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At least 1,225 records · Page 68Linked to original sources
PubMed2006

Characterization and distribution of an arginine vasotocin receptor in mouse.

A cDNA, which has a high homology with teleost Platichthys flesus [Arg8] vasotocin (AVT) receptor (GenBank: AK033957), was found in mouse genome database. Analyses of the deduced amino acid sequence revealed that a cDNA has several features of AVT receptor. We tentatively named it as a mouse vasotocin receptor (MVTR). A two-electrodes voltage clamp technique was applied to characterize the MVTR expressed in Xenopus laevis oocytes. AVT induced Ca2+-dependent Cl- currents in Xenopus oocytes injected with MVTR cRNA. On the other hand, [Arg8] vasopressin, oxytocin and isotocin did not induce such currents. RT-PCR showed that MVTR mRNA was specifically expressed in the brain. In situ hybridization analysis demonstrated significant expression of MVTR mRNA in suprachiasmatic nucleus, arcuate nucleus and medial habenular nucleus of mouse brain. These results suggest that MVTR may mediate a variety of physiological functions in mouse.

Amino Acid Sequence↗
PubMed2004

Fluorescent detection of differentially expressed cDNA using SYBR gold nucleic acid gel stain.

We describe herein a modified differential gene display (DGD) technique that can be rapidly and simply performed and that eliminates the need for radioactivity by fluorescent visualization of complementary deoxyribonucleic acid (cDNA) bands with SYBR gold nucleic acid gel stain. To streamline the DGD procedure, a number of modifications were employed. Ribonucleic acid isolated from differentially treated populations of human trabecular bone-derived mesenchymal progenitor cells was reverse-transcribed into cDNA using oligo-dT primer, and subsequent amplification of differentially expressed cDNAs was done using arbitrary 25-mer primers and oligo-dT9 30-mer primers. Moderate-sized nondenaturing 6% polyacrylamide gels (30 x 20 cm) of 1.5-mm thickness were used for easier handling and increased sample loading capacity. Gels were subjected to electrophoresis overnight, stained with SYBR gold, and visualized and photographed using a commercially available gel imager. DNA bands ranging in size from 100 to 400 bp were visualized directly on an ultraviolet transilluminator, excised from the gel, and reamplified. The cDNA amplicons were subcloned, sequenced, and gene sequences were identified by a Basic Local Alignment Search Tool of genomic databases. Overall, this rapid and functional method proved quite effective for identification of novel genes that may be of interest in studies of cartilage and bone differentiation.

DNA, Complementary↗
PubMed2003

PXO set of recombinant inbred strains of the rat: a new strain distribution pattern containing 448 markers.

A new PXO set of RIS represents a fixed F2 population derived from polydactylous (P) congenic strain SHR.Lx and oligodactylous (O) RI strain BXH2. The PXO strains were derived as a complementary set to current RIS (HXB, BXH) of the laboratory rat. All PXO strains are homozygous in the Lx allele and express different morphological phenotypes of the polydactyly-luxate syndrome (PLS) due to variable combinations of Lx modifying genes of either SHR or BN origin. The SDP is being built up by genotyping polymorphic microsatellite markers and several gene polymorphisms. The markers were ordered according to data from public mapping resources such as the Rat Genome Database (rgd.mcw.edu) and current SDP of the other RI strain sets (HXB, BXH). The resulting map corresponding to the common SDP of HXB, BXH RIS sets consists of 448 markers, from which 261 were proven to be polymorphic in the PXO set. The SDP of PXO strains with polymorphic markers arranged in approximately 5 cM intervals is ready for the association analysis and interval mapping in interconnection with the SDP of HXB/BXH strains.

Animals↗
PubMed2003

Toward computer-based cleavage site prediction of cysteine endopeptidases.

Identification of relevant substrates is essential for elucidation of in vivo functions of peptidases. The recent availability of the complete genome sequences of many eukaryotic organisms holds the promise of identifying specific peptidase substrates by systematic proteome analyses in combination with computer-based screening of genome databases. Currently available proteomics and bioinformatics tools are not sufficient for reliable endopeptidase substrate predictions. To address these shortcomings the bioinformatics tool 'PEPS' (Prediction of Endopeptidase Substrates) has been developed and is presented here. PEPS uses individual rule-based endopeptidase cleavage site scoring matrices (CSSM). The efficiency of PEPS in predicting putative caspase 3, cathepsin B and cathepsin L cleavage sites is demonstrated in comparison to established algorithms. Mortalin, a member of the heat shock protein family HSP70, was identified by PEPS as a putative cathepsin L substrate. Comparative proteome analyses of cathepsin L-deficient and wild-type mouse fibroblasts showed that mortalin is enriched in the absence of cathepsin L. These results indicate that CSSM/PEPS can correctly predict relevant peptidase substrates.

Animals↗
PubMed2004

Cloning and expression of a novel CREB mRNA splice variant in human testis.

Identification of genes specifically expressed in adult and fetal testis is important in furthering our understanding of testis development and function. In this study, a novel human transcript, designated human testis cAMP-responsive element-binding protein (htCREB), was identified by hybridization of adult and fetal human testis cDNA probes with a human cDNA microarray containing 9216 clones. The htCREB transcript (GenBank Accession no. AY347527) was expressed at 2.35-fold higher levels in adult human testes than in fetal testes. Sequence and ntBLAST analyses against the human genome database indicated that htCREB was a novel splice variant of human CREB. RT-PCR-based tissue distribution experiments demonstrated that the htCREB transcript was highly expressed in adult human testis and in healthy sperm, but not in testes from patients with Sertoli cell-only syndrome. Taken together, these results suggest that the htCREB transcript is chiefly expressed in germ cells and is most likely involved in spermatogenesis.

Alternative Splicing↗
PubMed2000

Genome mapping in ruminants and map locations for genes influencing reproduction.

Genetic maps provide a critical link between genes and phenotypes and are essential tools in the search for the genetic basis of variation in reproductive traits. Genes coding for hormones, growth factors, receptors, binding proteins, transcription factors and enzymes that influence the development and function of the reproductive axis have been assigned to genetic maps of ruminants and locations can be found in the respective genome databases. In addition, comparative information on gene structure and map location will help define the functions of essential genes. Gene locations from other species can be used because of extensive comparative links among mammalian gene maps. Large-scale projects to sequence genes and the ability to map these genes in parallel in radiation hybrid panels of different species will greatly improve the maps and our ability to translate between them. Cloning the genes responsible for genetic differences in fertility and fecundity in ruminants is likely to provide valuable clues to understanding ovarian function and germ cell development.

Animals↗
PubMed2005

Proteomics for biodefense applications: progress and opportunities.

The increasing threat of bioterrorism and continued emergence of new infectious diseases has driven a major resurgence in biomedical research efforts to develop improved treatments, diagnostics and vaccines, as well as increase the fundamental understanding of the host immune response to infectious agents. The availability of multiple mass spectrometry platforms combined with multidimensional separation technologies and microbial genomic databases provides an unprecedented opportunity to develop these much needed resources. An overview of current proteomic strategies applied to microbes and viruses considered potential bioterrorism agents is presented. The emerging area of immunoproteomics as applied to the development of new vaccine targets is also summarized. These powerful research approaches can generate a multitude of potential new protein targets; however, translating these proteomic discoveries to useful counter-bioterrorism products will require large collaborative research efforts across multiple basic science and clinical disciplines. A translational proteomic research paradigm illustrating this approach using influenza virus as an example is discussed.

Adult↗
PubMed2005

Cloning and hypothalamic distribution of the chicken thyrotropin-releasing hormone precursor cDNA.

In this paper we report the cloning of the chicken preprothyrotropin-releasing hormone (TRH) cDNA and the study of its hypothalamic distribution. Chicken pre-proTRH contains five exact copies of the TRH progenitor sequence (Glu-His-Pro-Gly) of which only four are flanked by pairs of basic amino acids. In addition, the amino acid sequence contains three sequences that resemble the TRH progenitor sequence but seem to have lost their TRH-coding function during vertebrate evolution. The amino acid sequence homology of preproTRH between different species is very low. Nevertheless, when the tertiary structures are compared using hydrophobicity plots, the resemblance between chicken and rat prepro-TRH is striking. The cloning results also showed that the chicken preproTRH sequence includes neither a rat peptide spacer 4 (Ps4) nor a Ps5 connecting peptide. Comparison of the cDNA sequence with the chicken genome database revealed the presence of two introns, one in the 5' untranslated region, and another downstream from the translation start site. This means that the gene structure of chicken preproTRH resembles the gene stucture of this precursor in mammals. Based on the cDNA sequence, digoxigenin-labelled probes were produced to study the distribution of preproTRH in the chicken brain. By means of in situ hybridization, preproTRH mRNA was detected in the chicken paraventricular nucleus (PVN) and in the lateral hypothalamus (LHy).

Amino Acid Sequence↗
PubMed2004

Safety concerns related to hematopoietic stem cell gene transfer using retroviral vectors.

Endogenous retroviruses have developed efficient methods during their life cycle for stable integration into the host genome. Because of this ability, retroviral vectors were designed with the goal of gene transfer into hematopoietic stem cells (HSCs). The ability to genetically modify HSCs provides a vehicle for durable expression of potentially therapeutic transgenes in all lineages of mature blood cells for the lifetime of the patient. Combined with bone marrow transplant, retroviral gene transfer has many potential applications for a wide range of blood diseases. Advances in the development of oncoretroviral vectors based on murine leukemia viruses (MLV) and more recent development of human immunodeficiency virus (HIV)-based vectors have greatly increased the gene transfer efficiency. Optimization of methods for gene transfer using MLV-based vectors has substantially improved marking levels in mice, with lower levels in large animals and in human clinical trials. With advances in gene transfer technology has also come renewed concern about insertional mutagenesis and activation of oncogenes. Advanced techniques for integration site analysis combined with sequence comparison using mouse and human genome databases has now made it possible to begin to understand the spectrum of possible integration sites for both MLV- and HIV-based vectors. Furthermore, other studies have shown positive and negative dosage-dependent effects of transgene expression in mouse and human cells. Therefore, vector design and safety testing are at the forefront of the field of gene therapy and this review discusses recent developments.

Animals↗
PubMed2004

Molecular biology of pyridine nucleotide and nicotine biosynthesis.

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous coenzyme in oxidation-reduction reactions. Recent animal and fungal studies show that it also plays important roles in transcriptional regulation, longevity, and age-associated diseases. NAD is synthesized de novo from aspartic acid in E. coli or from tryptophan in animals, by way of quinolinic acid. Although the number of biochemical studies on NAD is very limited, a bioinformatic search of genome databases suggests that Arabidopsis (dicots) synthesizes NAD from aspartic acid whereas rice (monocots) may utilize both aspartate and tryptophan as starting amino acids. The salvage pathway recycles the breakdown products of NAD metabolism. In tobacco, an intermediate in the de novo NAD synthetic pathway supplies the pyridine ring moiety of nicotine alkaloids. Gene expression studies in tobacco suggest that part of the NAD pathway is coordinately regulated with nicotine biosynthesis.

Animals↗
PubMed1992

Characterization of the primate-specific repetitive DNA element MER1.

Computer analyses of the 3'-flanking DNA sequence of the human elastase I gene revealed a significant degree of similarity with seven human gene sequences in the GenBank and EMBL databases. Genomic Southern analysis indicates that the shared nucleotide sequences are a primate-specific family of short interspersed elements. These elements are members of MER1 sequences (medium reiteration frequency sequences). The consensus sequence of MER1 repeats spans 543 nucleotides and contains several inverted repeats. Since the copy number of MER1 elements seems to be much smaller than that of Alu and L1 repeats, MER1 elements may provide useful landmarks marks for human genome mapping.

Animals↗
PubMed2022

An Exosomal miRNA Biomarker for the Detection of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains a difficult tumor to diagnose and treat. To date, PDAC lacks routine screening with no markers available for early detection. Exosomes are 40-150 nm-sized extracellular vesicles that contain DNA, RNA, and proteins. These exosomes are released by all cell types into circulation and thus can be harvested from patient body fluids, thereby facilitating a non-invasive method for PDAC detection. A bioinformatics analysis was conducted utilizing publicly available miRNA pancreatic cancer expression and genome databases. Through this analysis, we identified 18 miRNA with strong potential for PDAC detection. From this analysis, 10 (MIR31, MIR93, MIR133A1, MIR210, MIR330, MIR339, MIR425, MIR429, MIR1208, and MIR3620) were chosen due to high copy number variation as well as their potential to differentiate patients with chronic pancreatitis, neoplasms, and PDAC. These 10 were examined for their mature miRNA expression patterns, giving rise to 18 mature miRs for further analysis. Exosomal RNA from cell culture media was analyzed via RTqPCR and seven mature miRs exhibited statistical significance (miR-31-5p, miR-31-3p, miR-210-3p, miR-339-5p, miR-425-5p, miR-425-3p, and miR-429). These identified biomarkers can potentially be used for early detection of PDAC.

Humans↗
PubMed2001

Immunological and biochemical characterization of streptococcal pyrogenic exotoxins I and J (SPE-I and SPE-J) from Streptococcus pyogenes.

Recently, we described the identification of novel streptococcal superantigens (SAgs) by mining the Streptococcus pyogenes M1 genome database at Oklahoma University. Here, we report the cloning, expression, and functional analysis of streptococcal pyrogenic exotoxin (SPE)-J and another novel SAg (SPE-I). SPE-I is most closely related to SPE-H and staphylococcal enterotoxin I, whereas SPE-J is most closely related to SPE-C. Recombinant forms of SPE-I and SPE-J were mitogenic for PBL, both reaching half maximum responses at 0.1 pg/ml. Evidence from binding studies and cell aggregation assays using a human B-lymphoblastoid cell line (LG-2) suggests that both toxins exclusively bind to the polymorphic MHC class II beta-chain in a zinc-dependent mode but not to the generic MHC class II alpha-chain. The results from analysis by light scattering indicate that SPE-J exists as a dimer in solution above concentrations of 4.0 mg/ml. Moreover, SPE-J induced a rapid homotypic aggregation of LG-2 cells, suggesting that this toxin might cross-link MHC class II molecules on the cell surface by building tetramers of the type HLA-DRbeta-SPE-J-SPE-J-HLA-DRbeta. SPE-I preferably stimulates T cells bearing the Vbeta18.1 TCR, which is not targeted by any other known SAG: SPE-J almost exclusively stimulates Vbeta2.1 T cells, a Vbeta that is targeted by several other streptococcal SAgs, suggesting a specific role for this T cell subpopulation in immune defense. Despite a primary sequence diversity of 51%, SPE-J is functionally indistinguishable from SPE-C and might play a role in streptococcal disease, which has previously been addressed to SPE-C.

Amino Acid Sequence↗
PubMed2001

Human thymic stromal lymphopoietin preferentially stimulates myeloid cells.

The sequence of a novel hemopoietic cytokine was discovered in a computational screen of genomic databases, and its homology to mouse thymic stromal lymphopoietin (TSLP) suggests that it is the human orthologue. Human TSLP is proposed to signal through a heterodimeric receptor complex that consists of a new member of the hemopoietin family termed human TSLP receptor and the IL-7R alpha-chain. Cells transfected with both receptor subunits proliferated in response to purified, recombinant human TSLP, with induced phosphorylation of Stat3 and Stat5. Human TSLPR and IL-7Ralpha are principally coexpressed on monocytes and dendritic cell populations and to a much lesser extent on various lymphoid cells. In accord, we find that human TSLP functions mainly on myeloid cells; it induces the release of T cell-attracting chemokines from monocytes and, in particular, enhances the maturation of CD11c(+) dendritic cells, as evidenced by the strong induction of the costimulatory molecules CD40 and CD80 and the enhanced capacity to elicit proliferation of naive T cells.

Amino Acid Sequence↗
PubMed2005

Cloning, characterization and expression analysis of interleukin-10 from the zebrafish (Danio rerion).

Cytokines are proteins produced by many different cells of the immune system and play a significant role in initiating and regulating the inflammatory process. In this research, an important cytokine, interleukin-10 (IL-10) gene, has been identified and characterized from zebrafish (Danio rerio) genome database. Zebrafish IL-10 is located within a 2690 bp fragment and contains five exons and four introns, sharing the same organization with mammalian IL-10 genes. An open reading frame of 543 bp was found to encode a putative 180 amino acid protein with a signal peptide of 22 amino acids, which shares 29.7-80.9 % homology with amino acid sequences of other known IL-10. The signature motif of IL-10 is also conserved in zebrafish IL-10. The predicted transcript was finally confirmed by sequencing of cDNA clones. Multi-tissue reverse transcriptase PCR (RT-PCR) was performed to examine the tissue distribution and expression regulation of this gene in seven organs of normal and lipopolysaccharide (LPS) stimulation zebrafish. The results demonstrated that this gene was expressed slightly in normal kidney, gill and gut, no expression was detected in other four tissues. The expression was clearly upregulated after LPS stimulation. Using the ideal zebrafish model, further study of IL-10 characterization and function may provide insight on the understanding of the innate immune system.

Amino Acid Sequence↗
PubMed1999

[A preliminary study of human Y chromosome specific short tandem repeat loci].

OBJECTIVE: To understand the allele structure and reveal genetic polymorphism of Y chromosome specific short tandem repeat (Y-specific STR) loci in Chinese Han population. METHODS: The authors used a set of five Y-specific STR loci which were tetrameric tandem repeat loci chosen from the Genome Database. EDTA-blood specimens were collected from the unrelated individuals. DNA was extracted by Chelex method and amplified by the polymerase chain reaction (PCR). The PCR products were analyzed using both the PAGE horizontal electrophoresis with discontinuous buffer system and the automated fluorescence detection approach. RESULTS: The authors observed that the alleles at the five Y-specific STR loci were composed of some complex repeat structures. They successfully prepared a set of human allele ladders for the typing of the five Y-specific STRs and demonstrated the polymorphisms at the five Y-specific STR loci in Chinese Han population. CONCLUSION: Y-specific STRs are good genetic markers for the purpose of analysis of genetic relationship between populations. This preliminary study not only reveals allele frequencies and haplotype distribution of Y-specific STR in Chinese Han population, but also indicates a reference population for detecting male migration events and for reconstructing paternal history.

Chromosome Mapping↗
PubMed1999

[A preliminary study of human Y chromosome specific short tandem repeat loci]

OBJECTIVE: To understand the allele structure and reveal genetic polymorphism of Y chromosome specific short tandem repeat (Y-specific STR) loci in Chinese Han population. METHODS: The authors used a set of five Y-specific STR loci which were tetrameric tandem repeat loci chosen from the Genome Database. EDTA-blood specimens were collected from the unrelated individuals. DNA was extracted by Chelex method and amplified by the polymerase chain reaction (PCR). The PCR products were analyzed using both the PAGE horizontal electrophoresis with discontinuous buffer system and the automated fluorescence detection approach. RESULTS: The authors observed that the alleles at the five Y-specific STR loci were composed of some complex repeat structures. They successfully prepared a set of human allele ladders for the typing of the five Y-specific STRs and demonstrated the polymorphisms at the five Y-specific STR loci in Chinese Han population. CONCLUSION: Y-specific STRs are good genetic markers for the purpose of analysis of genetic relationship between populations. This preliminary study not only reveals allele frequencies and haplotype distribution of Y-specific STR in Chinese Han population, but also indicates a reference population for detecting male migration events and for reconstructing paternal history.

Journal Article↗
PubMed2000

Tandem duplication/deletion in a maternally derived chromosome 9 supernumerary derivative resulting in 9p trisomy and partial 9q tetrasomy.

A 19-week stillborn female fetus with bilateral cleft palate, horseshoe kidney, bicornuate uterus, low-set ears, and intrauterine growth retardation (IUGR) was found to have a supernumerary derivative chromosome 9 (der(9)) with an apparent tandem duplication in the long arm. PCR analysis at five polymorphic loci confirmed the duplication and showed an adjacent deletion, while whole chromosome FISH demonstrated only chromosome 9 to be involved. Further FISH studies of der(9) found the 9qh region to be duplicated, telomeric sequences to be intact, and subtelomeric sequences to be absent. Thus, the fetus was determined to be trisomic for 9pter-->9q12 and 9q34.3-->9qter, tetrasomic for 9q12--> 9q33, and disomic for 9q33-->9q34.3. These results are consistent with a tandem duplication of 9q12-->9q33 and adjacent distal deletion as designated by the karyotype, 47,XX,+der(9)dup(9) (q12q33)del(9) (q33q34.3).ish der(9)(WCP9+,D9Z1x2,STP9q-, AHT+) de novo. In addition to characterizing der(9), the combined PCR and cytogenetic studies refined the Genome Database Map of three loci (D9S907, D9S155, and D9S302) approximately to the distal 9q33 region. Without the attempt to refine breakpoints by PCR analysis, the deletion in distal 9q would not have been detected. Tandem direct duplication/deletion chromosomes have been reported in fewer cases than inverted duplication/deletions. We propose mechanisms of origin, consistent with those for recurrent inter stitial microdeletion and microduplication syndromes, shown to arise by recombination at homologous, flanking DNA sequences.

Aneuploidy↗