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H Kong

Publications and source records attributed to H Kong.

At least 55 records · Page 3Linked to original sources
PubMed1995

Characterization of the cloned human mu opioid receptor.

Opioid drugs exert a wide spectrum of physiological and behavioral effects, including effects on pain perception, mood, motor control and autonomic functions. The effects of opioids are mediated via a family of membrane-bound receptors, of which the most extensively characterized are the mu, delta and kappa receptors. We have now cloned the human homolog of the mu opioid receptor and, in the present study, we have examined its pharmacological profile. The human mu receptor has high affinities for several alkaloids of high abuse potential as well as a variety of peptide and nonpeptide drugs characterized previously as mu-selective, but not delta- or kappa-selective. Most importantly, the human mu receptor has higher affinity for morphine and methadone than does the rat mu receptor, despite the fact that these receptors are 95% identical at the amino acid level. The labeling of the receptor by agonist was decreased by nonhydrolyzable GTP analogs and by pertussis toxin treatment of cells expressing the human mu receptor, consistent with the coupling of the receptor to guanine nucleotide binding proteins. The human mu receptor functionally couples to the inhibition of adenylyl cyclase in a stereospecific and naloxone-reversible manner. We have also investigated the distribution of mRNAs encoding the mu receptor in human brain by Northern analysis, which demonstrates the existence of multiple transcripts of 13.5, 11, 4.3 and 2.8 kb, which were highly expressed in the hypothalamus, thalamus and subthalamic nucleus, more moderately expressed in the amygdala and caudate nucleus and which demonstrated lowest levels of expression in the hippocampus, substantia nigra and corpus callosum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Agonists and antagonists bind to different domains of the cloned kappa opioid receptor.

Opium and its derivatives are potent analgesics that can also induce severe side effects, including respiratory depression and addiction. Opioids exert their diverse physiological effects through specific membrane-bound receptors. Three major types of opioid receptors have been described, termed delta, kappa, and mu. The recent molecular cloning of these receptor types opens up the possibility to identify the ligand-binding domains of these receptors. To identify the ligand-binding domains of the kappa and delta receptors, we have expressed in COS-7 cells the cloned mouse delta and kappa receptors and chimeric delta/kappa and kappa/delta receptors in which the NH2 termini have been exchanged. The opioid antagonist naloxone binds potently to wild-type kappa receptor but not to wild-type delta receptor. The kappa/delta chimera bound [3H]naloxone with high affinity. In contrast, the kappa-specific agonist [3H]U-69,593 did not bind to the kappa/delta chimera. These findings indicate that selective agonists and antagonists interact with different recognition sites in the kappa receptor and localize the antagonist-binding domain to the NH2 terminus. Consistent with the results of radioligand-binding studies, the kappa/delta chimera did not mediate kappa-agonist inhibition of cAMP formation. In contrast, the delta/kappa chimera did mediate kappa-agonist inhibition of cAMP formation, but this effect was not blocked by naloxone. Furthermore, a truncated kappa receptor lacking its NH2 terminus was able to mediate agonist inhibition of cAMP accumulation in a naloxone-insensitive manner. This result further indicates that the NH2 terminus of the kappa receptor contains the selective antagonist-binding domain. The ability to dissociate agonist- and antagonist-binding sites will facilitate the development of more specific kappa agonists, which could have analgesic properties devoid of side effects.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1994

Expression of mu opioid receptor mRNA in rat brain: an in situ hybridization study at the single cell level.

The mu (mu) opioid receptors, which mediate the effects of morphine, are widely distributed in brain. We have examined the distribution of mRNA encoding a mu opioid receptor in rat brain with in situ hybridization histochemistry at the single-cell level to obtain information about the cell types synthesizing this receptor. Only neurons, not glia, were labeled in discrete brain regions. High levels of labeling were detected in the thalamus, striosomes of the caudate-putamen, globus pallidus, and brain regions involved in nociception, arousal, respiratory control, and, possibly, addiction. The general distribution of the receptor mRNA paralleled that of mu opioid binding sites with some notable exceptions. These include the cerebral cortex, which contains binding sites, but very few labeled neurons. No labeling was observed in the cerebellum, a region devoid of mu binding sites. Three main findings emerged from these experiments: 1) the mRNA was present in regions mediating both the therapeutic (analgesia) and the unwanted (respiratory depression, addiction) effects of morphine, 2) the mRNA was very densely expressed by neurons known to receive dense enkephalin-containing inputs, and 3) the dissociation between the presence of binding sites and absence of mRNA in some brain regions supports a presynaptic localization of mu opioid receptors in these areas. Alternatively, other subtypes of mu opioid receptors may be encoded by a different mRNA. These results provide new insights into the receptor types and neuronal circuits involved in the effects of endogenous opioids and morphine.

Animals↗
PubMed1994

Characterization of BcgI, a new kind of restriction-modification system.

The BcgI restriction enzyme from Bacillus coagulans is unusual in that it cleaves on both sides of its recognition site, CGAN6TGC, releasing a fragment that includes the site and several bases on each side. We report the organization and nucleotide sequences of the genes for the BcgI restriction-modification system and the properties of the proteins that they encode. The system comprises two adjacent, similarly oriented genes. The proximal gene, bcgIA, codes for a 637-amino acid protein (molecular mass = 71.6 kDa) that resembles certain m6A-specific DNA-methyltransferases, particularly those that constitute the modification subunits of type I restriction-modification systems. The distal gene, bcgIB, codes for a 341-amino acid protein (molecular mass = 39.2 kDa) that resembles none of the sequences in the sequence data bases. The two genes overlap by several nucleotides. Alone, neither protein restricts or modifies DNA, but, together, they form a complex in the proportion A2B that does both. DNA binding assays showed that the DNA-protein complex can be formed only in the presence of both subunits, suggesting that the association of inactive subunits generates the active BcgI enzyme that can bind DNA and then either cleaves or methylates at target site.

Adenine↗
PubMed1994

Differential expression of messenger RNAs for somatostatin receptor subtypes SSTR1, SSTR2 and SSTR3 in adult rat brain: analysis by RNA blotting and in situ hybridization histochemistry.

The messenger RNAs encoding three somatostatin receptor subtypes, SSTR1, SSTR2 and SSTR3, were detected in rat by RNA blotting and in situ hybridization histochemistry to identify the sites of synthesis and expression of these somatostatin receptor subtypes. RNA blotting revealed that SSTR1 messenger RNA of 3.8 kilobases was highly expressed in cerebral cortex, hippocampus, midbrain and hypothalamus. In situ hybridization histochemistry revealed that SSTR1 messenger RNA was localized to discrete layers of the cerebral cortex, the piriform cortex and the dentate gyrus of the hippocampus. SSTR1 messenger RNA was expressed at low levels in the cerebellum and pituitary and was not detectable in striatum or other peripheral organs. At least two SSTR2 messenger RNAs were detected by RNA blotting of 2.4 and 2.8 kilobases which correspond to the size of the spliced and unspliced forms of this receptor messenger RNA. SSTR2 messenger RNA detected by in situ hybridization is diffusely expressed in cerebral cortex and amygdala but is discretely localized to dentate gyrus in the hippocampus, medial habenula and ventromedial and dorsomedial nuclei and arcuate nucleus of the hypothalamus. The levels of SSTR2 messenger RNA are very low in the cerebellum and were not observed in the striatum or peripheral tissues other than the pituitary or adrenal gland. A single SSTR3 messenger RNA of 4.0 kilobases was seen in hippocampus, cerebral cortex, midbrain, hypothalamus and pituitary. However, the tissue with the highest levels of SSTR3 messenger RNA is the cerebellum with messenger RNA localized to the granule cell layer. The expression of the three different somatostatin receptor messenger RNAs are distinct but overlapping. Such distinct expression may contribute to the selective biological roles of the receptor subtypes.

Animals↗
PubMed1994

Molecular mechanisms of agonist-induced desensitization of the cloned mouse kappa opioid receptor.

Prolonged exposure of opioid receptors to agonists can cause desensitization, a cellular event linked to tolerance. Although evidence exists for mu and delta opioid receptor desensitization, much less information is available concerning the in vitro regulation of kappa opioid receptors because no cell lines exist that specifically express this class of opioid receptor. Recently we have cloned the mouse kappa opioid receptor. After expression in COS-7 cells, this protein exhibits the pharmacological specificity of a kappa 1 receptor and mediates agonist inhibition of cAMP formation. Continuous exposure of COS-7 cells expressing the kappa receptor to the agonist trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate salt (U50,488) reduces the specific binding of the kappa-selective agonist [3H]U69,593. Furthermore, the potency of U50,488 to inhibit the binding of the opiate antagonist [3H]naloxone to the kappa receptor is reduced. However, total specific binding of [3H]naloxone is not altered, indicating that short-term (2-4 hr) agonist treatment of the kappa receptor reduces the affinity of the receptor for agonists but does not reduce the density of kappa receptors. The reduction in affinity of the kappa receptor for agonists is dependent on the time of agonist exposure and is reversible. The reduced affinity of the receptor for agonists is associated with kappa receptor desensitization, because kappa receptor-mediated inhibition of cAMP formation is lost in cells pretreated with U50,488. The desensitization of the kappa receptor is dependent on the time and concentration of agonist treatment, is blocked by the kappa-selective antagonist nor-binaltorphimine and is reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1994

Pharmacological characterization of the cloned kappa-, delta-, and mu-opioid receptors.

Opioid drugs, such as morphine, and the endogenous opioid peptides, namely the enkephalins, endorphins, and dynorphins, exert a wide spectrum of physiological and behavioral effects, including effects on pain perception, mood, motor control, and autonomic functions. These effects are mediated via membrane-bound receptors, of which the best characterized are the kappa, delta, and mu receptors. The existence of these distinct types of opioid receptors has recently been confirmed by molecular cloning. In the present study, we have examined the pharmacological profiles of the cloned kappa, delta, and mu receptors using a battery of widely employed opioid agents. Our results suggest that the cloned kappa and mu receptors have pharmacological characteristics similar to those of the endogenously expressed kappa 1 and mu receptors, respectively. The cloned delta receptor displays a pharmacological profile consistent with that of a delta 2 receptor. Opioid agents with abuse potential possess high affinities for the mu receptor. The availability of the cloned receptors will facilitate the identification and development of more specific and selective compounds with greater therapeutic potential and fewer undesirable side effects.

Analgesics↗
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

A unique restriction endonuclease, BcgI, from Bacillus coagulans.

We have purified and characterized a new restriction endonuclease, BcgI, which has properties unlike those of the three recognized classes of restriction enzymes. BcgI was isolated from Bacillus coagulans, and it recognizes the sequence CGAN6TGC. BcgI cleaves double stranded DNA on both strands upstream and downstream of the recognition sequence, so that the recognition sequence is released as a 34-base pair fragment with 2-base 3'-extensions. Mg++ and S-adenosylmethionine are required for cleavage. Sinefungin, a structural analogue of AdoMet which generally inhibits methylase activity, can replace AdoMet in the cleavage reaction. The apparent binding constant (Kappd) for AdoMet is about 100 nM, while the KappD for sinefungin is about 500 nM.

Bacillus↗
PubMed1993

Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities.

We have isolated, cloned, and characterized a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis, the Tli DNA polymerase (also referred to as Vent DNA polymerase). The enzyme is extremely thermostable, having a half-life of 8 h at 95 degrees C and about 2 h at 100 degrees C. Pseudo-first-order kinetics at 70 degrees C reveal an extremely low Km for a primed M13mp18 substrate (0.1 nM), coupled with a relatively high Km for dNTPs (50 microM). Accompanying extension rates are on the order of 1000 nucleotides/min. Synthesis by the polymerase is largely distributive, adding an average of 7 nucleotides/initiation event. This distributive synthesis can generate products of at least 10,000 bases. Tli DNA polymerase contains a 3'-->5' exonuclease activity that enhances the fidelity of replication by the enzyme (Mattila, P., Korpela, J., Tenkanen, T. and Pitkanen, K. (1991) Nucleic Acids Res. 19, 4967-4973). A 2-amino acid substitution within the conserved exonuclease domain abolishes both double and single strand-dependent exonuclease activity, without altering kinetic parameters for polymerization on a primed single-stranded template. Strand displacement activity by the mutated and unmutated forms increases with increasing temperature and is enhanced in the exonuclease-deficient form of the enzyme.

Archaea↗
PubMed1993

Splice variant of the somatostatin receptor 2 subtype, somatostatin receptor 2B, couples to adenylyl cyclase.

The diverse biological actions of somatostatin (SRIF) are mediated by a family of receptors, of which five have been cloned and characterized. One of the SRIF receptor subtypes, SSTR2, has been shown to exist in two forms. SSTR2A and SSTR2B are 369 and 346 amino acids in size, respectively, and differ in length and amino acid sequence in their intracellularly located carboxyl termini. SSTR2A and SSTR2B are generated by alternative splicing of SSTR2 mRNA. We previously characterized mouse SSTR2A and showed that it could be distinguished from other cloned SRIF receptor subtypes by its high affinity for MK-678 and its lack of coupling to adenylyl cyclase. To determine whether the properties of mouse SSTR2A and SSTR2B differ, we have expressed both in COS-7 cells and characterized their ligand-binding properties and ability to couple to adenylyl cyclase. The two receptors exhibited similar affinities for a number of SSTR2-selective agonists such as MK-678. Pretreatment with SRIF of COS-7 cells expressing each receptor reduced high affinity agonist binding to both SSTR2A and SSTR2B, indicating that both receptors can be regulated. Furthermore, agonist binding to both receptors was reduced by GTP analogs and Na+, indicating that they both associate with G proteins. As shown previously, SSTR2A could not mediate SRIF inhibition of forskolin-stimulated cAMP formation. In contrast, SSTR2B was coupled to adenylyl cyclase and was able to mediate SRIF inhibition of forskolin-stimulated cAMP formation. Thus, SSTR2A and SSTR2B differ in their ability to couple to adenylyl cyclase. Because SSTR2A and SSTR2B differ only in the length and amino acid sequence of their carboxyl termini, these findings imply that the carboxyl-terminal 15 residues of SSTR2B may be involved in coupling this receptor to adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1993

Mutation of an aspartate at residue 89 in somatostatin receptor subtype 2 prevents Na+ regulation of agonist binding but does not alter receptor-G protein association.

Sodium ions have been shown to reduce the binding of agonists to a number of G protein-linked receptors. They are believed to do so by interacting with aspartate residues in the second membrane-spanning region of these receptors to cause G protein uncoupling, resulting in a diminished affinity of the receptors for agonists. To investigate Na+ regulation of agonist binding to somatostatin receptors, Na+ was tested for its effect on the binding of agonists to cloned somatostatin receptor type 1 (SSTR1) and somatostatin receptor type 2 (SSTR2) stably expressed in Chinese hamster ovary cells. Na+ reduced agonist binding to SSTR2 but not to SSTR1. Because high affinity agonist binding to SSTR1 does not depend on G protein coupling but agonist binding to SSTR2 is reduced by guanosine-5'-(beta, gamma-imido)triphosphate and pertussis toxin treatment, the selective Na+ effect on SSTR2 is consistent with previous findings with other receptors showing that Na+ uncouples receptors from G proteins, thereby reducing the affinity of the receptors for agonists. Conversion of Asp89 to Asn89 in SSTR2 resulted in a mutant receptor whose affinity for agonists was not altered by Na+, indicating that Asp89 is involved in mediating the effects of Na+ on agonist binding to SSTR2. However, the affinities of the mutant and wild-type receptors for somatostatin were the same, and both guanosine-5'-O-(gamma-thio)triphosphate and pertussis toxin treatment reduced agonist binding to the mutant and wild-type receptors. These findings differ from the results of similar mutagenesis studies on other G protein-linked receptors, in that the mutant and wild-type SSTR2 forms associate with G proteins in similar ways. These results indicate that Asp89 acts in a novel manner to regulate agonist binding and G protein interaction with SSTR2.

Animals↗
PubMed1993

Characterization of cloned somatostatin receptors SSTR4 and SSTR5.

The recent molecular cloning of the genes and cDNAs encoding multiple somatostatin (SRIF) receptor subtypes has allowed for the individual expression of these receptors in mammalian cells and characterization of their respective pharmacological profiles. Previously, we fully described and compared the pharmacological properties of the first three SRIF receptor subtypes, SRIF receptor type (SSTR)1, SSTR2, and SSTR3. In the present study, we have investigated the properties of the newly cloned SRIF receptor subtypes SSTR4 and SSTR5 with regard to pharmacological profiles, the regulation of high affinity agonist binding to these receptors by stable GTP analogues, Na+, or prior exposure to agonists, and the inhibition of forskolin-stimulated cAMP accumulation mediated by these receptors. We labeled SSTR4 and SSTR5 expressed in Chinese hamster ovary (CHO-K1) and COS-1 cells, respectively, with the metabolically stable SRIF analogue 125I-CGP 23996. Radioligand binding competition studies were performed using SRIF analogues of differing structures, including hexapeptide analogues similar to MK-678, octapeptide analogues similar to SMS 201-995, pentapeptide analogues similar to c[Ahep-Phe-D-Trp-Lys-Thr(Bzl)], and linear SRIF analogues. SSTR4 bound compounds in all structural classes with high to moderate affinities, and several compounds were identified that are > 100-fold selective for SSTR4, compared with the other cloned SRIF receptors, including the linear SRIF analogue BIM-23052 and the CGP 23996-like SRIF analogue L-362,855. In contrast, SSTR5 bound very few SRIF analogues with high affinity. Both receptors could be regulated by prior exposure to agonist. In addition, agonist binding to SSTR4 was reduced by stable GTP analogues, Na+, and pertussis toxin, but agonist binding to SSTR5 was not affected by these treatments. SSTR4 is efficiently coupled to the inhibition of adenylyl cyclase activity, whereas SSTR5 appears not to couple to this cellular effector system. Such differences between the cloned SRIF receptors provide useful strategies for identifying regions of these receptor subtypes that may be involved in ligand-binding specificities and G protein and cellular effector system coupling. The identification of subtype-selective SRIF analogues may lead to more specific therapeutic interventions.

Amino Acid Sequence↗
PubMed1993

Lipoperoxidative damage in experimental rabbits with atherosclerosis.

The extent of lipoperoxidation in experimental rabbits with atherosclerosis was determined dynamically during the experimental period of 65 days. Lipoperoxide (LPO) levels and selenium-dependent glutathions peroxidase (SeGSHPx) activities in liver, aorta, heart muscle, plasma erythrocyte (RBC) and platelet were examined on the 65th day. The results showed that the potential anti-lipoperoxidation in the atherosclerotic rabbits was decreased significantly, and the tissues were suffered from lipoperoxidative damage. It seems that there is a close relationship between lipoperoxidative damage and the development of atherosclerosis.

Animals↗
PubMed1989

Female gene expression in the seminal vesicle of mice after prenatal exposure to diethylstilbestrol.

Previous studies from our laboratory on the feminization of the male mouse reproductive tract after prenatal exposure to diethylstilbestrol (DES) showed that the mRNA for the major estrogen-inducible uterine secretory protein, lactoferrin (LF), was constitutively expressed in the seminal vesicle of male mice exposed prenatally to DES, but not in the seminal vesicle of control mice. After castration, treatment with 17 beta-estradiol (20 micrograms/kg.day) for 3 days induced the LF mRNA in the seminal vesicle of both control and prenatally DES-exposed mice; however, the levels in DES-treated tissues were approximately 6-fold higher than those in control tissue. This report describes the presence of LF in seminal vesicle tissues and secretions of prenatally DES-exposed mice, as determined by immunohistochemistry and Western blot analysis. Further, these data are correlated with immunolocalization of the estrogen receptor in the seminal vesicle tissue. We conclude that the seminal vesicle of prenatally DES-exposed male mice has acquired two key characteristics of female tissues, namely LF production/regulation and estrogen receptor localization/distribution similar to that in uterine tissues.

Animals↗