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Biomedical subjects

B J Hoffman

Publications and source records attributed to B J Hoffman.

At least 91 records · Page 5Linked to original sources
PubMed1995

Endoscopic ultrasound and clinical competence.

The last few years have seen substantial growth in technology, research, and performance of endoscopic ultrasound. Unfortunately, opportunities for training in EUS have not kept pace with this growth. To improve the breadth and quality of training in EUS, resources will need to be channeled in two directions. One is the development of endoscopic ultrasound training programs in academic centers. These would provide the expertise and the number of patients necessary to train selected GI fellows in EUS. The second direction is toward development of systems for retraining of physicians already in practice. We believe that this would require the development of EUS simulation systems combined with a short, intense, hands-on training in centers of excellence. How fast these training systems will develop is dependent upon market forces. If the information provided by EUS can be proven to reduce costs and if the results improved patient's quality of life, the development of training programs for EUS would accelerate markedly.

Clinical Competence↗
PubMed1994

Adenosine A3 receptors regulate serotonin transport via nitric oxide and cGMP.

Many antidepressants inhibit 5-hydroxytryptamine (5HT) transport resulting in increased 5HT levels in the synapse. However, physiological regulation of neurotransmitter uptake has not been demonstrated. We have examined the effect of receptor-activated second messengers on the 5HT transporter in rat basophilic leukemia cells (RBL 2H3). Here, we show that activation of an A3 adenosine receptor results in an increase of 5HT uptake in RBL cells, due to an increase in maximum velocity (Vmax). The A3 adenosine receptor-stimulated increase in transport is blocked by inhibitors of nitric oxide synthase and by a cGMP-dependent kinase inhibitor. In fact, compounds that generate nitric oxide (NO) and the cGMP analog 8-bromo-cGMP mimicked the effect of A3 receptor stimulation, suggesting that the elevation in transport occurs through the generation of the gaseous second messenger NO and a subsequent elevation in cGMP. Additionally, the 5HT transporter is differentially regulated by second messengers since direct activation of protein kinase C by phorbol esters decreases 5HT uptake by decreasing Vmax. Our results suggest that the changes in transport are due to a direct modification of the 5HT transporter, possibly by phosphorylation, which appears to alter the rate at which transport occurs. As the 5HT transporter in RBL cells is identical to that in neurons, our results suggest that analogous mechanisms may operate in the brain.

Adenosine↗
PubMed1994

Conducting states of a mammalian serotonin transporter.

We have studied permeation at a cloned rat 5-HT transporter expressed in Xenopus oocytes. [3H]5-HT uptake and [125I]RTI-55 binding yield a turnover rate of approximately 1/s that does not depend on membrane potential. However, in voltage-clamp experiments, three distinct currents results from 5-HT transporter expression. First, a steady-state, voltage-dependent transport-associated current is induced by 5-HT application. Second, a transient inward current is activated by voltage jumps to high negative potentials in the absence of 5-HT and is blocked by 5-HT itself. Third, a small leakage current is observed in the absence of 5-HT. All the observed currents are blocked by inhibitors of 5-HT uptake but are differentially affected by Na+, Li+, K+, Ba2+, Cs+, Cl-, and amiloride. The conducting states of the 5-HT transporter may reflect the existence of a permeation pathway similar to that of ionic channels.

Amiloride↗
PubMed1994

Expression cloning of a serotonin transporter: a new way to study antidepressant drugs.

Tricyclic antidepressants revolutionized the treatment of depression. These results and the monoamine-depleting effect of reserpine have contributed to the proposal that an imbalance in monoamines is a causal factor in depression. Most antidepressants act to concentrate monoamines in the synapse either by blocking metabolism via monoamine oxidase or by inhibiting reuptake by plasma membrane transporters. We have used a novel cDNA expression cloning strategy to isolate cDNAs for the antidepressant-sensitive serotonin transporter and for a reserpine-sensitive vesicular monoamine transporter which is critical for packaging serotonin, dopamine, norepinephrine, epinephrine and histamine into synaptic vesicles and secretory granules. In addition, we have isolated a dopamine transporter. The three papers summarized here describe the molecular characteristics of three proteins critical for monoamine neurotransmission and which are targets for antidepressant and psychostimulant drugs. The cloning of the serotonin and dopamine transporters and of the CNS vesicular transporter provide new tools to examine how post-translational and transcriptional regulation of these transporters effect uptake, storage and release of monoamines in normal and disease states. In addition to providing substrates for further drug discovery, isolation of human homologues should be useful for assessing the possible genetic bases of depression.

Animals↗
PubMed1994

Potential problems in using [35S]-dATP-tailed oligonucleotides for detecting mRNAs in certain cells of the immune system.

In this study we examined the cause of unusually intense signals obtained in immune cells by in situ hybridization histochemistry using 35S-labeled oligonucleotides. We verified that the phenomenon is an amplification of a specific signal due to a series of chemical interactions after the probe binds to a specific mRNA in the tissue. The presence of oxidative enzymes in the tissue seems to be necessary for this reaction to occur. Therefore, most cells of the immune system (e.g., macrophages, neutrophil and eosinophil leukocytes), being rich in oxidative enzymes, will show some signal amplification. The intensification of the signal can be avoided if MgCl2 is substituted for CoCl2 in the synthesis of [35S]-thiophosphate-labeled probes, if 2,3-dimercaptopropanol [British anti-Lewisite (BAL)] is added to the hybridization buffer, or if [33P]-phosphate is used instead of [35S]-thiophosphate in the labeling of the probes.

Adenosine Triphosphate↗
PubMed1994

Infectious esophagitis and the primary care physician.

The various clinical presentations of infectious esophagitis have been discussed. The physician approach to patients with suspected infectious esophagitis is based on whether the patient has an underlying immune problem. Symptomatic patients with thrush and AIDS should be empirically treated but most other patients should be referred to endoscopy. Considering the AIDS epidemic, any patient without known immune deficiency who is diagnosed with infectious esophagitis should be screened for an immunodeficiency disorder.

AIDS Serodiagnosis↗
PubMed1994

Pharmacological heterogeneity of the cloned and native human dopamine transporter: disassociation of [3H]WIN 35,428 and [3H]GBR 12,935 binding.

Controversy exists as to whether the functional state of the dopamine (DA) transporter is identical to sites mediating the specific binding of selective DA transporter radioligands. Therefore, we compared the pharmacological profile of numerous dopamine transport substrates and inhibitors on [3H]DA uptake with the binding of [3H]WIN 35,428 and [3H]GBR 12,935 to COS-7 cells transiently expressing the cloned human DA transporter. [3H]DA uptake and [3H]WIN 35,428 binding was specific, saturable, and to a single class of binding sites with an estimated Km/Vmax of approximately 2 microM and 6 pmol/min/10(5) cells for DA uptake and Kd/Bmax values of approximately 10 nM and 113 fmol/10(5) cells for [3H]WIN 35,428. [3H]DA uptake was inhibited in a concentration-dependent and uniphasic manner by dopaminergic agents with an appropriate rank order of potency for the DA transporter. Although most uptake blockers inhibited [3H]WIN 35,428 binding in a uniphasic manner, WIN 35,428, Lu 19,005, D-amphetamine, and DA clearly displayed the presence of both high and low affinity components. Comparison of the Ki values for the inhibition of [3H]DA uptake with [3H]WIN 35,428 binding reveals that, for uptake blockers and D-amphetamine, it is the high affinity component that shares pharmacological identity with effects on DA uptake (r = 0.9985), whereas for DA it is the low affinity site. In striking contrast, however, [3H]GBR 12,935 binding to COS-7 cells could not be made to exhibit a pharmacological profile indicative of the DA transporter and suggests that the site regulating functional [3H]DA uptake may not be identical with sites labeled by [3H]GBR 12,935 in these cells. Moreover, these sites appear unrelated to those previously described in native membranes as "piperazine acceptor" or P450 proteins. Comparison of Ki values and rank order of potency for the inhibition of [3H]WIN 35,428 or [3H]GBR 12,935 binding to human caudate membranes reveals pharmacological homology, but not identity, with that of the cloned DA uptake process. Taken together, these data suggest that 1) [3H]WIN 35,428 recognizes two sites of the DA transporter, of which only one appears to represent the functional state of the protein, and 2) [3H]WIN 35,428 and [3H]GBR 12,935 do not appear to bind the same functional form/state of the DA transporter. Whether the nonidentity of binding sites is a manifestation of some post-translational regulatory event (e.g., phosphorylation/accessory binding protein) or caused by the existence of multiple molecular forms of the DA transporter is currently unknown.

Amino Acid Sequence↗
PubMed1993

Calcineurin inhibits desensitization of cloned rat 5-HT1C receptors.

Functional responses to stimulation of rat 5-HT1C receptors expressed in A9 cells were studied using whole cell voltage clamp recording technique. Stimulation of 5-HT1C receptors with serotonin (5-HT) evoked calcium-dependent outward currents of 109 pA in cells clamped at -50 mV. Pretreatment with the protein kinase C (PKC) activator phorbol myristic acetate (PMA) reduced the 5-HT-induced current amplitude by 46% of the control value. Inclusion of inositol triphosphate (IP3) in the pipette solution induced an outward current of 84 pA. The IP3-induced response was not affected by 60 min pretreatment with PMA. In the presence of the PKC antagonist calphostin C, 60 min treatment with PMA (10(-6) mol/l) reduced the 5-HT response only by 8%. In cells preincubated with PMA, injection of the calcium/calmodulin dependent serine proteinphosphatase calcineurin gradually increased the 5-HT-induced responses by 34%. In A9 cells which were incubated 24 h with the 5-HT1C receptor agonist meta chlorophenylpiperazine hydrochloride (mCPP), 5-HT-induced responses were reduced by 23% of the vehicle pretreated control value. Injection of calcineurin in mCPP treated cells enhanced the 5-HT-induced response by 24%. The results suggest that in A9 cells rat 5-HT1C receptors are desensitized after phosphorylation by PKC. This desensitization can be counteracted by calcineurin-induced dephosphorylation.

Animals↗
PubMed1993

Characterization of functional responses in A9 cells transfected with cloned rat 5-HT1C receptors.

Functional responses to stimulation of rat 5-HT1C receptors expressed in A9 cells were studied using whole cell voltage clamp and calcium recording techniques. Stimulation of 5-HT1C receptors evoked outward currents clamped at -50 mV. The outward currents were reduced when GTP was excluded from the intracellular recording solution or when GDP-beta-S was added. 8-Bromo cyclic AMP (5 mmol/l) neither produced an effect per se nor affected the 5-HT-induced outward current in A9 cells, thus excluding cAMP as a second messenger involved in 5-HT1C receptor activation. Phorbol myristic acetate (PMA; 10 mumol/l) did not affect the electrical activity of the transfected A9 cells but reduced the 5-HT-induced current amplitude to 71 +/- 9% of the control value (n = 12). This indicates that activation of protein kinase C does not play a direct role in the 5-HT-induced response in these cells. The 5-HT induced currents mainly involved potassium ions, although a small contribution of chloride ions was also observed. The 5-HT-induced current was inhibited by the K+ channel blocking agents tetraethylammonium (1 mmol/l), apamin (0,5 mumol/l) and 4-aminopyridine (5 mmol/l). The 5-HT-induced currents recorded at -50 mV were unaffected by removal of extracellular calcium, but inclusion of the calcium chelator BAPTA (5 mmol/l) in the intracellular solutions abolished the current. Measurement with the calcium indicator Fluo-3 revealed a 5-HT-induced increase in intracellular calcium which was not affected by removal of extracellular calcium but declined after repeated stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Glial cell-specific expression of the serotonin 2 receptor gene: selective reactivation of a repressed promoter.

The 5' flanking region of the 5-HT2 receptor gene has been cloned, sequenced and its transcriptional regulatory functions analyzed. The promoter lacks an identifiable TATA motif, and utilizes at least 11 clustered start sites. Promoter function was analyzed by transient assays in rat C6 glioma cells, which were shown to express the endogenous 5-HT2 receptor gene, as well as in rat CREF and human HeLa cells which do not express the endogenous gene. The basal promoter functioned equally well in all three cell lines; and a repression domain, located upstream of the basal promoter, inhibited activity of the promoter in all three cell lines. A far upstream cell specific activator domain restored promoter activity in C6 glioma cells, but did not reactivate the silenced promoter in CREF or HeLa cells. The upstream activator domain, repressor domain and basal promoter functioned in concert to achieve cell type specific expression. The activator domain did not direct C6 glioma cell specific expression in the absence of the repressor domain or in constructs carrying a heterologous basal promoter. These results indicate that glial cell expression of the 5-HT2 receptor gene is achieved through a cell type specific reactivation of a repressed promoter.

Amino Acid Sequence↗
PubMed1993

Two glycine transporter variants with distinct localization in the CNS and peripheral tissues are encoded by a common gene.

We have isolated a cDNA encoding a high affinity, Na+/Cl(-)-dependent glycine transporter, GLYT-2, which is distinct from another glycine transporter, GLYT-1. While the 3' sequences of these two cDNAs are identical, the 5' noncoding regions and the N-termini are completely different. GLYT-1 is found only in the white matter of the CNS, while GLYT-2 is found in the gray matter of the CNS as well as in macrophages and mast cells in peripheral tissues. Our findings suggest that tissue-specific alternative splicing or alternative promoter usage from a single gene results in two mRNA products encoding similar but distinct glycine transporters. The anatomic distribution of GLYT-2 mRNA supports the emerging status of glycine as a supraspinal neurotransmitter and suggests that glycine may function as a chemical messenger outside the CNS.

Amino Acid Sequence↗
PubMed1993

Use of endoscopic ultrasonography for evaluation of painless jaundice.

Precise preoperative evaluation and staging of cases of painless jaundice is extremely important. Many of the patients are older and may have multiple medical problems that increase their operative risk. Assessment of the distal common bile duct, ampulla of Vater, and head of the pancreas is particularly difficult. Various imaging modalities are available but are not sensitive enough to detect small lesions or local invasion. Endoscopic ultrasonography is a new technique that places the transducer closer to the organ being evaluated, adding a new dimension to defining tumor invasion and extension.

Adenocarcinoma↗
PubMed1993

Evaluation of metaiodobenzylguanidine uptake by the norepinephrine, dopamine and serotonin transporters.

Metaiodobenzylguanidine (MIBG) is taken up by sympathetic neurons, but the precise mechanism of uptake has not been elucidated. Uptake of monoamines by presynaptic neurons is mediated by plasma membrane proteins, the monoamine transporters. The human norepinephrine transporter (hNET), the bovine dopamine transporter (bDAT) and the rat serotonin transporter (r5HTT) have been cloned, sequenced and expressed in various cell lines. This study involves the measurement of MIBG uptake by cell lines that have been transfected with complementary DNAs encoding these monoamine transporters. At 20 nM MIBG, hNET transfected cells demonstrate a ninefold greater uptake of MIBG than nontransfected cells. MIBG uptake in hNET transfected cells is inhibited by 3 x 10(-6) M norepinephrine (87% inhibition) and by hNET transport inhibitors: 10(-7) M desipramine (94% inhibition) and 10(-7) M mazindol (97% inhibition). hNET transfected cells exhibit a Km for MIBG transport of 264 nM. Percent nonspecific uptake rises with increasing concentrations of MIBG while specific uptake is saturable. There is no significant uptake by bDAT or r5HTT. The NET appears to be responsible for the specific uptake of MIBG.

3-Iodobenzylguanidine↗
PubMed1992

Expression cloning of a reserpine-sensitive vesicular monoamine transporter.

A cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [3H]serotonin accumulation by CV-1 cells expressing the cDNA clone suggested sequestration by an intracellular compartment. In cells permeabilized with digitonin, uptake was ATP dependent with an apparent Km of 1.3 microM. Uptake was abolished by the proton-translocating ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and with tri-(n-butyl)tin, an inhibitor of the vacuolar H(+)-ATPase. The rank order of potency to inhibit uptake was reserpine > tetrabenazine > serotonin > dopamine > norepinephrine > epinephrine. Direct comparison of [3H]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[125I]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of approximately 75 kDa. The mRNA that encodes MAT was present specifically in monoamine-containing cells of the locus coeruleus, substantia nigra, and raphe nucleus of rat brain, each of which expresses a unique plasma membrane reuptake transporter. The MAT cDNA clone defines a vesicular monoamine transporter representing a distinct class of neurotransmitter transport molecules.

Affinity Labels↗
PubMed1992

A human serotonin 1D receptor variant (5HT1D beta) encoded by an intronless gene on chromosome 6.

An intronless gene encoding a serotonin receptor (5HT1D beta) has been cloned and functionally expressed in mammalian fibroblast cultures. Based on the deduced amino acid sequence, the gene encodes a 390-amino acid protein displaying considerable homology, within putative transmembrane domains (approximately 75% identity) to the canine and human 5HT1D receptors. Membranes prepared from CHO cells stably expressing the receptor bound [3H]serotonin with high affinity (Kd 4 nM) and displayed a pharmacological profile consistent, but not identical, with that of the characterized serotonin 5HT1D receptor. Most notably, metergoline and serotonergic piperazine derivatives, as a group, display 3- to 8-fold lower affinity for the 5HT1D beta receptor than for the 5HT1D receptor, whereas both receptors display similar affinities for tryptamine derivatives, including the antimigraine drug sumatriptan. Northern blot analysis revealed an mRNA of approximately 5.5 kilobases expressed in human and monkey frontal cortex, medulla, striatum, hippocampus and amygdala but not in cerebellum, olfactory tubercle, and pituitary. The 5HT1D beta gene maps to human chromosome 6. The existence of multiple neuronal 5HT1D-like receptors may help account for some of the complexities associated with [3H]serotonin binding patterns in native membranes.

Amino Acid Sequence↗
PubMed1992

Molecular pharmacological differences in the interaction of serotonin with 5-hydroxytryptamine1C and 5-hydroxytryptamine2 receptors.

5-Hydroxytryptamine (5HT)1C and 5HT2 receptors appear to be closely related, from a molecular viewpoint, displaying similar second messenger systems and a high degree of sequence homology. However, there are striking differences in the interactions of 5HT with 5HT1C and 5HT2 receptors; 5HT is generally more potent in stimulating responses mediated through 5HT1C receptors than responses mediated through 5HT2 receptors. Also [3H]5HT labels 5HT1C receptors and not 5HT2 receptors. In order to explore more fully the molecular rationale for these differences, radioligand binding studies were performed in rat, human, and porcine brain and choroid plexus tissues and in mammalian cells transfected with rat 5HT1C or 5HT2 receptors; second messenger studies (inositol phosphate accumulation) were performed in the transfected cells. The second messenger studies confirmed the approximately 10-fold higher potency of 5HT in stimulating intracellular responses through 5HT1C receptors (EC50 = 8.3 nM) than in stimulating intracellular responses through 5HT2 receptors (EC50 = 101 nM). An agonist radioligand selective for the 5HT1C and 5HT2 receptors, 2,5-dimethoxy-(4-[125I]iodo)phenylisopropylamine, was used, as well as [3H]5HT, [3H]mesulergine (antagonist radioligand for 5HT1C receptors), and [3H]ketanserin (antagonist radioligand for 5HT2 receptors). Computer-assisted analyses of the binding data revealed two agonist affinity states for the 5HT1C receptor. The agonist high affinity state of the receptor was modifiable by guanyl nucleotides. The proportion of agonist high affinity states, relative to the total receptor population, was approximately 10% for both receptors. The apparent higher affinity of 5HT for the radiolabeled 5HT1C receptors was due to the higher affinity 5HT displayed for the agonist low affinity state of the 5HT1C receptor, compared with the affinity of 5HT for the agonist low affinity state of the 5HT2 receptor. The correspondence between the higher affinity of 5HT for the agonist low affinity state of the 5HT1C receptor, relative to the 5HT2 receptor, and the higher potency of 5HT in stimulating 5HT1C responses indicates that 5HT interacts with the agonist low affinity state of the 5HT1C and 5HT2 receptors in initiating its biological effects. These observations indicate that guanine nucleotide-binding protein (G protein)-coupled receptors can exhibit high affinity for neurotransmitters in both the free receptor and the G protein-coupled states and that receptors exhibiting this property may represent a novel subfamily of G protein-coupled receptors.

3T3 Cells↗