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Biomedical subjects

J D Neill

Publications and source records attributed to J D Neill.

At least 19 recordsLinked to original sources
PubMed1992

Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor.

Gonadotropin releasing hormone is a hypothalamic decapeptide that stimulates the release of gonadotropic hormones from the anterior pituitary gland. Therapeutically, the human pituitary GnRH receptor is the target of agonists used in the suppression of prostate cancer. Here we report the isolation of a cDNA representing this receptor. It encodes a protein with a transmembrane topology similar with that of other G protein-coupled, 7-transmembrane receptors. Binding studies of the cloned receptor demonstrate high affinity and pharmacological properties similar with the native human pituitary GnRH receptor. Northern blot and reverse transcriptase/PCR analysis revealed that its mRNA is expressed in pituitary, ovary, testis, breast, and prostate but not in liver and spleen. Availability of a human GnRH receptor cDNA should permit the design of improved analogs for therapeutic applications.

Amino Acid Sequence↗
PubMed1992

Differential expression of angiotensin II receptor subtype mRNAs (AT-1A and AT-1B) in the brain.

Two highly similar rat angiotensin II, type 1 receptor cDNAs (AT1) have been described that probably are encoded by separate genes. AT1A subtype mRNA was expressed in vascular smooth muscle whereas AT1B mRNA was expressed in adrenal and pituitary. Here we measured the two AT1 subtype mRNAs in brain using reverse transcriptase/polymerase chain reactions. AT1B mRNA was predominant in subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), the two regions that mediate angiotensin II-induced drinking behavior, and also in cerebellum. AT1A mRNA was predominant in the hypothalamus. Thus, the two AT1 receptor subtypes established to reside in peripheral tissues also are found in the central nervous system where the AT1B subtype may mediate drinking behavior.

Animals↗
PubMed1992

Angiotensin II type-1 receptor subtype cDNAs: differential tissue expression and hormonal regulation.

A rat angiotensin, type 1A (AT1A) receptor cDNA was cloned recently and shown to be a member of the 7-transmembrane, G-protein coupled family of receptors. Here, we report the cloning, sequencing, and expression of a previously unsuspected second form of the type 1 receptor (AT1B) in the rat which exhibits high similarity with the AT1A receptor relative to amino acid sequence (95% identity), binding of angiotensin II analogs, and utilization of Ca+2 as its intracellular second messenger. The adrenal and pituitary gland express primarily AT1B mRNA whereas vascular smooth muscle and lung express primarily AT1A mRNA. Estrogen treatment suppressed AT1B but not AT1A mRNA levels in the pituitary gland. Thus, the unexpected existence of two putative AT1 receptor genes appears to be related to the differential regulation of their expression rather than to different functional properties of the encoded receptor proteins.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of the capsid protein gene of two serotypes of San Miguel sea lion virus: identification of conserved and non-conserved amino acid sequences among calicivirus capsid proteins.

The San Miguel sea lion viruses, members of the calicivirus family, are closely related to the vesicular disease of swine viruses which can cause severe disease in swine. In order to begin the molecular characterization of these viruses, the nucleotide sequence of the capsid protein gene of two San Miguel sea lion viruses (SMSV), serotypes 1 and 4, was determined. The coding sequences for the capsid precursor protein were located within the 3' terminal 2620 bases of the genomic RNAs of both viruses. The encoded capsid precursor proteins were 79,500 and 77,634 Da for SMSV 1 and SMSV 4, respectively. The SMSV 1 protein was 47.7% and SMSV 4 was 48.6% homologous to the feline calicivirus (FCV) capsid precursor protein while the two SMSV capsid precursors were 73% homologous to each other. Six distinct regions within the capsid precursors (denoted as regions A-F) were identified based on amino acid sequence alignment analysis of the two SMSV serotypes with FCV and the rabbit hemorrhagic disease virus (RHDV) capsid protein. Three regions showed similarity among all four viruses (regions B, D and F) and one region showed a very high degree of homology between the SMSV serotypes but only limited similarity with FCV (region A). RHDV contained only a truncated region A. A fifth region, consisting of approximately 100 residues, was not conserved among any of the viruses (region E) and, in SMSV, may contain the serotype-specific determinants. Another small region (region C) contained between 15 and 27 amino acids and showed little sequence conservation. Region B showed the highest degree of conservation among the four viruses and contained the residues which had homology to the picornavirus VP3 structural protein. An open reading frame, found in the 3' terminal 514 bases of the SMSV genomes, encoded small proteins (12,575 and 12,522 Da, respectively for SMSV 1 and SMSV 4) of which 32% of the conserved amino acids were basic residues, implying a possible nucleic acid-binding function.

Amino Acid Sequence↗
PubMed1991

Localization of receptors for luteinizing hormone-releasing hormone in pancreatic and mammary cancer cells.

Previous work showed that hamster and human pancreatic tumors but not normal pancreata exhibit low-affinity cell-membrane receptors for luteinizing hormone-releasing hormone (LHRH). Although the regression of experimental pancreatic cancers induced by treatment with LHRH agonists or antagonists could be explained in part by the creation of sex-steroid deficiency, direct effects mediated by LHRH receptors might also play a role. Here, we demonstrate that pancreatic tumor cells do exhibit high-affinity binding sites for LHRH, but only in their nuclei; low-affinity sites are associated with the cell membranes. These binding sites appear to be LHRH receptors since electron microscopic immunohistochemical studies show that an antibody to the LHRH receptor reacted with sites in the nucleus of pancreatic tumor cells. Immunoreactive sites in the nucleus also were found in a restricted set of normal hamster pituitary cells thought to be luteinizing hormone-secreting cells and in MXT mouse mammary tumor cells. Such nuclear receptors may be involved in the transmission of the direct action of LHRH analogues on the tumor cells, resulting in the enhancement of programmed cell death.

Animals↗
PubMed1991

Nucleotide sequence and expression of the capsid protein gene of feline calicivirus.

The sequence of the 3'-terminal 2,486 bases of the feline calicivirus (FCV) genome was determined. This region of the FCV genome, from which the 2.4-kb subgenomic RNA is derived, contained two open reading frames. The larger open reading frame, found in the 5' end of the subgenomic mRNA, contained 2,004 bases encoding a polypeptide of 73,467 Da. The smaller open reading frame, encoded in the 3' end of the mRNA, was composed of 318 bases, encoding a polypeptide of 12,185 Da. The AUG initiation codon of the second open reading frame overlapped the UGA termination codon of the first, with the sequence AUGA. The nucleotide sequence of the region containing this overlap resembles the -1 frameshift sequences of the retroviruses. The 5' end of the 2.4-kb subgenomic RNA was mapped by primer extension analysis. There were two apparent transcription initiation points, both of which were 5' to the AUG initiation codon of the large open reading frame. Transcription from these sites yielded RNA transcripts with 5' nontranslated leader regions of 17 and 18 bases. The total length of the 2.4-kb subgenomic RNA was 2,375 bases (from the 5'-most start site) excluding the poly(A) tail. Edman degradation of the purified capsid protein of FCV showed that the capsid protein was encoded by the large open reading frame. Western immunoblot analysis of FCV-infected cells using a feline anti-FCV antiserum demonstrated that translation of the capsid protein was detectable at 3 h postinfection and continued to accumulate until 8 h postinfection, the last time examined.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of a region of the feline calicivirus genome which encodes picornavirus-like RNA-dependent RNA polymerase, cysteine protease and 2C polypeptides.

A region of the feline calicivirus (FCV) genome was sequenced which encodes polypeptides that are similar by amino acid alignment analysis to several picornavirus polypeptides. These polypeptides include the 2C polypeptide, the 3C cysteine protease and the 3D RNA-dependent RNA polymerase. The 2C-like region of the FCV genome encodes a GxxGxGKT nucleotide binding motif as well as amino acids which have been shown to be conserved in the picornavirus 2C polypeptides. The FCV RNA-dependent RNA polymerase also shows regions of similarity with picornavirus RNA polymerase sequences including the GDD sequence which is thought to be in or near the active site of the polymerase. The FCV cysteine protease-like sequences have the lowest degree of similarity with picornavirus cysteine proteases of the three regions aligned. However, the cysteine and histidine residues thought to be in the active site of the protease are present and are surrounded by amino acids conserved in the picornavirus cysteine proteases. The order of the polypeptides encoded in the FCV genome is the same as in the picornaviruses with the RNA-dependent RNA polymerase being located at the C-terminus of the FCV polyprotein. However, there is an approximately 40,000 dalton region between the FCV 2C- and the cysteine protease-like polypeptides which has no similarity to any known picornavirus protein. A striking difference between the organization of these sequences in FCV and the picornaviruses is that in the FCV genome, these non-structural polypeptides are encoded near the 5' end of the genomic RNA. Termination of the reading frame encoding these polypeptides occurs approximately 2400 bases from the 3' end of the genomic RNA as compared to 71 bases in the poliovirus genomic RNA.

Amino Acid Sequence↗
PubMed1986

Antibodies to the binding site of the receptor for luteinizing hormone-releasing hormone (LHRH): generation with a synthetic decapeptide encoded by an RNA complementary to LHRH mRNA.

A molecular recognition code has been hypothesized to exist in which ligands and their binding sites are encoded on complementary segments of genomic DNA. We have tested this hypothesis by generating a rabbit antibody to a synthetic decapeptide (complementary peptide) encoded by an RNA complementary to the mRNA for luteinizing hormone-releasing hormone (LHRH) and determining whether this antibody recognizes the LHRH receptor. When the antibody was used for immunoperoxidase staining of enzymatically dispersed rat anterior pituitary cells, only those that contained and secreted luteinizing hormone (i.e., the gonadotropes) were recognized. This staining could be abolished by preincubation with the complementary peptide or with an LHRH agonist, suggesting that the antibody is specific to the complementary peptide and is directed at the binding site of the receptor. Further evidence that the antibody recognizes the LHRH receptor was obtained in immunoblot experiments on solubilized receptors from pituitary glands. Immunoperoxidase staining with the antibody revealed two bands at 60 kDa and 51 kDa, which are values similar to those previously obtained for the LHRH receptor in photoaffinity-labeling experiments. The staining of these bands was inhibited by preincubation with the complementary peptide or an LHRH agonist. The antibody as well as the complementary peptide to LHRH also suppressed LHRH-stimulated luteinizing hormone release in a quantitative reverse hemolytic plaque assay, presumably by binding to the LHRH receptor and by binding LHRH, respectively. These findings suggest that the synthetic decapeptide whose sequence is specified by the complementary RNA to LHRH mRNA is sufficiently similar to an LHRH binding site that the peptide not only binds LHRH but was also recognized by the immune system as such a site. These findings provide strong support for the hypothesis that recognition molecules are encoded by complementary segments of genomic DNA.

Amino Acid Sequence↗
PubMed1979

Observations on some properties of a long-acting preparation of propranolol.

1. The effects of 160 mg of propranolol and 160 mg of a long-acting (LA) formulation of propranolol were studied in healthy subjects. 2. Both drugs reduced an exercise tachycardia but the peak was less and the 24 h effect greater after long-acting propranolol than after propranolol. 3. These differences were maintained on repeated dosing for 8 days. 4. In contrast to single doses of 400 mg of sotalol, 160 mg of oxprenolol and 160 mg of slow-release oxprenolol, the peak effect of long-acting propranolol was less and that at 24 h was greater.

Adult↗
PubMed1979

Dopamine levels in hypophysial stalk plasma of the rat during surges of prolactin secretion induced by cervical stimulation.

Tonic hypothalamic inhibition of PRL release is partially explainable by dopamine secretion into hypophysial portal blood. However, the probable existence of other PRL-inhibiting factors as well as PRL-releasing factors opens to question the role of dopamine in the dynamic regulation of PRL secretion. We investigated this question in the present study by measuring dopamine concentrations in hypophysial stalk blood of the rat during the surgess of PRL secretion induced by cervical stimulation. Urethane anesthesia, necessary for the surgery attendant to stalk blood collection, did not suppress the surge of PRL secretion induced by cervical stimulation 16-24 h previously. Increases in plasma PRL levels during such surges were 4- to 5-fold above baseline. Dopamine concentrations in hypophysial stalk plasma were 36% lower in cervically stimulated than in control rats during the diurnal and nocturnal PRL surges. However, dopamine levels were not different during the interval between the surges, a time at which PRL levels are similar in stimulated and control rats. To determine if the observed 36% decrease in dopamine levels might account for the associated 4- to 5-fold rise in PRL levels during surges, we treated rats with alpha-methyl-p-tyrosine to block endogenous dopamine secretion and then infused dopamine at various rates to achieve plasma dopamine concentrations throughout the physiological range. These dopamine levels significantly but incompletely suppressed PRL levels, and a 36% decrease in administered dopamine was associated with only an approximate 1.5-fold increase in plasma PRL levels. Thus, it is unlikely that changes in dopamine secretion alone can account for the increased release of PRL engendered by cervical stimulation.

Animals↗