf. yasumotoi) and a hitherto unreported phylotype Gambierdiscus sp. type 3. The distributions of type 3 and G. cf. yasumotoi were restricted to the temperate and the subtropical area, respectively. On the other hand, type 1, type 2 and G. australes occurred from the subtropical to the temperate area, with a tendency that type 1 and G. australes were dominant in the subtropical area, whereas type 2 was dominant in the temperate area. By using mouse bioassay, type 1, type 3 and G. australes exhibited mouse toxicities.

Conclusions/Significance

This study revealed a surprising diversity of Japanese Gambierdiscus and the distribution of five species/phylotypes displayed clear geographical patterns in Japanese coastal areas. The SSU rDNA and the LSU rDNA D8–D10 as genetic markers are recommended for further use.