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C T Bond

Publications and source records attributed to C T Bond.

35 records · Page 2Linked to original sources
PubMed1995

Contributions of the C-terminal domain to gating properties of inward rectifier potassium channels.

Two inward rectifier potassium channels, the G protein-dependent GIRK1 and the G protein-independent BIR10, display large differences in rectification and macroscopic kinetics. A chimeric channel was constructed in which the putative intracellular carboxy-terminal domain of the G protein-dependent channel replaced the corresponding domain of the G protein-independent channel. The chimeric channel conducted potassium ions without the requirement of activated G proteins, yet displayed activation and deactivation kinetics and rectification properties similar to those of the G protein-dependent channel. The results demonstrate that structural elements in the C-terminus can independently control gating but not G protein signal transduction. The voltage dependence, time course, and kinetics of gating suggest a mechanism in which the pore may be occluded by reversible interactions with charged residues in the C-terminus.

Amino Acid Sequence↗
PubMed1995

A neuron-specific enhancer targets expression of the gonadotropin-releasing hormone gene to hypothalamic neurosecretory neurons.

The molecular mechanisms specifying gene expression in individual neurons of the mammalian central nervous system have been difficult to study due to the cellular complexity of the brain and the absence of cultured model systems representing differentiated central nervous system neurons. We have developed clonal, differentiated, neuronal tumor cell lines of the hypothalamic GnRH-producing neurons by targeting tumorigenesis in transgenic mice. These cells (GT1 cells) provide a model system for molecular studies of GnRH gene regulation. Here we present the identification and characterization of a neuron-specific enhancer responsible for directing expression of the rat GnRH gene in GT1 hypothalamic neurons. This approximately 300 base pair (bp) upstream region (-1571 to -1863) confers enhancer activity to a short -173-bp GnRH promoter or to a heterologous promoter only in GT1 cells. The enhancer is bound by multiple GT1 nuclear proteins over its entire length. Deletion of more than 30 bp from either end dramatically reduces activity, and even large internal fragments carrying seven of the eight DNAse I-protected elements show decreased activity. Scanning replacement mutations demonstrate that several of the internal elements are required for activity of the enhancer. Thus, the GnRH gene is targeted to hypothalamic neurons by a complex multicomponent enhancer that relies on the interaction of multiple nuclear-protein binding enhancer elements.

Animals↗
PubMed1994

Cloning and functional expression of a rat heart KATP channel.

Potassium channels that are ATP-sensitive (KATP) couple membrane potential to the metabolic status of the cell. KATP channels are inhibited by intracellular ATP and are stimulated by intracellular nucleotide diphosphates. KATP channels are important regulators of secretory processes and muscle contraction, and are targets for therapeutic treatment of type II diabetes by the inhibitory sulphonylureas and for hypertension by activators such as pinacidil. In cardiac tissue, KATP channels are central regulators of post-ischaemic cardioprotection. Electrophysiological and pharmacological characteristics vary among KATP channels recorded from diverse tissues suggesting extensive molecular heterogeneity. A complementary DNA encoding a KATP channel was isolated from rat heart using the polymerase chain reaction. We report here that the expressed channels possess all of the essential features of native cardiac KATP channels, including sensitivity to intracellular nucleotides. In addition the cloned channels are activated by the potassium channel opener, pinacidil, but are not inhibited by the sulphonylurea, glibenclamide.

Adenosine Triphosphate↗
PubMed1994

A second gene for gonadotropin-releasing hormone: cDNA and expression pattern in the brain.

In vertebrates, the gonadotropin-releasing hormone (GnRH) decapeptide is secreted from hypothalamic nerve terminals to regulate reproduction via control of synthesis and release of pituitary gonadotropins. Only one GnRH peptide has been found in mammals, with one exception, although numerous other vertebrate species express more than one of the eight known decapeptide forms as shown by immunocytochemical labeling of distinct cell groups in the brain. However, neither the functional nor the evolutionary relationships among these GnRH forms are clear, because only one preprohormone gene sequence from any species has been reported. The most ubiquitous alternative form of GnRH is [His5,Trp7,Tyr8]GnRH (also referred to as chicken-II), which differs from the mammalian sequence at amino acids 5, 7, and 8. This peptide has been shown to have the most potent releasing-hormone activity, although immunocytochemical staining has suggested it is synthesized only in the mesencephalon. Here we report the cloning and expression pattern of the gene for the precursor of this form from the teleost fish Haplochromis burtoni. This is the second GnRH-encoding gene to be characterized in this species. The newly discovered preprohormone gene differs from that previously reported in two ways. First, whereas the original gene predicts only a single associated peptide, this one predicts two associated peptides, both of which appear to be unique. Second, the gene for [His5,Trp7,Tyr8]GnRH is expressed in only one cell group in the mesencephalon. In contrast, the previously reported gene is expressed only in the terminal nerve. The striking differences between the preprohormone structure and localization suggest that the genes coding for the two known GnRH forms in H. burtoni did not arise from a recent duplication event. Interestingly, neither of the two genes found to date in this species is expressed in cells which project from the hypothalamus to the pituitary, suggesting that yet a third gene coding for GnRH may exist.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of a family of inward rectifier potassium channels.

Five new members of the two-transmembrane domain potassium channel family have been identified from rat brain, heart and skeletal muscle. The channel mRNAs are differentially expressed and found in both the central nervous system and periphery. Expression of two of these channels in Xenopus oocytes gave rise to inwardly rectifying potassium currents which were voltage-dependently blocked by barium and cesium. Voltage command pulses negative to Ek evoked inward currents which rapidly reached a peak amplitude and relaxed to a steady-state level. The quantity of current relaxation differed in the two channels and was increased at more negative potentials. The degree of current rectification was also different for the two channels. The results demonstrate the existence of a large and widely expressed family of inward rectifier potassium channel subunits with distinct tissue distributions and functional properties.

Amino Acid Sequence↗
PubMed1993

The min K channel underlies the cardiac potassium current IKs and mediates species-specific responses to protein kinase C.

A clone encoding the guinea pig (gp) min K potassium channel was isolated and expressed in Xenopus oocytes. The currents, gpIsK, exhibit many of the electrophysiological and pharmacological properties characteristic of gpIKs, the slow component of the delayed rectifier potassium conductance in guinea pig cardiac myocytes. Depolarizing commands evoke outward potassium currents that activate slowly, with time constants on the order of seconds. The currents are blocked by the class III antiarrhythmic compound clofilium but not by the sotalol derivative E4031 or low concentrations of lanthanum. Like IKs in guinea pig myocytes, gpIsK is modulated by stimulation of protein kinase A and protein kinase C (PKC). In contrast to rat and mouse IsK, which are decreased upon stimulation of PKC, myocyte IK and gpIsK in oocytes are increased after PKC stimulation. Substitution of an asparagine residue at position 102 by serine (N102S), the residue found in the analogous position of the mouse and rat min K proteins, results in decreased gpIsK in response to PKC stimulation. These results support the hypothesis that the min K protein underlies the slow component of the delayed rectifier potassium current in ventricular myocytes and account for the species-specific responses to stimulation of PKC.

Amino Acid Sequence↗
PubMed1992

Calcium-activated potassium channels expressed from cloned complementary DNAs.

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.

Amino Acid Sequence↗
PubMed1992

Partial characterization of the gonadotropin-releasing hormone (GnRH) gene transcript in the rat ovary.

It has been hypothesized that GnRH or a GnRH-like peptide is produced in the rat ovary, but the presence of GnRH in the ovary has not been unequivocally demonstrated. This study was undertaken to determine whether the GnRH gene is expressed in the rat ovary and to compare the GnRH gene transcripts from the ovary and the hypothalamus. Twelve samples of total RNA from ovaries of individual rats were screened by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of GnRH gene transcripts. Fragments of GnRH cDNA were amplified using pairs of specific primers. GnRH transcripts were detected in all the ovaries examined, and differed from hypothalamic GnRH transcripts in two ways: first, in the ovaries a greater proportion of GnRH transcripts contained intronic sequences; second, the major transcription start utilized in the ovary differed from that used in the hypothalamus. Although fully processed GnRH gene transcripts were detected by RT-PCR in both, ovary and hypothalamus, they were not detected in the ovary by Northern blot. The GnRH probe hybridized specifically to the predicted 0.6 kb transcript in the hypothalamus, and to a 3.3 kb transcript in the ovary. We conclude that in the ovary, most GnRH gene transcripts retain intronic sequences.

Animals↗
PubMed1991

Characterization of complementary DNA encoding the precursor for gonadotropin-releasing hormone and its associated peptide from a teleost fish.

Reproductive maturity among male African cichlids Haplochromis burtoni is cued by a series of environmental and social interactions and is mediated physiologically by GnRH. A cDNA clone encoding the precursor for GnRH was isolated from this teleost. The molecular architecture of the predicted prohormone is analogous to that of the previously characterized mammalian forms; however, the predicted sequence of the associated peptide is strikingly different. Attempts to isolate a putative second precursor using low stringency hybridization were not successful despite evidence that a second related decapeptide exists in at least some teleost species.

Amino Acid Sequence↗
PubMed1989

In situ hybridization detection of marked differences in pre-proopiomelanocortin messenger ribonucleic acid content of individual corticotropes and melanotropes.

Recently, heterogeneity of POMC mRNA content between intermediate lobe melanotropes of the rat pituitary gland was demonstrated by in situ hybridization of tissue sections. In the present study the heterogeneity of POMC mRNA content in dispersed rat pituitary cells has been investigated. Acutely dispersed cells from adult male rat anterior or neurointermediate lobe tissues were adhered to poly-L-lysine-coated coverslips. The cells were fixed and then hybridized with 35S-labeled POMC or 1B15 (cyclophilin) cRNA. Parallel studies measuring constitutively expressed cellular 1B15 mRNA content were undertaken to ensure that the apparent single cell differences in POMC mRNA were not inherent to the in situ hybridization procedure. When classified by image analysis, extensive differences in silver grain densities were seen over POMC mRNA-containing cells from both lobes. To determine if mRNA in polysomal configurations was less accessable for hybridization with probes than naked mRNA, cells were preincubated with pactamycin, a potent inhibitor of ribosomal initiation of protein synthesis. Pactamycin had no effect on these results. Thus, there appears to be large differences in POMC mRNA content between individual pituitary cells expressing the same gene product.

Adrenocorticotropic Hormone↗
PubMed1989

Combined immunohistochemistry for gonadotropin-releasing hormone (GnRH) and pro-GnRH, and in situ hybridization for GnRH messenger ribonucleic acid in rat brain.

Experiments were performed to explore the distribution of neurons containing pro-GnRH and GnRH in the rat brain and to determine the correspondence of immunoreactive peptides and pro-GnRH mRNA. Using avidin-biotin immunohistochemistry on free floating vibratome sections it was found that pro-GnRH- and GnRH-containing cells exhibited a similar distribution within the preoptic area-basal hypothalamus region. Within individual neurons pro-GnRH was primarily detected in the cell soma and proximal fibers, whereas the decapeptide was present in cells, fibers, and nerve terminals. Combined avidin-biotin immunohistochemistry for GnRH or pro-GnRH peptides and in situ hybridization for pro-GnRH mRNA using a cRNA probe revealed that the peptides and mRNA could be detected in the same cells. In both male and female rats pro-GnRH mRNA was localized primarily in GnRH-containing cells; however, not all immunoreactive GnRH neurons contained detectable levels of pro-GnRH mRNA, and not all neurons containing pro-GnRH mRNA contained GnRH peptides. In proestrous females a close correlation existed between the total number of neurons containing GnRH and those containing pro-GnRH mRNA (r = 0.84-0.9), while in intact male rats the correlation was not as high (r = 0.56). These results document the distribution of pro-GnRH and GnRH in the rat preoptic area-basal hypothalamus and describe the extent of colocalization of GnRH peptide and pro-GnRH mRNA in proestrous females and intact male rats. Further work will determine how each of the molecular components is regulated during different reproductive states.

Animals↗
PubMed1989

The rat gonadotropin-releasing hormone: SH locus: structure and hypothalamic expression.

The rat GnRH gene as expressed in the central nervous system is comprised of four exons and three introns and spans 4.5 kilobases of genomic DNA. Recently it has been shown that the DNA strand opposite that which is transcribed to produce GnRH mRNA is transcribed in heart to produce a set of transcripts, SH RNAs, which share significant exonic sequences with the GnRH gene. The nucleotide sequence of this locus and approximately 3 kilobases on either side has been determined. Northern analysis of hypothalamic RNA probed with GnRH and SH strand specific probes demonstrate that both GnRH and SH RNAs are present within the preoptic hypothalamus. The cap sites for GnRH and SH transcripts have been localized using polymerase chain reaction technology. Results from these experiments indicate that in the preoptic hypothalamus GnRH transcription initiates from three sites. The majority of GnRH transcripts is spliced efficiently and gives rise to the major class of GnRH mRNA. A second spliced population is present in lower abundance, while a third population is not spliced. The SH gene contains at least two distinct promoters, from which two populations of transcripts are derived containing unique 5'-sequences spliced to a common 3'-region.

Animals↗
PubMed1988

Heterogeneity of motilin immunoreactivity in mammalian tissue.

Motilin is a 22 amino acid peptide first isolated and sequenced from porcine gut. The use of antisera directed to the synthetic porcine gut motilin sequence has produced conflicting results as to regional distribution and histological localization of motilin in mammalian brain and gut. Motilin immunoreactivity has been detected (by RIA) in brain regions where no immunostaining is discernible. Variations in the patterns of staining are also observed with different antisera. These discrepancies have been explained by postulating species-, tissue-, and region-specific variations in peptide immunoreactivity, and variable cross reactivities of the independent antisera with these forms. The use of cloned porcine cDNA encoding gut prepromotilin in Northern blot analysis of brain regions expressing motilin-like immunoreactivity has also failed to reveal a homologous message, questioning the true nature of the immunoreactive material in the brain. Physiological studies, however, have suggested central roles for motilin in a variety of CNS (feeding behavior, bladder control, cerebral and brain stem modulation, pituitary growth hormone release) and gastrointestinal (gastric emptying, intestinal motility) functions. The motilin immunoreactive material detected in brain may be encoded by a distinct non-homologous gene, and still share amino acid homologies with the motilin sequence. Molecular biological characterization of the cell systems which contain motilin and motilin-like immunoreactivity should allow a better definition of their roles in these tissues.

Adrenal Medulla↗
PubMed1988

Characterization of complementary deoxyribonucleic acid for precursor of porcine motilin.

Cloned cDNAs encoding the precursor protein for motilin and a novel peptide, motilin-associated peptide, were isolated from a library derived from porcine intestinal mucosa mRNA. Nucleotide sequence analysis predicts a precursor protein of 119 amino acids including a signal peptide in direct linkage with the 22 amino acid sequence for motilin, and a 70 amino acid peptide of unknown function. The putative bioactive moieties are separated by Lys-Lys, dibasic residues that serve as substrates for cleavage by proteolytic maturation enzymes in many polyprotein precursors. While there is an abundant literature detailing a spectrum of tissues and cell types which express motilin like immunoreactivity, analysis of mRNA derived from many of these tissues suggests that the mRNA for the mucosal motilin precursor is only transcribed in this tissue. The nature of the immunoreactive material in the central nervous system and other peripheral tissues remains to be determined.

Animals↗
PubMed1987

Two mammalian genes transcribed from opposite strands of the same DNA locus.

This report describes the characterization of a genomic locus in the rat that encodes overlapping genes occupying both strands of the same piece of DNA. One gene (strand) encodes gonadotropin-releasing hormone (GnRH). A second gene, SH, is transcribed from the other DNA strand to produce RNA of undefined function. The RNAs transcribed from each DNA strand are spliced and polyadenylated, and share significant exon domains. GnRH is expressed in the central nervous system while SH transcripts are present in the heart. Thus, the genome of a mammalian organism encodes two distinct genes by using both strands of the same DNA.

Animals↗
PubMedDate not supplied

Heterooligomeric assembly of inward-rectifier K+ channels from subunits of different subfamilies: Kir2.1 (IRK1) and Kir4.1 (BIR10).

Activities of strong inward-rectifier K+ channels composed of Kir2. 1(84 M), Kir2.1(84T) and Kir4.1 subunits and weak inward-rectifier K+ channels composed of Kir4.1(E158N) subunits were measured from giant inside-out patches of Xenopus laevis oocytes. The conductance/voltage (g/V) relationship for block by intracellular spermine (SPM) was biphasic for both Kir2.1 channel types while it was monophasic for both Kir4.1 channel types. The release of blocking Mg2+ ions was slow for Kir2.1(84T) but virtually instantaneous for Kir2.1(84M) and both Kir4.1 channel types. Coexpression of Kir2.1(84T) and Kir4.1(E158N) resulted in heterooligomeric channels which were strongly rectifying, with a g/V relationship for SPM-evoked block that was significantly different from that of either parental homooligomeric channel type. Block by intracellular Mg2+ was markedly stronger than that for Kir4.1(E158N) channels, while release of the block was almost instantaneous, similar to that for Kir4.1(E158N) channels. This suggests preferential formation of a particular heterooligomer such as was recently proposed for subunits within the Kir3.0 family.

Animals↗