Preferential Myosin Heavy Chain Isoform B Expression May Contribute to the Faster Velocity of Contraction in Veins versus Arteries

Smooth muscle myosin heavy chains occur in 2 isoforms, SMA (slow) and SMB (fast). We hypothesized that the SMB isoform is predominant in the faster-contracting rat vena cava compared to thoracic aorta. We compared the time to half maximal contraction in response to a maximal concentration of endothelin-1 (ET-1; 100 nM), potassium chloride (KCl; 100 mM) and norepinephrine (NE; 10 µM). The time to half maximal contraction was shorter in the vena cava compared to aorta (aorta: ET-1 = 235.8 ± 13.8 s, KCl = 140.0 ± 33.3 s, NE = 19.8 ± 2.7 s; vena cava: ET-1 = 121.8 ± 15.6 s, KCl = 49.5 ± 6.7 s, NE = 9.0 ± 3.3 s). Reverse-transcription polymerase chain reaction supported the greater expression of SMB in the vena cava compared to aorta. SMB was expressed to a greater extent than SMA in the vessel wall of the vena cava. Western analysis determined that expression of SMB, relative to total smooth muscle myosin heavy chains, was 12.5 ± 4.9-fold higher in the vena cava compared to aorta, while SMA was 4.9 ± 1.2-fold higher in the aorta than vena cava. Thus, the SMB isoform is the predominant form expressed in rat veins, providing one possible mechanism for the faster response of veins to vasoconstrictors

Erratum: Corrigendum: Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution

International Chicken Genome Sequencing Consortium. The Original Article was published on 09 December 2004. Nature432, 695–716 (2004). In Table 5 of this Article, the last four values listed in the ‘Copy number’ column were incorrect. These should be: LTR elements, 30,000; DNA transposons, 20,000; simple repeats, 140,000; and satellites, 4,000. These errors do not affect any of the conclusions in our paper. Additional information. The online version of the original article can be found at 10.1038/nature0315

Alignment of the linkage map, physical map, and sequence of the chicken genome

Four BAC libraries constructed at Texas A&M University and at the Children's Hospital of Oakland Research Institute providing altogether ~28X coverage of the chicken genome have been screened with most of the suitable genes and markers that are found on the chicken genetic linkage map (Schmid et al., 2000). This has provided the means to align the chicken BAC contig physical map (Ren et al., 2003) with the linkage map and assign most contigs to their appropriate location on a chicken chromosome. It also has aided in assembly of the first draft sequence of the chicken genome. Our screening approach of choice is overgo hybridization, using four-dimensional pooling (36 probes at a time, one degree of redundancy), chosen from a matrix of 216 probes per screen. All BACs assigned to chicken genes and markers have been incorporated into a database available online (https://www.msu.edu/~dodgson/resources/resources.htm#bacdata). As of October 2003, the database includes nearly 5000 BAC assignments for 577 markers across almost all chicken chromosomes and linkage groups. On-going overgo hybridization is being used to do fine structure comparative mapping between the human and chicken genomes and to close gaps detected in the first round chicken sequence assembly. Overgo hybridization provides a cost-effective, high throughput method for integration of physical maps and linkage maps for domestic animal (and plant) species. Supported by the USDA/CSREES (Project numbers: 99-35205-8566 and 2001-52100-11225)

Integration of chicken linkage and physical maps and sequence alignment using overgo hybridization

Research is underway to align the chicken genetic linkage map, defined primarily by segregation of DNA-based markers, with the BAC contig physical map (Ren et al., in press). This aids the assembly of BAC and genome sequence contigs and alignment of contigs with chicken chromosomes. Efforts to date have focused on the use of overgo oligonucleotide probes, ca. 40 nucleotide double-stranded DNAs, hybridized to BAC filter arrays. Four BAC libraries are now in use (BamHI, EcoRI, and HindIII insert libraries, Texas A&M, and CHORI-261, Children's Hospital of Oakland Research Institute, all using DNA from a single UCD001 inbred Jungle Fowl). A 6x6x6 matrix of 216 probes is employed, with each probe designed based on DNA sequence information for a specific mapped marker or gene. Hybridization is done with pools of 36 probes at a time, such that one probe is uniquely contained in a combination of 3 pools, each from a different dimension (row, column or plate). In our hands, a fourth, redundant dimension of six additional pools is desirable. All BACs assigned to chicken genes and markers have been incorporated into a database available online (http://poultry.mph.msu.edu/resources/Resources.htm#bacdata). As of October 2003, the database includes nearly 5000 BAC assignments for 577 markers across almost all chicken chromosomes and linkage groups. Overgo hybridization provides a cost-effective, high throughput method for integration of physical maps, sequences and linkage maps for domestic animal (and plant) species. Supported, in part, by the USDA/CSREES (Project numbers: 99-35205-8566 and 2001-52100-11225

PAHs Target Hematopoietic Linages in Bone Marrow through Cyp1b1 Primarily in Mesenchymal Stromal Cells but Not AhR: A Reconstituted In Vitro Model

7,12-Dimethylbenz(a)anthracene (DMBA) rapidly suppresses hematopoietic progenitors, measured as colony forming units (CFU), in mouse bone marrow (BM) leading to mature cell losses as replenishment fails. These losses are mediated by Cyp1b1, independent of the AhR, despite induction of Cyp1b1. BM mesenchymal progenitor cells (MPC) may mediate these responses since basal Cyp1b1 is minimally induced. PreB colony forming unit activity (PreB CFU) is lost within 24 hours in isolated BM cells (BMC) unless cocultured with cells derived from primary MPC (BMS2 line). The mouse embryonic OP9 line, which provides more efficient coculture support, shares similar induction-resistant Cyp1b1 characteristics. This OP9 support is suppressed by DMBA, which is then prevented by Cyp1b1 inhibitors. OP9-enriched medium partially sustains CFU activities but loses DMBA-mediated suppression, consistent with mediation by OP9 Cyp1b1. PreB CFU activity in BMC from Cyp1b1-ko mice has enhanced sensitivity to DMBA. BMC gene expression profiles identified cytokines and developmental factors that are substantially changed in Cyp1b1-ko mice. DMBA had few effects in WT mice but systematically modified many clustered responses in Cyp1b1-ko mice. Typical BMC AhR-responsive genes were insensitive to Cyp1b1 deletion. TCDD replicated Cyp1b1 interventions, suggesting alternative AhR mediation. Cyp1b1 also diminishes oxidative stress, a key cause of stem cell instability

Oxidative Stress Alters miRNA and Gene Expression Profiles in Villous First Trimester Trophoblasts

The relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in preeclampsia (PE) is not fully elucidated. We investigated the impact of oxidative stress on miRNAs and mRNA expression profiles of genes associated with PE in villous 3A first trimester trophoblast cells exposed to H2O2 at 12 different concentrations (0-1 mM) for 0.5, 4, 24, and 48 h. Cytotoxicity, determined using the SRB assay, was used to calculate the IC50 of H2O2. RNA was extracted after 4 h exposure to H2O2 for miRNA and gene expression profiling. H2O2 exerted a concentration- and time-dependent cytotoxicity on 3A trophoblast cells. Short-term exposure of 3A cells to low concentration of H2O2 (5% of IC50) significantly altered miRNA profile as evidenced by significant changes in 195 out of 595 evaluable miRNAs. Tool for annotations of microRNAs (TAM) analysis indicated that these altered miRNAs fall into 43 clusters and 34 families, with 41 functions identified. Exposure to H2O2 altered mRNA expression of 22 out of 84 key genes involved in dysregulation of placental development. In conclusion, short-term exposure of villous first trimester trophoblasts to low concentrations of H2O2 significantly alters miRNA profile and expression of genes implicated in placental development

A Comprehensive Haplotype Analysis of the XPC Genomic Sequence Reveals a Cluster of Genetic Variants Associated with Sensitivity to Tobacco-Smoke Mutagens

The impact of single-nucleotide polymorphisms (SNPs) of the DNA repair gene XPC on DNA repair capacity (DRC) and genotoxicity has not been comprehensively determined. We constructed a comprehensive haplotype map encompassing all common XPC SNPs and evaluated the effect of Bayesian-inferred haplotypes on DNA damage associated with tobacco smoking, using chromosome aberrations (CA) as a biomarker. We also used the mutagen-sensitivity assay, in which mutagen-induced CA in cultured lymphocytes are determined, to evaluate the haplotype effects on DRC. We hypothesized that if certain XPC haplotypes have functional effects, a correlation between these haplotypes and baseline and/or mutagen-induced CA would exist. Using HapMap and single nucleotide polymorphism (dbSNP) databases, we identified 92 SNPs, of which 35 had minor allele frequencies ≥ 0.05. Bayesian inference and subsequent phylogenetic analysis identified 21 unique haplotypes, which segregated into six distinct phylogenetically grouped haplotypes (PGHs A–F). A SNP tagging approach used identified 11 tagSNPs representing these 35 SNPs (r2 = 0.80). We utilized these tagSNPs to genotype a population of smokers matched to nonsmokers (n = 123). Haplotypes for each individual were reconstituted and PGH designations were assigned. Relationships between XPC haplotypes and baseline and/or mutagen-induced CA were then evaluated. We observed significant interaction among smoking and PGH-C (p = 0.046) for baseline CA where baseline CA was 3.5 times higher in smokers compared to nonsmokers. Significant interactions among smoking and PGH-D (p = 0.023) and PGH-F (p = 0.007) for mutagen-induced CA frequencies were also observed. These data indicate that certain XPC haplotypes significantly alter CA and DRC in smokers and, thus, can contribute to cancer risk