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Biomedical subjects

G I Bell

Publications and source records attributed to G I Bell.

At least 163 records · Page 9Linked to original sources
PubMed1993

Molecular genetics of myeloid leukemia: identification of the commonly deleted segment of chromosome 20.

A deletion of the long arm of chromosome 20 [del(20q)] is a recurring abnormality in malignant myeloid disorders. The occurrence of the del(20q) in a broad spectrum of myeloid disorders suggests that the loss of genetic material on 20q could provide a proliferative advantage to myeloid cells, possibly through the loss of a tumor-suppressor gene. We have examined a series of patients with the del(20q) using fluorescence in situ hybridization (FISH) with unique sequence probes that map along the length of 20q, and have delineated a segment that is deleted in 95% of all patients examined (18 of 19). In addition, we have shown that the deletions are interstitial rather than terminal. This region of deletion extends from 20q11.2 to q12, and is flanked by the RPN2 (proximal) and D20S17 loci (distal). The SRC and ADA genes are located within the commonly deleted segment. Our findings emphasize the importance of FISH and other molecular mapping techniques in defining such a region. The delineation of a commonly deleted segment in 20q11.2-q12 will facilitate the identification of candidate tumor-suppressor genes on 20q.

Adult↗
PubMed1993

A single residue, aspartic acid 95, in the delta opioid receptor specifies selective high affinity agonist binding.

The enkephalins, dynorphins, and endorphins are endogenous opioids which function as neurotransmitters, neuromodulators, and hormones and are involved in the perception of pain, modulation of behavior, and regulation of autonomic and neuroendocrine function. Pharmacological studies have defined three classes of opioid receptors, designated as delta, kappa, and mu. To investigate mechanisms by which agonists and antagonists interact with the delta opioid receptor, we have substituted aspartic acid 95 in the transmembrane segment 2 of the cloned mouse delta opioid receptor with an asparagine (D95N). The D95N mutant receptor had reduced affinity for delta receptor-selective agonists such as enkephalin, [D-Pen2,D-Pen5]enkephalin and [D-Ser2,Leu5]enkephalin-Thr6 such that it did not bind these peptides even at micromolar concentrations. The binding of delta-selective non-peptide agonists was also reduced. In contrast, the delta receptor-selective antagonists, such as naltrindole, the benzofuran analog of naltrindole, and 7-benyllidenenaltrexone, bound equally well to the wild-type and mutant receptor. Similarly, non-selective opioid agonists such as bremazocine and buprenorphine, which interact with delta, kappa, and mu opioid receptors, showed no difference in binding to the wild-type and mutant delta receptor. The D95N mutant remained coupled to G proteins, and the receptor was functionally active since it mediated agonist inhibition of cAMP accumulation. These results indicate that selective agonists and antagonists bind differently to the delta receptor and show that Asp-95 contributes to high affinity delta-selective agonist binding. The identification of a key residue involved in selective agonist binding to the delta opioid receptor will facilitate the development of novel therapeutic reagents that can be used for the treatment of chronic pain and other conditions.

Animals↗
PubMed1993

Cloning and functional comparison of kappa and delta opioid receptors from mouse brain.

While trying to identify new members of the somatostatin receptor family of G protein-coupled receptors, we isolated cDNAs from a mouse brain library encoding two related receptor-like proteins, designated msl-1 and msl-2, of 380 and 372 amino acids, respectively. There was 61% identity and 71% similarity between the sequences of msl-1 and msl-2. Among members of the G protein-coupled receptor superfamily, the sequences of both msl-1 and msl-1 were most closely related to those of the somatostatin receptors (SSTRs), having approximately 35% identity with the sequence of SSTR1. Transient expression in COS-1 cells showed that msl-1 and msl-2 did not bind somatostatin. Rather they bound opioids selectively and with high affinity and had the pharmacological properties of kappa and delta opioid receptors, respectively. Indeed, the sequence of msl-2 was identical to that of a delta opioid receptor recently cloned by other workers. Functional characterization of kappa/msl-1 and delta/msl-2 opioid receptors showed that they were coupled to G proteins and mediated opioid receptor class-specific agonist inhibition of forskolin-stimulated cAMP formation. RNA blotting studies and in situ hybridization histochemistry showed that kappa opioid receptor mRNA was expressed at high levels in brain in the neocortex, hippocampus, amygdala, medial habenula, hypothalamus (arcuate and paraventricular nuclei), locus ceruleus, and parabrachial nucleus, suggesting that this receptor may play a role in arousal and regulation of autonomic and neuroendocrine functions.

Amino Acid Sequence↗
PubMed1993

Gi alpha 3 and G(o) alpha selectively associate with the cloned somatostatin receptor subtype SSTR2.

The interaction of the SRIF receptor subtype SSTR2 with pertussis toxin-sensitive G proteins was investigated using an immunoprecipitation approach employing peptide-directed antisera against Gi alpha and G(o) alpha. Antisera directed against either the COOH terminus of Gi alpha or Go alpha uncoupled SSTR2-G protein complexes from CHO cells stably expressing the cloned receptor indicating that both G proteins form complexes with SSTR2. Chinese hamster ovary cells primarily express Gi alpha 3 and G(o) alpha 2 immunoreactivity, with much lower levels of the other pertussis toxin-sensitive G proteins. Antiserum against Gi alpha 3 uncoupled SSTR2/G protein complexes to a similar extent as Gi alpha common antiserum while antisera against Gi alpha 1 and Gi alpha 2 did not. These findings indicate that SSTR2 expressed in Chinese hamster ovary cells predominantly associates with Gi alpha 3 and G(o) alpha 2. In HEK 293 cells which endogenously express low densities of SSTR2 and similar levels of Gi alpha 1 and Gi alpha 3 immunoreactivity but no G(o) alpha, only antiserum directed against Gi alpha 3 immunoprecipitated SSTR2-G protein complexes, indicating that in these cells SSTR2 primarily associates with Gi alpha 3. SRIF can not inhibit forskolin-stimulated cAMP formation in wild-type HEK 293 cells nor in HEK 293 cells transfected with SSTR2. In contrast, SRIF can inhibit cAMP formation in HEK 293 cells expressing the cloned SRIF receptor SSTR3, which requires the presence of Gi alpha 1 to functionally couple to adenylyl cyclase. The lack of efficient association of SSTR2 with Gi alpha 1 may be the cause of its inability to mediate inhibition of cAMP formation. Differences in the G protein-coupling domains of the cloned SRIF receptors may be responsible for their differences in G protein association and ability to effect various signaling pathways.

Adenylate Cyclase Toxin↗
PubMed1993

Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues.

Inositol 1,4,5-trisphosphate (IP3) functions as a second messenger for many neurotransmitters, hormones and growth factors. It causes the release of Ca2+ from intracellular stores by binding to specific receptors that are coupled to Ca2+ channels. Recent studies have shown that there is a family of IP3 receptors, and the complete sequences of two members of this family and partial sequences of two others have been reported. We have determined the complete sequence of a third IP3 receptor, designated IP3R-3, and characterized its pharmacological properties and sites of expression. Rat IP3R-3 is 2670 amino acids in size, has 62 and 64% identity with IP3R-1 and IP3R-2, and is predicted to have a similar structure including a region of eight potential membrane-spanning segments at its COOH terminus, which presumably functions as a Ca2+ channel. Expression of recombinant rat IP3R-3 in COS-7 cells showed that it bound IP3 as well as inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate. Immunohistocytochemical studies of cells expressing recombinant IP3R-3 indicated that it has a preferential cellular distribution in the endoplasmic reticulum. RNA and protein blotting studies indicate that IP3R-3 is expressed in a number of different cultured cell lines including insulin-secreting RINm5F cells. The IP3R-3 is also expressed in adult pancreatic islets, kidney, gastrointestinal tract, and brain. Reverse transcriptase-polymerase chain reaction amplification of IP3R-1, -2, and -3 mRNAs in adult rat pancreatic islets indicated that IP3R-3 was the predominant subtype expressed in this tissue and thus may be responsible for mediating the effects of IP3 on insulin secretion.

Amino Acid Sequence↗
PubMed1993

Hormonal/metabolic regulation of the human GLUT4/muscle-fat facilitative glucose transporter gene in transgenic mice.

To examine the hormonal/metabolic as well as tissue-specific expression of the GLUT4/muscle-fat facilitative glucose transporter gene, we have generated several transgenic mouse lines expressing a human GLUT4 mini-gene which extends 5.3 kilobases (kb) upstream of transcription start and terminates within exon 10. This construct (hGLUT4-11.5) was expressed in a tissue-specific pattern identical to the endogenous mouse GLUT4 gene. The transcription initiation sites of the transgenic construct were similar to the GLUT4 gene expressed in human tissues. To investigate the hormonal/metabolic-dependent regulation of GLUT4, the transgenic animals were made insulin-deficient by streptozotocin (STZ) treatment. In these animals, STZ-induced diabetes resulted in a parallel decrease in endogenous mouse GLUT4 mRNA and the transgenic human GLUT4 mRNA in white adipose tissue, brown adipose tissue, and cardiac muscle. Similarly, insulin treatment of the STZ-diabetic animals restored both the endogenous mouse and transgenic human GLUT4 mRNA levels. To further define cis-regulatory regions responsible for this hormonal/metabolic regulation, the same analysis was performed on transgenic animals which carry 2.4 kb of the human GLUT4 5'-flanking region fused to a CAT reporter gene (hGLUT4[2.4]-CAT). This reporter construct responded similarly to the human GLUT4 mini-gene demonstrating that the element(s) controlling hormonal/metabolic regulation and tissue specificity all reside exclusively within 2.4 kb of the transcriptional initiation site.

Adipose Tissue↗
PubMed1993

Human somatostatin receptor genes: localization to human chromosomes 14, 17, and 22 and identification of simple tandem repeat polymorphisms.

The genes encoding three different human somatostatin receptor subtypes, designated SSTR1, SSTR2, and SSTR3, were mapped to chromosomes 14, 17, and 22, respectively, by analyzing their segregation in a panel of reduced human-hamster somatic cell hybrids. These assignments were confirmed independently using fluorescence in situ hybridization to metaphase chromosomes and the genes were further localized to 14q13, 17q24, and 22q13.1, respectively. Highly informative simple tandem repeat DNA polymorphisms were identified in SSTR1 and SSTR2.

Base Sequence↗
PubMed1993

Human somatostatin receptor genes: localization of SSTR5 to human chromosome 20p11.2.

The gene encoding the somatostatin receptor subtype designated as SSTR5 was mapped to human chromosome 20p11.2 by using fluorescence in situ hybridization to metaphase chromosomes. Fluorescence in situ hybridization using a probe for SSTR5 in combination with probes for neuroendocrine convertase-2 (NEC2), thrombomodulin (THBD), and brain glycogen phosphorylase (PYGB) established a physical order for these loci of 20pter-NEC2-SSTR5-THBD-PYGB-cen.

Chromosome Mapping↗
PubMed1993

Human insulin receptor substrate-1 gene (IRS1): chromosomal localization to 2q35-q36.1 and identification of a simple tandem repeat DNA polymorphism.

The protein designated as insulin receptor substrate-1 (IRS-1) is a major substrate for the insulin receptor tyrosine kinase. Since post-receptor defects in the insulin signalling pathway are a common feature of Type 2 (non-insulin-dependent) diabetes mellitus, we have cloned the human IRS-1 gene in order to study the role of genetic variation in this gene in the pathogenesis of diabetes mellitus. As a first step in these studies, we have mapped the IRS-1 gene to chromosome 2, bands q35-q36.1 and identified a simple tandem repeat DNA polymorphism in this gene that will be useful for genetic studies.

Animals↗
PubMed1993

Characterization of a third simple tandem repeat polymorphism in the human glucokinase gene.

Recent studies have implicated mutations in the glucokinase gene as a cause of early-onset Type 2 (non-insulin-dependent) diabetes mellitus. Two simple tandem repeat DNA polymorphisms have been identified in this gene and used for genetic studies. However, their heterozygosity is relatively low and not all families are informative for linkage with these markers. Here we report the characterization of a third simple tandem repeat polymorphism that can be used for genetic studies.

Base Sequence↗
PubMed1993

Human hexokinase II: localization of the polymorphic gene to chromosome 2.

Type 2 (non-insulin-dependent) diabetes mellitus is characterized by decreased levels of glucose 6-phosphate in skeletal muscle. It has been suggested that the lower concentrations of glucose 6-phosphate contribute to the defect in glucose metabolism noted in muscle tissue of subjects with Type 2 diabetes or subjects at increased risk of developing Type 2 diabetes. Lower levels of glucose 6-phosphate could be due to a defect in glucose uptake, or phosphorylation, or both. Hexokinase II is the isozyme of hexokinase that is expressed in skeletal muscle and is responsible for catalysing the phosphorylation of glucose in this tissue. The recent demonstration that mutations in another member of this family of glucose phosphorylating enzymes, glucokinase, can lead to the development of Type 2 diabetes prompted us to begin to examine the possible role of hexokinase II in the development of this genetically heterogeneous disorder. As a first step, we have cloned the human hexokinase II gene (HK2) and mapped it to human chromosome 2, band p13.1, by fluorescence in situ hybridization to metaphase chromosomes. In addition, we have identified and characterized a simple tandem repeat DNA polymorphism in HK2 and used this DNA polymorphism to localize this gene within the genetic linkage map of chromosome 2.

Alleles↗
PubMed1993

Type 2 (non-insulin-dependent) diabetes mellitus associated with a mutation of the glucokinase gene in a Japanese family.

Mutations were screened for in the glucokinase gene of 25 Japanese patients with Type 2 (non-insulin-dependent) diabetes mellitus. Each exon was scanned by electrophoresis of enzymatically amplified DNA segments under non-denaturing conditions and variants were sequenced. A variant pattern was detected in exon 5 of one patient. Direct sequencing of this exon revealed a single nucleotide substitution in codon 188 (GCT-->ACT) of one of two alleles resulting in the mutation of Ala188-->Thr, an invariant residue in the sequence of all mammalian glucokinases and hexokinases. This mutation was not found in 40 normal control subjects. The proband had been diagnosed with Type 2 diabetes at the age of 62 years. Four other members of her family have the same mutation and all have Type 2 diabetes or impaired glucose tolerance. The youngest age at diagnosis of Type 2 diabetes in these other members was 13 years, suggesting that her pedigree was maturity-onset diabetes of the young (MODY). All subjects with the Thr188 mutation show a decreased insulin secretory response during oral glucose tolerance testing. Mutations in the glucokinase gene associated with Type 2 diabetes have been previously identified in Caucasian (French and British) subjects. This study indicates that mutations in this gene are also implicated in the development of Type 2 diabetes in Asians. Further studies are required to determine the frequency of mutations in glucokinase among Japanese patients with Type 2 diabetes.

Adolescent↗
PubMed1993

Molecular biology of somatostatin receptors.

The neuropeptide somatostatin (SRIF) has diverse physiological actions in the brain and endocrine organs. A family of SRIF receptors has recently been cloned that may mediate the distinct biological effects of SRIF. These receptors have a high degree of amino acid sequence similarity among themselves, but their sequences are different from any other receptors, indicating that they represent a distinct neurotransmitter receptor subfamily. The availability of the cloned receptors will now allow for detailed structure-function analysis of SRIF receptors and will facilitate development of subtype-selective agonists and antagonists that could be useful in the treatment of central nervous system and endocrine disorders.

Amino Acid Sequence↗
PubMed1993

Molecular biology of opioid receptors.

Opium and its derivatives are potent analgesics that also have many other pharmacological effects in the nervous system. These agents and the endogenous opioid peptides exert their effects by interacting with high-affinity receptors. Complementary DNAs encoding the delta, kappa and mu opioid receptors have been isolated and characterized. These receptors, which are members of the superfamily of seven-transmembrane spanning receptors, share a high degree of amino acid sequence similarity with approximately 50% of the residues being identical. The cloned opioid receptors mediate agonist inhibition of cyclic AMP formation and have pharmacological properties similar to the endogenous proteins. The cloning of these receptors will facilitate the development of new clinically useful compounds as well as studies of the molecular basis of tolerance and drug addiction.

Amino Acid Sequence↗
PubMed1993

cDNA sequence and localization of polymorphic human cytosolic phosphoenolpyruvate carboxykinase gene (PCK1) to chromosome 20, band q13.31: PCK1 is not tightly linked to maturity-onset diabetes of the young.

Complementary DNA clones encoding human cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) (PEPCK)] were isolated from a human kidney cDNA library. The nucleotide sequence of the 2.7 kb insert of one of these clones indicates that human PEPCK is a protein of 622 amino acids whose sequence shows 90% identity with that of the cognate rat enzyme. The human PEPCK gene (PCK1) was isolated by hybridization using a fragment of the hPEPCK cDNA as a probe. PCK1 was mapped to human chromosome 20 using DNA from a panel of reduced human-hamster somatic cell hybrids. This assignment was confirmed using fluorescence in situ chromosomal hybridization which localized PCK1 to chromosome 20, band q13.31. A simple tandem repeat DNA polymorphism in the 3'-untranslated region of the mRNA was characterized and used to localize PCK1 relative to the gene responsible for a form of non-insulin-dependent (Type 2) diabetes mellitus called maturity-onset diabetes of the young (MODY). Linkage studies showed that PCK1 is not tightly linked to MODY in one large pedigree and exclude this diabetes candidate gene as the cause of MODY in this family.

Alleles↗