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At least 523 records · Page 29Linked to original sources
PubMed1992

An alternatively processed mRNA from the avian c-erbB gene encodes a soluble, truncated form of the receptor that can block ligand-dependent transformation.

At least four major transcripts are produced by the avian c-erbB/epidermal growth factor receptor gene. cDNAs corresponding to the smallest one, a 2.6-kb transcript, were isolated from an adult chicken liver cDNA library. Sequence analysis revealed that the 3' end of one cDNA clone diverged from the known sequence of the extracellular ligand-binding domain (LBD) of the full-length receptor. A genomic DNA subfragment that contained this unique 3' divergent end was isolated. Sequence analysis of this genomic DNA fragment revealed that the 2.6-kb c-erbB transcript is produced by alternative processing. Translation of this 2.6-kb transcript would produce a secreted, truncated receptor molecule which contains the amino-terminal three-fourths of the extracellular LBD of the native receptor. COS1 cells and primary chicken embryo fibroblast cells were transfected with expression vectors that contained the 2.6-kb c-erbB cDNA. Conditioned medium from these transfected cells contained a 70-kDa protein that was specifically immunoprecipitated by a polyclonal antiserum directed against the LBD of the avian c-erbB gene product. The 70-kDa truncated receptor could be coimmunoprecipitated from conditioned medium of transfected COS1 cells that was supplemented with recombinant human transforming growth factor alpha (TGF alpha) by a monoclonal antibody against human TGF alpha. Additionally, transfected chicken embryo fibroblast cells that overexpressed the 70-kDa truncated receptor were blocked in their ability to form TGF alpha-dependent colonies in soft agar. These data suggest that the secreted, truncated receptor encoded by the 2.6-kb c-erbB transcript can bind to TGF alpha and may play an important growth-regulatory function in vitro.

Amino Acid Sequence↗
PubMed2001

Molecular cloning and functional analysis of mouse C-terminal kinesin motor KifC3.

Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. To study the molecular mechanism of intracellular transport involving microtubule-dependent motors, a cDNA encoding a new kinesin-like protein called KifC3 was cloned from a mouse brain cDNA library. Sequence and secondary structure analysis revealed that KifC3 is a member of the C-terminal motor family. In contrast to other mouse C-terminal motors, KifC3 is apparently ubiquitous and may have a general role in intracellular transport. To understand the in vivo function of the KifC3 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC3 gene. Homozygous mutants of the KifC3 gene are viable, reproduce normally, and apparently develop normally. These results suggest that KifC3 is dispensable for normal development and reproduction in the mouse.

Amino Acid Sequence↗
PubMed1995

Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

Amino Acid Sequence↗
PubMed2001

Eel urea transporter is localized to chloride cells and is salinity dependent.

Urea transporters (UTs) in the ureotelic vertebrates have been well-characterized, but little is known about those of the ammonotelic teleost fishes. To clarify the physiological roles of UTs in the ammonotelic teleosts, we determined the structure, tissue and cellular localizations, and regulation of expression of eel UT (eUT) by cDNA cloning, Northern analysis, and immunohistochemistry. A full-length cDNA (approximately 1.9 kb) coding for a UT of 486 amino acid residues was isolated from a seawater eel gill cDNA library. Sequence comparison with those of other species indicated that the eUT is a short isoform with 10 transmembrane spans and has longer NH2- and COOH-terminal cytoplasmic tails compared with the mammalian counterparts. Northern blot analysis demonstrated high expression of eUT mRNA confined in the gill and a substantial increase of its levels when eels were transferred from freshwater to seawater. Immunohistochemistry showed that eUT is localized on the basolateral membranes of the chloride cells, establishing, at the cellular level, the site of urea excretion in the eel, an ammonotelic teleost.

Amino Acid Sequence↗
PubMed2002

Molecular cloning, expression, and function of osteoclastic calcineurin Aalpha.

This study explores the role of the calmodulin- and Ca(2+)-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin Aalpha and Abeta cDNA from a rabbit osteoclast library. Sequence analysis revealed an approximately 95 and 86% homology between the amino acid and the nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat, and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin Aalpha mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aalpha expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 x 10(-7) M) and FK506 (5 x 10(-9) and 5 x 10(-7) M). We then constructed a fusion protein comprising calcineurin Aalpha and TAT, a 12-amino acid-long arginine-rich sequence of the human immunodeficiency virus protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Similarly, unfolding of the TAT-calcineurin Aalpha fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin Aalpha and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aalpha-positive osteoclasts revealed a concentration-dependent (10-200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinin did not produce significant effects on bone resorption or cell number. The study suggests the following: 1) the 61-kDa protein phosphatase calcineurin Aalpha can be effectively tranduced into osteoclasts by using the TAT-based approach, and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.

Amino Acid Sequence↗
PubMed2003

Systematic screening of sheep skin cDNA libraries for microsatellite sequences.

65,000 sheep skin cDNA clones were gridded in high density on to nylon membranes and screened for (CA)n and (GA)n repeat containing clones. 296 dinucleotide repeat-containing clones were identified with approximately 85% non-redundancy. Clones were single-pass 5' sequenced and we compared the Expressed Sequence Tag (EST) sequences to the Swiss-Prot database to ascertain their identity and/or putative function. We then aligned the ESTs against the human genomic sequence to determine the locations of human orthologous sequences. Finally, we developed a subset of polymorphic microsatellite markers and positioned them on the ovine linkage map.

Animals↗
PubMed1997

Differentially regulated epithelial expression of an Eph family tyrosine kinase (fHek2) during tracheal surface airway and submucosal gland development.

A ferret model was used to evaluate the potential role of an Eph family tyrosine kinase (fHek2) in tracheal development of surface airway epithelium and submucosal glands. A partial 2.6-kb cDNA fragment of fHek2 was isolated from a ferret tracheal/lung cDNA library. Sequence analysis demonstrated that this gene is the ortholog to the previously cloned human Hek2 gene. In situ hybridization analysis of fHek2 mRNA expression on ferret tracheal developmental time points revealed an expression pattern within a subset of surface airway epithelial cells which remained relatively constant throughout tracheal development (from -2 d in utero to adult). In contrast, developing tracheal submucosal glands at 3-day postnatal time points demonstrated little fHek2 mRNA expression. However, expression of fHek2 significantly increased more than 4-fold over the course of gland development to adulthood. These findings, which demonstrate a uniquely regulated pattern of fHek2 mRNA expression between surface airway epithelium and submucosal glands, have implications on regulatory processes which control differentiation and/or maturation of secretory structures in the lung. Such findings may be useful in further delineating the mechanisms which control cellular differentiation in the lung and how these processes are abnormally regulated in hypersecretory diseases such as chronic bronchitis, asthma, and cystic fibrosis.

Amino Acid Sequence↗
PubMed2006

Intrahost variations in the envelope receptor-binding domain (RBD) of HTLV-1 and STLV-1 primary isolates.

Four primate (PTLV), human (HTLV) and simian (STLV) T-cell leukemia virus types, have been characterized thus far, with evidence of a simian zoonotic origin for HTLV-1, HTLV-2 and HTLV-3 in Africa. The PTLV envelope glycoprotein surface component (SUgp46) comprises a receptor-binding domain (RBD) that alternates hypervariable and highly conserved sequences. To further delineate highly conserved motifs in PTLV RBDs, we investigated the intrahost variability of HTLV-1 and STLV-1 by generating and sequencing libraries of DNA fragments amplified within the RBD of the SUgp46 env gene. Using new and highly cross-reactive env primer pairs, we observed the presence of Env quasispecies in HTLV-1 infected individuals and STLV-1 naturally infected macaques, irrespective of the clinical status. These intrahost variants helped us to define highly conserved residues and motifs in the RBD. The new highly sensitive env PCR described here appears suitable for the screening of all known variants of the different PTLV types and should, therefore, be useful for the analysis of seroindeterminate samples.

Animals↗
PubMed1992

Characterization of the short isoform of the growth hormone receptor synthesized by rat adipocytes.

Two mRNA transcripts that are believed to be alternately spliced products of the GH receptor gene have been reported in a variety of rat tissues. The smaller (1.2 kilobases) transcript was cloned from an adipocyte library, sequenced, and found to encode a protein identical to the soluble GH-binding protein (GHBP) in plasma. An assay that is specific for the short isoform of the GH receptor, often referred to as the GHBP, has been developed using a rabbit antiserum that recognizes the unique amino acid sequence at its carboxyl end. The assay depends upon immunoprecipitation of a complex consisting of [125I]human GH, the binding protein, antiserum, and protein-A cross-linked to agarose beads. To validate the assay, samples of rat plasma were analyzed and found to contain sufficient binding protein to bind 1.46 pmol (32 ng) GH/ml, with an affinity of 2.7 x 10(9) M-1. In adipocyte extracts, binding protein activity was sufficient to bind 61 fmol GH/g tissue, with an affinity of 2.3 x 10(9) M-1. The binding protein was found primarily in the particulate fraction of adipocytes, and it is estimated that adipocytes contain approximately 7000 copies of the binding protein/cell. Only 10% of the binding activity was present in the high speed supernatant of adipocyte homogenates, and soluble binding protein did not appear to be released into the incubation medium when adipocytes were incubated in vitro. A 50-kilodalton (kDa) 35S-labeled protein that may be a glycosylated form of the binding protein was immunoprecipitated from both the soluble and particulate fractions of adipocyte extracts by the antiserum, and addition of the synthetic peptide antigen blocked immunoprecipitation of this protein. A 150-kDa protein in the high speed supernatant fraction was also specifically immunoprecipitated by the antiserum. Although it is unlikely to be a glycosylated form of the binding protein, it may cross-react with the antiserum or perhaps be coprecipitated, because it interacts with the binding protein. In addition, 38- and 42-kDa bands were specifically immunoprecipitated from the detergent-treated particulate fraction of adipocyte extracts that were enriched for the binding protein by adsorption to immobilized GH. We conclude that 1) adipocytes synthesize the short isoform of the GH receptor, and that this protein is primarily associated with a membrane fraction of the cells; and 2) the GHBP expressed in adipocytes is not released into the incubation medium and differs in size from the GHBPs in rat plasma.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1998

Molecular cloning and characterization of the porcine calcitonin gene-related peptide receptor.

Calcitonin gene-related peptide (CGRP) receptors (CGRP-Rs) are widely distributed throughout the central and peripheral nervous systems. A novel CGRP-R was identified from a porcine lung complementary DNA library. Sequence analysis indicated that the CGRP-R is 462 amino acids in length and shares 93% sequence identity with the human CGRP-R. Northern blot analysis indicated a messenger RNA species of 5.4 kilobases, which is abundantly expressed in the lung. Ligand binding studies of the cloned CGRP-R expressed in human embryonic kidney (HEK-293) cells showed the presence of high affinity receptor for CGRP with a Kd of 38.5 pM. The pharmacological profiles of various ligands competing for [125I]CGRP binding to the expressed receptor were in accordance with those for the natural receptor. Binding of [125I]CGRP to the expressed receptor was decreased in the presence of a nonhydrolyzable analog of GTP, guanosine 5' (gamma-thio)-triphosphate. In functional studies, CGRP stimulated the activation of adenylyl cyclase with an EC50 of 2.5 nM. The linear analog of CGRP, diacetoamidomethyl cysteine CGRP, did not affect adenylyl cyclase activity on its own or in the presence of CGRP. Furthermore, the CGRP receptor antagonists, CGRP-(8-37)alpha, inhibited the CGRP-mediated response in a competitive manner. Collectively, the binding and functional data demonstrate that we have cloned a porcine CGRP type 1 receptor. The availability of the CGRP-R complementary DNA will allow us to examine its participation in pathophysiological processes.

Amino Acid Sequence↗
PubMed1993

Heterogeneity in the expression of fibroblast growth factor receptors during limb regeneration in newts (Notophthalmus viridescens).

Two closely related fibroblast growth factor receptors, FGFR1 and FGFR2, have been cloned from a newt (Notophthalmus viridescens) limb blastema cDNA library. Sequence analysis revealed that we have isolated both the bek and KGFR variants of FGFR2. These two variants differ only in the second half of the last of their three Ig-like domains. The expression patterns of FGFR1 and FGFR2 during limb regeneration have been determined by in situ hybridization. During the preblastema stages of regeneration, FGFR2 expression is observed in the basal layer of the wound epithelium and in the cells of the periosteum. As regeneration progresses to the blastema stages, FGFR2 expression continues to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. From the early bud to the mid-bud blastema stage, FGFR1 expression is observed throughout the blastema mesenchyme but, unlike FGFR2, is distinctly absent from the wound epithelium. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. During these later stages of regeneration, the wound epithelium hybridization to the FGFR2 probe is no longer observed. The expression patterns of these receptors suggest that FGFR1 and FGFR2 have distinct roles in limb regeneration, despite their sharing a number of the FGF ligands. Further investigation regarding the potential sources of the FGF ligands will help establish the role that FGFs and FGFRs play in limb regeneration.

Amino Acid Sequence↗
PubMed1993

A reversibly palmitoylated resident protein (p63) of an ER-Golgi intermediate compartment is related to a circulatory shock resuscitation protein.

The recently identified 63 kDa membrane protein, p63, is a resident protein of a membrane network interposed in between rough ER and Golgi apparatus. To characterize p63 at the molecular level a 2.91 kb cDNA encoding p63 has been isolated from a human placenta lambda gt10 cDNA library. Sequence analysis of tryptic peptides prepared from isolated p63 confirmed the identify of the cloned gene. The translated amino acid sequence consists of 601 amino acids (65.8 kDa) with a single putative membrane-spanning region and a N-terminal cytoplasmic domain of 106 amino acids. The human p63 cDNA exhibits a high level of sequence identify to the pig hepatic cDNA 3AL (accession number M27092) whose expression is enhanced after resuscitation from circulatory shock. An additional remarkable feature of p63 is that it becomes reversibly palmitoylated when intracellular protein transport is blocked by the drug brefeldin A. Overexpression of p63 in COS cells led to the development of a striking tubular membrane network in the cytoplasm. This suggests that the protein may be determinant for the structure of the p63 compartment.

Amino Acid Sequence↗
PubMed1995

Cloning of the dopamine-1A (D1A) receptor gene expressed in porcine renal epithelial cells.

We sought to determine the molecular identify of the dopamine-1 (D1) receptor expressed in the porcine renal epithelial cell line LLC-PK1. We first isolated a partial cDNA by the reverse transcription-polymerase chain reaction procedure and then used the partial cDNA to isolate positive overlapping clones from a porcine genomic DNA library. Sequence analysis of the gene revealed that the longest open-reading frame encoded a 446 amino acid protein that was 95% identical to the human D1A receptor. Expression studies in mammalian cells were also consistent with the clones encoding a D1 receptor. Northern blot hybridizations with LLC-PK1 poly (A+) RNA were strongly positive. The porcine D1A gene has two exons and a short intron in the 5' untranslated region. The 5' flanking region lacks a TATA and CAAT box but is high in GC content (68%) and contains multiple Sp1 binding sites. The 5' flanking region also contains numerous other cis-acting elements for transcription factors. These results indicate that the D1A receptor is the major D1 receptor expressed in LLC-PK1 cells and further suggest that LLC-PK1 cells may be a useful model to study the regulation of renal D1A receptor gene transcription.

Amino Acid Sequence↗
PubMed2026

Uncovering the diagnostic potential of seminal fluid beyond fertility: cfDNA methylation analysis for the detection of clinically significant prostate cancer.

Research on the potential use of seminal fluid as a liquid biopsy for prostate cancer detection has been limited due to challenges associated with acquisition of this bodily fluid in clinical studies. Here we sought to expand on our previous analysis, which demonstrated high levels of prostate-derived cell free DNA (cfDNA) in seminal fluid in presumed healthy individuals, to a much larger cohort that included participants with prostate cancer. A total of 279 men scheduled for prostate biopsy were enrolled over 4 months across 12 sites. Prior to their biopsy, participants mailed a seminal fluid sample collected at home to the laboratory, from which cfDNA was extracted and underwent methylation analysis. Consistent with our earlier study in healthy individuals, we observed an abundance of high molecular weight (HMW) cfDNA in all samples. Tissue-of-origin deconvolution revealed that granulocytes and sperm were the principal contributors to seminal fluid cfDNA, while prostate-derived cfDNA was present at abundances readily detectable with current technologies. The nucleosomal fraction was very pronounced in some but not all samples and was determined to be correlated with the relative sperm signal. The sperm signal was also observed to be associated with an increase in small insert sizes (< 125 bp) in the sequenced libraries. Unsupervised clustering revealed two distinct populations driven by the abundance of sperm and granulocytes. Since summarizing at the genomic region level confounded tissues of different origins, fragment-level DNA methylation features were used to characterize and quantify the prostate cancer related signal, and features associated with clinically significant prostate cancer were identified. This study expands on our previous work to further characterize seminal fluid and highlights its potential as a promising liquid biopsy medium for the detection and monitoring of clinically significant prostate cancer.

Humans↗
PubMed1991

Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase.

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Study of a region on yeast chromosome XIII that complements pet G199 mutants (COX7) and carries a new non-essential gene.

The mutants of Saccharomyces cerevisiae assigned to complementation group G199 are deficient in mitochondrial respiration and lack a functional cytochrome oxidase complex. Recombinant plasmids capable of restoring respiration were cloned by transformation of mutants of this group with a yeast genomic library. Sequencing indicated that a 2.1-kb subclone encompasses the very end (last 11 amino acids) of the PET111 gene, the COX7 gene and a new gene (YMR255W) of unknown function that potentially codes for a polypeptide of 188 amino acids (about 21.5 kDa) without significant homology to any known protein. We have shown that the respiratory defect corresponding to group G199 is complemented by plasmids carrying only the COX7 gene. The gene YMR255W was inactivated by one-step gene replacement and the disrupted strain was viable and unaffected in its ability to grow in a variety of different test media such as minimal or complete media using eight distinct carbon sources at three pH values and temperatures. Inactivation of this gene also did not affect mating or sporulation.

Amino Acid Sequence↗
PubMed2006

Anopheles gambiae (Diptera: Culicidae) oviposition in response to agarose media and cultured bacterial volatiles.

Anopheles gambiae s.s. Giles accepted a range of agarose gels, varying from 0.5 to 8% (wt:vol), for oviposition; laid more eggs on 0.5% agarose gels than on moist filter paper or on drier agarose; and laid equal numbers of eggs on 0.5% agarose gels and distilled water. Larvae hatched on agarose gel substrates and crawled onto the surface of moist agarose gels, but they tended only to burst the egg cap and remain within the egg case on drier gels. A mixture of cultured bacteria, originating from a natural larval habitat of An. gambaiae s.s. in western Kenya, was classified by 16S rDNA sequence analysis, and then this mixture or individual colonies from it was used as odor sources in ovipositional experiments with agarose substrates. Of 61 sequences from a mixed clone sequence library, most (78%) were Pseudomonas strains, whereas the remainder were Stenotrophomonas, Enterobacter, Pantoea, Klebsiella, Acinetobacter, Aeromonas, and Bacillus. Oviposition was significantly reduced when mixtures of bacterial colonies of these strains, or a lawn of colonies of a field isolate of S. maltophilia, was presented. Oviposition was neither reduced nor enhanced when field isolates of Pseudomonas putida or Pseudomonas alcaligenes colonies were presented. These results suggest that gravid An. gambiae females are sensitive to bacterial-derived odors emanating from cultured bacteria from natural larval habitats and that some bacterial odors may be repellent.

Animals↗