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At least 325 records · Page 18Linked to original sources
PubMed2004

T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages.

The fecal microbiota of two healthy Swedish infants was monitored over time by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Principal component analysis (PCA) of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during breast-feeding and a major change occurred after weaning. The two infants had different sets of microbiota at all sampling time points. 16S rDNA clone libraries were constructed and the predominant terminal restriction fragments (T-RFs) were identified by comparing T-RFLP patterns in the fecal community with that of corresponding 16S rDNA clones. Sequence analysis indicated that the infants were initially colonized mostly by members of Enterobacteriaceae, Veillonella, Enterococcus, Streptococcus, Staphylococcus and Bacteroides. The members of Enterobacteriaceae and Bacteroides were predominant during breast-feeding in both infants. However, Enterobacteriaceae decreased while members of clostridia increased after weaning. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes.

Base Sequence↗
PubMed2004

Extra-embryonic vasculature development is regulated by the transcription factor HAND1.

The basic helix-loop-helix (bHLH) transcription factor HAND1 (also called eHAND) is expressed in numerous tissues during development including the heart, limbs, neural crest derivatives and extra-embryonic membranes. To investigate the role of Hand1 during development, we generated a Hand1 knockout mouse. Hand1-null mice survived to the nine somite stage at which time they succumbed to numerous developmental defects. One striking defect in Hand1-null embryos was the accumulation of hematopoietic cells between the yolk sac and the amnion because of defects in the yolk sac vasculature. In Hand1-null yolk sacs, vasculogenesis occurs but vascular refinement was arrested. Analysis of angiogenic genes in extra-embryonic membranes showed that most are expressed at normal levels in Hand1-null embryos but several, including Vegf, Ang1 and ephrin B2, and gene components of the Notch pathway are upregulated. In the absence of Hand1 the expression of the bHLH factor Hand2 is also enhanced. Although HAND1 and HAND2 share many structural features, and Hand2 is required for vasculature development in yolk sacs, enhanced expression of Hand2 is insufficient to compensate for the loss of Hand1. The most striking aspect of the vascular defect in Hand1 mutant yolk sacs is the abnormal distribution of smooth muscle cells. During normal angiogenesis, vascular smooth muscle precursors are recruited to the peri-endothelial tissue before differentiation, however, in Hand1 null yolk sacs, smooth muscle cells are not recruited but differentiate in clusters distributed throughout the mesoderm. These data indicate that Hand1 is required for angiogenesis and vascular smooth muscle recruitment in the yolk sac.

Animals↗
PubMed1998

Dexamethasone but not indomethacin inhibits human phagocyte nicotinamide adenine dinucleotide phosphate oxidase activity by down-regulating expression of genes encoding oxidase components.

We investigated the effects of dexamethasone or indomethacin on the NADPH oxidase activity, cytochrome b558 content, and expression of genes encoding the components gp91-phox and p47-phox of the NADPH oxidase system in the human monocytic THP-1 cell line, differentiated with IFN-gamma and TNF-alpha, alone or in combination, for up to 7 days. IFN-gamma and TNF-alpha, alone or in combination, caused a significant up-regulation of the NADPH oxidase system as reflected by an enhancement of the PMA-stimulated superoxide release, cytochrome b558 content, and expression of gp91-phox and p47-phox genes on both days 2 and 7 of cell culture. Noteworthy was the tremendous synergism between IFN-gamma and TNF-alpha for all studied parameters. Dexamethasone down-regulated the NADPH oxidase system of cytokine-differentiated THP-1 cells as assessed by an inhibition on the PMA-stimulated superoxide release, cytochrome b558 content, and expression of the gp91-phox and p47-phox genes. The nuclear run-on assays indicated that dexamethasone down-regulated the NADPH oxidase system at least in part by inhibiting the transcription of gp91-phox and p47-phox genes. Indomethacin inhibited only the PMA-stimulated superoxide release of THP-1 cells differentiated with IFN-gamma and TNF-alpha during 7 days. None of the other parameters was affected by indomethacin. We conclude that dexamethasone down-regulates the NADPH oxidase system at least in part by inhibiting the expression of genes encoding the gp91-phox and p47-phox components of the NADPH oxidase system.

Anti-Inflammatory Agents↗
PubMed2005

TLR-2 is involved in airway epithelial cell response to air pollution particles.

Primary cultures of normal human airway epithelial cells (NHBE) respond to ambient air pollution particulate matter (PM) by increased production of the cytokine IL-8, and the induction of several oxidant stress response genes. Components of ambient air PM responsible for stimulating epithelial cells have not been conclusively identified, although metal contaminants, benzo[a]pyrene and biological matter have been implicated. Stimulation of IL-8 release from NHBE with coarse (PM(2.5-10)), fine (PM2.5), and UF particle fractions has shown that the coarse particle fraction has the greatest effect on the epithelial cells as well as alveolar macrophages (AM). Since this fraction concentrates fugitive dusts and particle-associated microbial matter, it was hypothesized that NHBE may recognize PM through microbial pattern recognition receptors TLR2 and TLR4, as has been previously shown with AM. NHBE were shown to release IL-8 when exposed to a Gram-positive environmental isolate of Staphylococcus lentus, and lower levels when exposed to Gram-negative Pseudomonas spp. Comparison of TLR2 and TLR4 mRNA expression in NHBE and AM showed that NHBE express similar levels of TLR2 mRNA as the AM, but expressed very low levels of TLR4. When NHBE were stimulated with PM(2.5-10), PM2.5, and UF PM, in the presence or absence of inhibitors of TLR2 and TLR4 activation, a blocking antibody to TLR2 inhibited production of IL-8, while TLR4 antagonist E5531 or the LPS inhibitor Polymixin B had no effect. Furthermore, effects on expression of TLR2 and TLR4 mRNA, as well as the stress protein HSP70 was assessed in NHBE exposed to PM. TLR4 expression was increased in these cells while TLR2 mRNA levels were unchanged. Hsp70 was increased by PM(2.5-10) > PM2.5 > UF PM suggesting the possibility of indirect activation of TLR pathway by this endogenous TLR2/4 agonist.

Adolescent↗
PubMed1999

Study on the formation of specialized inter-Sertoli cell junctions in vitro.

An in vitro culture system using Sertoli cells was employed to assess the expression of component genes pertinent to occluding junctions (OJ) (such as zonula occludens-1, ZO-1), anchoring junctions (AJ) (such as N-cadherin and beta-catenin), and communicating gap junctions (GJ) (such as connexin 33, Cx33) when they are being formed in vitro. Freshly isolated Sertoli cells from 20-day-old rats with a purity of greater than 90% were cultured either at low- (2.5 x 10(4) cells/cm(2)) or high-cell density (0.6 x 10(6) cells/cm(2)) on Matrigel-coated dishes for 7 days in vitro to allow the establishment of specialized junctions. In low cell density Sertoli cell cultures, specialized OJ such as tight junctions did not form during the entire culture period when assessed by the transepithelial electrical resistance (TER). In high cell density cultures, there was an increase in ZO-1 expression in days 1 to 3 preceding the establishment of tight junctions by day 4. When Sertoli cells were cultured at both cell densities, there was a transient increase in Sertoli cell N-cadherin expression, which peaked by days 4-5, suggesting the time course for the establishment of AJ may overlap with the OJ. A significant increase in the expression of Sertoli cell beta-catenin was also detected by days 5-7 in the high but not low cell density cultures. The expression of Cx33 was also enhanced at days 4-5 in both high and low density cultures. These results suggest that OJ, AJ, and GJ are formed between Sertoli cells in high density cultures, whereas OJ cannot be formed in low density cultures. A full-length cDNA clone coding for rat testicular beta-catenin was also isolated. The deduced amino acid sequence of rat beta-catenin yielded a 781 amino acid polypeptide which displayed a 99.9% identity with the mouse homolog. Conditioned medium of germ cells induced a dose-dependent stimulation on Sertoli cell beta-catenin expression, suggesting germ cells may affect the N-cadherin/beta-catenin-mediated signal transduction pathway. In summary, this study illustrates several target genes can be used as molecular markers to monitor the inter-Sertoli junction formation. This system should be applicable to screen new male contraceptives in vitro targeted at the interference of junction formation by disrupting the timely expression of genes necessary for junction establishment and/or maintenance.

Amino Acid Sequence↗
PubMed2004

The keratins and their disorders.

Diseases caused by mutations in gene encoding keratin intermediate filaments (IF) are characterized by a loss of structural integrity in the cells expressing those keratins in vivo. This is manifested as cell fragility, compensatory epidermal hyperkeratosis, and keratin filament aggregation in some affected tissues. Keratin disorders are a novel molecular category including quite different phenotypes such as epidermolysis bullosa simplex (EBS), bullous congenital ichthyosiform erthroderma (BCIE), pachyonychia congenital (PC), steatocystoma multiplex, ichthyosis bullosa of Siemens (IBS), and white sponge nevus (WSN) of the orogenital mucosa.

Epidermis↗
PubMed2005

Molecular biology of the invertebrate dopamine receptors.

Dopamine is found in the nervous systems of both vertebrates and invertebrates. However, the specific actions of dopamine depend on the dopamine receptor type that is expressed in the target cell. As in mammals, different subtypes of dopamine receptors have been cloned and characterized from invertebrates, and these receptor subtypes have different structural and functional properties. Understanding how these receptors respond to dopamine and in which cells each receptor type is expressed is key to our understanding of the role of dopamine signaling. Comparison of the amino acid sequences and experimentally determined functional properties suggest that there are at least three distinct types of dopamine receptors in invertebrates. This review focuses on invertebrate dopamine receptors for which the genes have been isolated and identified, and examines our current knowledge of the functional and structural properties of these receptors, and their pharmacology and expression.

Amino Acid Sequence↗
PubMed2003

In vivo regulation of the chicken cardiac troponin T gene promoter in zebrafish embryos.

The chicken cardiac troponin T (cTnT) gene is representative of numerous cardiac and skeletal muscle-specific genes that contain muscle-CAT (MCAT) elements within their promoters. We examined the regulation of the chicken cTnT gene in vivo in zebrafish embryos, and in vitro in cardiomyocyte, myoblast, and fibroblast cultures. Defined regions of the cTnT promoter were linked to the green fluorescent protein (GFP) gene for in vivo analysis, and the luciferase gene for in vitro analysis. Injection of the cTnT promoter constructs into fertilized zebrafish eggs resulted in GFP expression in both heart and skeletal muscle cells reproducing the pattern of expression of the endogenous cTnT gene in the chicken embryo. Promoter deletion analysis revealed that the cis-regulatory regions responsible for cardiac and skeletal muscle-specific expression functioned in an equivalent manner in both in vitro and in vivo environments. In addition, we show that mutation of the poly-ADP ribose polymerase-I (PARP-I) binding site adjacent to the distal MCAT element in the chicken cTnT promoter produced a non-cell-specific promoter in vitro and in the zebrafish. Thus, the PARP-I transcriptional regulatory mechanism that governs muscle specificity of the chicken cTnT promoter is conserved across several chordate classes spanning at least 350 million years of evolution.

Animals↗
PubMed2005

Analysis of the regulation of lin-41 during chick and mouse limb development.

We have cloned the chicken and mouse orthologues of the Caenorhabditis elegans heterochronic gene lin-41. During limb development, lin-41 is expressed in three phases over developmental time and most notably is associated with the developing autopod. Using chicken and mouse mutants and bead implantations, we report that lin-41 is genetically and biochemically downstream of both the Shh and Fgf signaling pathways. In C. elegans, it is proposed that lin-41 activity is temporally regulated by miRNAs (let-7 and lin-4) that bind to complementary sites in the lin-41 3'-untranslated region (UTR). Taking a bioinformatics approach, we also report the presence of potential miRNA binding sites in the 3'-UTR of chicken lin-41, including sites for the chicken orthologues of both C. elegans let-7 and lin-4. Finally, we show that these miRNAs and others are expressed in the chick limb consistent with the hypothesis that they regulate chicken Lin-41 activity in vivo.

Amino Acid Sequence↗
PubMed2005

Reciprocal expression of lin-41 and the microRNAs let-7 and mir-125 during mouse embryogenesis.

In C. elegans, heterochronic genes control the timing of cell fate determination during development. Two heterochronic genes, let-7 and lin-4, encode microRNAs (miRNAs) that down-regulate a third heterochronic gene lin-41 by binding to complementary sites in its 3'UTR. let-7 and lin-4 are conserved in mammals. Here we report the cloning and sequencing of mammalian lin-41 orthologs. We find that mouse and human lin-41 genes contain predicted conserved complementary sites for let-7 and the lin-4 ortholog, mir-125, in their 3'UTRs. Mouse lin-41 (Mlin-41) is temporally expressed in developing mouse embryos, most dramatically in the limb buds. Mlin-41 is down-regulated during mid-embryogenesis at the time when mouse let-7c and mir-125 RNA levels are up-regulated. Our results suggest that mammalian lin-41 is temporally regulated by miRNAs in order to direct key developmental events such as limb formation.

Amino Acid Sequence↗
PubMed2007

Myocardin-related transcription factor B is required for normal mouse vascular development and smooth muscle gene expression.

Smooth muscle gene expression is required for the proper development and function of multiple organ systems. Expression of smooth muscle genes is critical for contractile function and tissue architectural integrity. One critical transcription factor for smooth muscle gene expression is the Serum Response Factor (SRF). SRF is expressed ubiquitously, but tissue-specific transcriptional regulation is conferred by its binding to cofactors such as myocardin. Myocardin-related transcription factor B (MRTF-B) is a member of a family of genes (Myocardin(Myocd),Myocardin-related transcription factor A(MRTF-A),MRTF-B) that provides tissue-specificity and potentiate SRF-dependent transcription. Unlike myocardin, which is expressed specifically in smooth and cardiac muscle, MRTF-B is expressed in a wide variety of tissues. To examine the function of MRTF-B, we generated mice containing an insertional mutation of MRTF-B. MRTF-B homozygous mutants die in late gestation with vascular defects and liver hemorrhage. At E9.5, MRTF-B is expressed strongly in the septum transversum mesoderm critical for development of the vitelline system that produces the liver sinusoids and portal venous system. MRTF-B deficiency results in defective smooth muscle gene expression in the liver sinusoids, vitelline veins, and yolk sac, which contributes significantly to the lethal phenotype. These data support our hypothesis that MRTF-B has a unique role in regulating smooth muscle genes important for liver, yolk sac, and portal vascular development.

Animals↗
PubMed2006

Recombinase-mediated cassette exchange to rapidly and efficiently generate mice with human cardiac sodium channels.

SCN5A encodes the predominant voltage-gated sodium channel isoform in human heart and nearly 100 variants have now been described and studied in vitro. However, development of animal models to analyze function of such large numbers of human gene variants represents a continuing challenge in translational medicine. Here, we describe the implementation of a two stage procedure, recombinase-mediated cassette exchange (RMCE), to efficiently and rapidly generate mice in which a full-length human cDNA replaces expression of the murine ortholog. In the first step of RMCE, conventional homologous recombination in mouse ES cells was used to replace scn5a exon 2 (that contains the translation start site) with a cassette acceptor that includes the thymidine kinase gene, flanked by loxP/inverted loxP sites. In the second step, the cassette acceptor site was replaced by the full-length wild-type human SCN5A cDNA by Cre/loxP-mediated recombination. The exchange event occurred in 7/29 (24%) colonies, and the time from electroporation to first homozygotes was only 8 months. PCR-restriction fragment length polymorphism (RFLP) showed that the murine isoform was replaced by the human one, and functional studies indicated that mice with human cardiac sodium channels have wild-type sodium current density, action potential durations, heart rates, and QRS durations. These data demonstrate that RMCE can be used to generate mice in which a targeted allele can be rapidly and efficiently replaced by variants of choice, and thereby can serve as an enabling approach for the functional characterization of ion channel and other DNA variants.

Animals↗
PubMed2006

Generation of conditional Cited2 null alleles.

Cited2 is a transcriptional co-factor that is widely expressed in both embryonic and extraembryonic cells during early development. It is essential for embryonic development with Cited2 null embryos showing abnormal development of organs including heart, neural tube, adrenal glands, and placenta (both in trophoblast derivatives and invading fetal vasculature), as well as having defects in the establishment of the left-right body axis. We report the generation of two conditional null alleles allowing Cre-recombinase-mediated somatic cell gene inactivation. Mice heterozygous or homozygous for these alleles are viable and fertile. Crossing conditional mutants with CMV-Cre transgenic mice produces an embryonic-lethal phenotype in the offspring indistinguishable from germline null mutants. We also demonstrate that conditional deletion results in lacZ expression under the control of the Cited2 promoter. These alleles are therefore useful genetic tools for dissecting the functions of Cited2 in the formation of different organs and patterning of the developing embryo. genesis

Alleles↗
PubMed2006

Conditional inactivation of the Cacna1a gene in transgenic mice.

Ca(v)2.1 (P/Q-type) voltage-gated calcium channels play an important role in neurotransmitter release at many brain synapses and at the neuromuscular junction. Mutations in the CACNA1A gene, encoding the pore forming alpha(1) subunit of Ca(v)2.1 channels, are associated with a wide spectrum of neurological disorders. Here we generated mice with a conditional, floxed, Cacna1a allele without any overt phenotype. Deletion of the floxed Cacna1a allele resulted in ataxia, dystonia, and lethality during the fourth week, a severe phenotype similar to conventional Ca(v)2.1 knockout mice. Although neurotransmitter release at the neuromuscular junction was not affected in the conditional mice, homozygous deletion of the floxed allele caused an ablation of Ca(v)2.1 channel-mediated neurotransmission that was accompanied by a compensatory upregulation of Ca(v)2.3 (R-type) channels at this synapse. Pharmacological inhibition of Ca(v)2.1 channels is possible, but the contributing cell-types and time windows relevant to the different Ca(v)2.1-related neurological disorders can only be reliably determined using Cacna1a conditional mice.

Animals↗
PubMed2006

Targeting iCre expression to murine progesterone receptor cell-lineages using bacterial artificial chromosome transgenesis.

Gene-targeting in embryonic stem cells has been the dominant genetic approach when engineering mouse models to query the physiologic importance of the progesterone receptor (PR). Although these models have been instrumental in disclosing the in vivo significance of the progesterone signaling pathway, generation of such mice exacts considerable expenditure of time, effort, and expense. Considering the growing list of new PR mouse models that are urgently required to address the next questions in progestin biology, bacterial artificial chromosome (BAC) recombineering in conjunction with transgenesis was evaluated as an alternative method to accelerate the creation of these models in the future. Using this approach, we describe the generation of three PR-BAC(iCre) transgenic lines in which improved Cre recombinase (iCre) was targeted in-frame, downstream, and under the control of the PR promoter contained within a BAC transgene. Crossing with the ROSA26R revealed that the PR-BAC(iCre) transgenic expresses active iCre only in cell-lineages that express the PR. The specificity of the PR-BAC(iCre) transgene not only underscores the importance of BAC-mediated transgenesis as a quick, easy, and affordable method by which to engineer the next generation of PR mouse models, but also provides a unique opportunity to investigate transcriptional control of PR expression as well as PR structure-function relationships in vivo.

Animals↗
PubMed2004

Efficient DNA cassette exchange in mouse embryonic stem cells by staggered positive-negative selection.

Recombinase-mediated cassette exchange (RMCE), when applied to mouse embryonic stem (ES) cells, promises to increase the ease with which genetic alterations can be introduced into targeted genomic loci in the mouse. However, existing selection strategies for identifying ES cells in which replacement DNA cassettes from a carrier plasmid have been exchanged correctly into a defined locus are suboptimal. Here, we report the generation in mouse ES cells of a loxed cassette acceptor (LCA) allele within the glucokinase (gk) gene locus. Using the gkLCA as a test allele, we developed a staggered positive-negative selection strategy that facilitates efficient identification of ES cell clones in which a DNA replacement cassette from a carrier plasmid has been exchanged correctly into the gkLCA allele. This selection strategy, by facilitating more efficient production of ES cell clones with various replacement DNA cassettes, should accelerate targeted repetitive introduction of gene modifications into the mouse.

Alleles↗
PubMed2004

Hypervolemic hypertension in mice with systemic inactivation of the (floxed) guanylyl cyclase-A gene by alphaMHC-Cre-mediated recombination.

To dissect the tissue-specific functions of atrial natriuretic peptide (ANP), we recently introduced loxP sites into the murine gene for its receptor, guanylyl cyclase-A (GC-A), by homologous recombination (tri-lox GC-A). For either smooth-muscle or cardiomyocyte-restricted deletion of GC-A, floxed GC-A mice were mated to transgenic mice expressing Cre-recombinase under the control of the smooth-muscle SM22 or the cardiac alphaMHC promoter. As shown in these studies, Cre-mediated recombination of the floxed GC-A gene fully inactivated GC-A function in a cell-restricted manner. In the present study we show that alphaMHC-Cre, but not SM22-Cre, with high frequency generates genomic recombinations of the floxed GC-A gene segments which were transmitted to the germline. Alleles with partial or complete deletions were readily recovered from the next generation, after segregation of the Cre-transgene. We took advantage of this strategy to generate a new mouse line with global, systemic deletion of GC-A. Doppler-echocardiographic and physiological studies in these mice demonstrate for the first time the tremendous impact of ANP/GC-A dysfunction on chronic blood volume homeostasis.

Animals↗
PubMed2004

Analysis of the role of the leucine zipper motif in regulating the ability of AFAP-110 to alter actin filament integrity.

AFAP-110 has an intrinsic ability to alter actin filament integrity as an actin filament crosslinking protein. This capability is regulated by a carboxy terminal leucine zipper (Lzip) motif. The Lzip motif facilitates self-association stabilizing the AFAP-110 multimers. Deletion of the Lzip motif (AFAP-110(Deltalzip)) reduces the stability of the AFAP-110 multimer and concomitantly increases its ability to crosslink actin filaments, in vitro, and to activate cSrc and alter actin filament integrity, in vivo. We sought to determine how the Lzip motif regulates AFAP-110 function. Substitution of the c-Fos Lzip motif in place of the AFAP-110 Lzip motif (AFAP-110(fos)) was predicted to preserve the alpha-helical structure while changing the sequence. To alter the structure of the alpha-helix, a leucine to proline mutation was generated in the AFAP-110 alpha-helical Lzip motif (AFAP-110(581P)), which largely preserved the sequence. The helix mutants, AFAP-110(Deltalzip), AFAP-110(fos), and AFAP-110(581P), demonstrated reduced multimer stability with an increased capacity to crosslink actin filaments, in vitro, relative to AFAP-110. An analysis of opposing binding sites indicated that the carboxy terminus/Lzip motif can contact sequences within the amino terminal pleckstrin homology (PH1) domain indicating an auto-inhibitory mechanism for regulating multimer stability and actin filament crosslinking. In vivo, only AFAP-110(Deltalzip) and AFAP-110(581P) were to activate cSrc and to alter cellular actin filament integrity. These data indicate that the intrinsic ability of AFAP-110 to crosslink actin filaments is dependent upon both the sequence and structure of the Lzip motif, while the ability of the Lzip motif to regulate AFAP-110-directed activation of cSrc and changes in actin filament integrity in vivo is dependent upon the structure or presence of the Lzip motif. We hypothesize that the intrinsic ability of AFAP-110 to crosslink actin filaments or activate cSrc are distinct functions.

Actin Cytoskeleton↗