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H Rhim

Publications and source records attributed to H Rhim.

53 records · Page 3Linked to original sources
PubMed1994

Wild-type and mutant HIV-1 and HIV-2 Tat proteins expressed in Escherichia coli as fusions with glutathione S-transferase.

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 encode related transcriptional activators known as Tat-1 and Tat-2, respectively, that are required for efficient viral replication. The Tat proteins have been studied extensively, and it appears that their mechanism of action is unique to the primate immunodeficiency viruses or a few distantly related lentiviruses. Here we describe a collection of 24 wild-type and mutant Tat-1 and Tat-2 proteins that are expressed in Escherichia coli as fusions with glutathione S-transferase (GST). The GST-Tat fusions can be used for biochemical studies after simple purification from E. coli lysates in a single step under nondenaturing conditions. The availability of these GST-Tat fusions should be useful to investigators examining biochemical properties of Tat-1 and Tat-2 proteins. E. coli cultures harboring GST-Tat fusions described here are available through the National Institute of Health AIDS Research and Reference Reagent Program.

Amino Acid Sequence↗
PubMed1994

Opioid receptors modulate diverse types of calcium channels in the nucleus tractus solitarius of the rat.

We have investigated the coupling between opioid receptors and different types of Ca2+ channels in neurons acutely isolated from the nucleus tractus solitarius (NTS) of the rat. Using fura-2-based imaging we found that Ca2+ transients evoked by depolarization with 50 mM KCl were suppressed by the mu-opioid receptor agonist D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO) and less effectively by the kappa-receptor agonist U-69,593. The delta-receptor agonist D-Pen2,D-Pen5-enkephalin (DPDPE) was ineffective. In whole-cell voltage-clamp recordings from these neurons, depolarizing voltage steps elicited high-threshold Ca2+ currents that could be distinguished pharmacologically into different components. Part of the current could be blocked by dihydropyridines, part by omega-conotoxin-GVIA and part by omega-agatoxin-IVA. This suggests that the neurons contained L-, N-, and P/Q-type Ca2+ channels. DAMGO and U-69,593 both blocked part of the Ca2+ current but DPDPE was ineffective. Perfusion of GTP-gamma-S into the cells produced a rapid rundown of the Ca2+ current and occluded further effects of the opioid agonists, suggesting the involvement of a G-protein in the coupling mechanism. Inhibition of L-channels did not alter the effect of DAMGO. On the other hand inhibition of N-channels occluded about 80% of the effect of DAMGO. Inhibition of the P/Q-current occluded the remainder of the DAMGO effect. Thus, it appears that activation of opioid receptors can inhibit N- and P/Q-type Ca2+ channels but not L-channels in these cells. It is likely that such effects are important in opioid-mediated inhibition of transmitter release in the brain.

Animals↗
PubMed1994

Construction and characterization of a potent HIV-2 Tat transdominant mutant protein.

The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) Tat proteins Tat-1 and Tat-2 stimulate transcription of the viral long terminal repeat (LTR) sequences and are required for efficient viral replication. A class of mutant Tat proteins, termed "transdominant mutants," has been described that possesses relatively low transactivation activity, yet is able to inhibit the function of wild-type Tat. These mutant proteins contain a nonfunctional TAR RNA-binding domain but apparently retain a functional activation domain. A potential limitation for therapeutic use of transdominant mutants described to date is their low but significant basal level of transactivation for the HIV-1 or HIV-2 LTRs. In order to make an improved transdominant mutant, we have constructed Tat-2 proteins that contain mutations in four contiguous arginines at residues 81 to 84 in the RNA-binding domain. Using purified proteins and in vitro RNA-binding assays, we verified that these mutant Tat-2 proteins are defective for TAR RNA binding. We also verified that these mutant Tat-2 proteins bind to a cellular protein kinase in vitro that we have previously shown to bind specifically to the Tat-1 and Tat-2 activation domain. Using plasmid cotransfection assays, we compared the phenotypes of these mutant Tat-2 proteins with the most potent Tat-1 transdominant mutant described to date. One Tat-2 mutant, named "R81-84A," was found to be equivalent to the Tat-1 mutant in ability to inhibit wild-type Tat transactivation of HIV-1 and HIV-2 LTRs. Moreover, the R81-84A mutant possessed a significantly lower basal level of transactivation than the Tat-1 mutant. The R81-84A Tat-2 mutant is therefore a promising reagent for future development as an anti-HIV agent. Additionally, our results suggest that wild-type Tat-2 transactivation of the HIV-2 LTR is especially sensitive to inhibition by transdominant mutants.

Amino Acid Sequence↗
PubMed1993

Rapid purification of monomer HIV-2 Tat protein expressed in Escherichia coli.

Human immunodeficiency viruses types 1 and 2 encode transactivator proteins, named Tat-1 and Tat-2, that stimulate transcription directed by the viral long terminal repeat sequences. The Tat-1 and Tat-2 proteins are related in protein sequence and mechanism of transactivation. We expressed Tat proteins by in vitro translation and found, as expected, that both Tat-1 and Tat-2 were monomers. Unexpectedly, we found that the Tat-1 and Tat-2 proteins displayed significantly different tertiary structures. As determined by velocity sedimentation and gel filtration, Tat-1 acted as a compact structure and Tat-2 acted as an extended, asymmetric structure. Additionally, we found that the in vitro-expressed Tat-2 protein was significantly less susceptible to aggregation than the Tat-1 protein. This observation led us to overexpress Tat-2 in Escherichia coli and develop a simple and rapid method to purify monomer Tat-2 to approximately 50% purity. These results suggest that large amounts of Tat-2 protein can be purified in suitable form for biochemical and biophysical studies of Tat protein structure.

Amino Acid Sequence↗
PubMed1993

TAR RNA binding properties and relative transactivation activities of human immunodeficiency virus type 1 and 2 Tat proteins.

Using gel shift assays, we found that the human immunodeficiency virus type 1 (HIV-1) Tat protein (Tat-1) bound both HIV-1 and HIV-2 TAR RNAs with similar high affinities. In contrast, the HIV-2 Tat protein (Tat-2) bound only TAR-2 RNA with high affinity. We conclude that the weak in vivo activity of Tat-2 on the HIV-1 long terminal repeat that has been observed previously is likely the result of low affinity for TAR-1 RNA. Additionally, TAR-2 RNA was found to contain multiple specific binding sites for Tat proteins. GAL4-Tat fusion proteins were analyzed to compare the relative transactivation activities of Tat-1 and Tat-2 in the absence of requirements for binding to TAR RNAs. The GAL4-Tat-2 protein was found to transactivate synthetic promoters containing GAL4 binding sites at levels severalfold higher than did the GAL4-Tat-1 protein.

Amino Acid Sequence↗
PubMed1993

Selective opioid agonists modulate afferent transmission in the rat nucleus tractus solitarius.

We examined the effects of agonists at mu, delta and kappa opioid receptors on neurons located in the nucleus tractus solitarius of the rat using whole-cell patch-clamp recordings in brainstem slices. The mu selective opioid agonist DAMGO hyperpolarized most neurons tested. This effect was associated with the activation of a K(+)-conductance. The effect of DAMGO tended to desensitize and was blocked by naloxone. Dynorphin A also produced this effect. However, the kappa-1-selective opioid agonist U-69593 and two delta-selective opioid agonists did not. DAMGO also depressed glutamate-mediated excitatory postsynaptic potentials and GABA-mediated evoked by stimulation of the tractus solitarius. Dynorphin A, U-69593 and delta-opioid agonists also reduced the excitatory postsynaptic potential, although they were less effective than DAMGO. The presynaptic inhibitory effects of DAMGO were also blocked by naloxone, but did not desensitize. These actions may help to explain the ability of opiates to modulate a variety of autonomic reflexes.

Action Potentials↗
PubMed1991

Bacterial lipopolysaccharide-stimulated GTPase activity in RAW 264.7 macrophage membranes.

The molecular mechanisms surrounding the toxicity and high mortality rate that accompany the release of bacterial lipopolysaccharide (LPS) are unclear, although its potent activity suggests that an amplification system is involved. Because previous studies suggest that a guanine-nucleotide-binding protein (G-protein) may participate in LPS action, we have evaluated the effects of LPS on GTPase activity in membranes isolated from macrophage (RAW 264.7) and fibroblast (B82L) cell lines. LPS induced substantial GTPase activation (200-300% above basal), and kinetic analyses indicated that the maximal LPS-stimulated increase in velocity is observed within 15 min, that it is a low-Km (for GTP) activity, that it can be enhanced by ammonium sulphate, and that it appears to be pertussis toxin-insensitive. Moreover, the LPS-enhanced GTPase activity was not antagonized by phosphatase/ATPase inhibitors such as p-nitrophenyl phosphate, ouabain, bafilomycin or N-ethylmaleimide, and in fact was potentiated by the addition of ATP or ADP. Conversely, the LPS precursor, lipid X, which can decrease the lethal effects of LPS, was found to dose-dependently inhibit the LPS-mediated stimulation of GTPase activity. Half-maximal inhibition was seen at the same lipid X/LPS ratio known to be effective in vivo, i.e. 1:1(w/w). These effects appear to be specific because other phospholipids, detergents and glycosides neither stimulated basal, nor inhibited LPS-induced, GTPase activity. These data suggest the involvement of a GTPase in LPS action, and indicate that lipid X may act to directly antagonize LPS at this level.

Adenosine Triphosphate↗
PubMed1991

Deletions in the tRNA(Lys) primer-binding site of human immunodeficiency virus type 1 identify essential regions for reverse transcription.

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA primer bound near the 5' end of the genomic RNA at a position termed the primer-binding site (PBS). The PBS is an 18-nucleotide region of the HIV-1 genome complementary to cellular tRNA(Lys). To further investigate the sequence requirements for the PBS in reverse transcription, deletions in the PBS were created and subcloned into a plasmid containing the infectious HIV-1 proviral genome. The mutations deleted the entire PBS (delta PBS) or the first 9 (delta 1-9), the second 9 (delta 10-18), or 12 (delta 7-18) nucleotides of the PBS. An additional mutation in the PBS was created in which the second nine nucleotides were deleted and nine additional nucleotides were substituted [Lys(1-9)]. The transfection of plasmids containing the wild-type or mutant proviral genomes into tissue culture cells resulted in expression of the HIV-1 gag and env gene products, as determined by immunoprecipitation using sera from AIDS patients. HIV-1 virus was released from the transfected cells, as determined by analysis of the supernatants for reverse transcriptase activity. The infectivity of the viruses derived from the transfection was examined by coculture experiments with SupT1 cells, which support high-level replication of HIV-1. The transfection of plasmids containing HIV-1 proviral genomes with the delta PBS and PBS (delta 1-9) mutations did not produce infectious virus. In contrast, the HIV-1 proviral genomes with the delta 10-18, delta 7-18, and Lys(1-9) mutations in the PBS produced infectious virus upon transfection, although the kinetics of appearance was significantly delayed for the mutant viruses compared with the wild type. To further explore the nature of this defect, the PBS region from integrated proviral genomes was amplified by polymerase chain reaction and individual DNA products were subcloned into M13mp19, followed by a sequence analysis of the PBS region from individual M13 phage clones. In each of the PBS regions examined, the 18-nucleotide PBS complementary to tRNA(Lys) was present. However, nucleotide deletions and insertions were found 3' to the PBS from the samples derived from the transfection of plasmids containing mutant proviral genomes. Upon reinfection, the revertant viruses maintained the deletions 3' to the PBS and had kinetics of replication similar to that of the wild-type virus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Per-rectal contrast material for abdominal CT: usefulness of administration with an automatic injector.

Adequate distention of the gastrointestinal tract is essential for the best quality image in abdominal computed tomography. We introduce a new technique for per-rectal administration of contrast material with the use of an automatic injector. With this technique, more contrast material can be inserted and thus adequately distend the colon, including the proximal colon.

Administration, Rectal↗
PubMedDate not supplied

Solid and papillary epithelial neoplasm of the pancreas with peritoneal metastasis and its recurrence: a case report.

Solid and papillary epithelial neoplasm of the pancreas is a low-grade malignant tumor, generally occurring as a slowly growing upper abdominal mass in a young female. With complete excision, the prognosis is good. Malignant behavior with metastasis does occur, but the incidence is very low. We report a young woman who had this neoplasm in the pancreatic tail with peritoneal seeding and its recurrence.

Adult↗
PubMedDate not supplied

Complications of radiofrequency ablation in hepatocellular carcinoma.

Radiofrequency thermal ablation has been accepted as a promising technique to treat unresectable liver tumors. However, any interventional procedure should be performed only if the procedure is safe, with minimal morbidity and mortality rates. Recently, three separate multicenter surveys have reported acceptable morbidity and mortality rates for a minimally invasive technique. The mortality rate ranged from 0.1% to 0.5%, the major complication rate ranged from 2.2% to 3.1%, and the minor complication rate ranged from 5% to 8.9%. The most common causes of death were sepsis and hepatic failure, and the most common complications were intraperitoneal bleeding, hepatic abscess, bile duct injury, hepatic decompensation, and grounding pad burns. Minor complications and side effects are more common than major complications, but most of them are transient and self-limiting. Several strategies for avoiding or limiting the impact of complications after radiofrequency ablation are recommended: (a) careful patient selection, (b) combined treatment with other techniques when appropriate, (c) selection of the most appropriate guiding modality and approach, and (d) early detection and appropriate management of any major complications. Knowledge of the broad spectrum of complications and relevant management enables the operator to minimize the incidence and effect of any complications that occur after radiofrequency ablation.

Carcinoma, Hepatocellular↗
PubMedDate not supplied

Peripheral cholangiocarcinoma with extensive tumor thrombi in the portal venous system: case report.

Peripheral cholangiocarcinoma is a primary liver tumor, with some features that distinguish it from hepatocellular carcinoma, especially the low incidence of vascular involvement relative to mass size. Radiologically, tumor thrombus seen as an expanding central filling defect is a rare finding of cholangiocarcinoma on computed tomography, even though microscopic invasion or vascular encasement has been reported in a few studies. We report a case of a peripheral cholangiocarcinoma with tumor thrombus expanding and spreading from the portal vein to the superior mesenteric vein.

Bile Duct Neoplasms↗
PubMedDate not supplied

Coexistent cystic teratoma of the omentum and ovary: report of two cases.

Benign cystic teratoma (dermoid cyst) of the omentum is a rare tumor, while ovarian cystic teratoma is one of the most common ovarian neoplasms. Seven cases of cystic teratoma of the omentum have been reported in association with an ovarian teratoma. We report two cases associated with an ovarian teratoma, which shows unusual dense rim calcification of the cystic wall.

Female↗
PubMedDate not supplied

Peritoneal lymphomatosis: CT findings.

BACKGROUND: Diffuse peritoneal tumor infiltration is well recognized on computed tomography (CT) and is usually associated with carcinomatosis. The purpose of this investigation was to analyze the CT findings of peritoneal spread from primary gastrointestinal lymphomas. METHODS: Abdominal CT scans in eight patients with peritoneal lymphomatosis were retrospectively reviewed. Patients were 12-75 years old (mean = 48 years); with six patients were male and two were female. Pathologic evidence of primary lymphoma was available by colonoscopic biopsy of the terminal ileum in seven cases and by gastroscopic biopsy of the stomach in one case. All patients had non-Hodgkin's lymphoma. We analyzed CT findings in view of presence or loculation of ascites, abnormal patterns of mesentery and omentum, presence of peritoneal enhancement, presence of low attenuation and location of lymph nodes, and primary gastrointestinal lymphoma. RESULTS: Although ascites was present in all patients, there was no loculation. The involvement of mesentery was present in seven patients, and the stellate pattern was the common type (4/7). The involvement of omentum was present in seven patients, and the common type was omental cake (3/7). Peritoneal enhancement was present in six patients. Enlarged lymph nodes were present in six patients, mainly at the retroperitoneum and mesentery, and showed centrally low attenuation in half the patients. CONCLUSION: Patterns of tumor involvement of mesentery, omentum, and peritoneum seen in peritoneal lymphomatosis are indistinguishable from those seen in peritoneal carcinomatosis or tuberculous peritonitis. However, ascites without any loculation or septation and diffuse distribution of enlarged lymph nodes were helpful signs of peritoneal lymphomatosis.

Adolescent↗
PubMedDate not supplied

Postadministration of oral contrast material for abdominal CT in patients with gastrectomy: modified drip ingestion method.

We introduce the modified drip ingestion method (MDIM) of administering oral contrast material for abdominal computed tomography (CT) in patients after gastrectomy. MDIM consists of the drip ingestion method, which we have recently reported, with additional ingestion of effervescent agent. MDIM in abdominal CT is a useful technique to improve the degree of distention of the gastrointestinal lumen including anastomotic sites in patients after gastrectomy.

Administration, Oral↗
PubMedDate not supplied

Minimally invasive treatment of malignant hepatic tumors: at the threshold of a major breakthrough.

Six existing minimally invasive techniques for the treatment of primary and secondary malignant hepatic tumors--radio-frequency ablation, microwave ablation, laser ablation, cryoablation, ethanol ablation, and chemoembolization--are reviewed and debated by noted authorities from six institutions from around the world. All of the authors currently believe that surgery remains the treatment of choice for patients with resectable hepatic tumors. However, the clinical results of each of the minimally invasive techniques presented have exceeded those obtained with conventional chemotherapy or radiation therapy. Thus, for nonsurgical patients, these techniques are becoming standard independent or adjuvant therapies. In addition, with continued improvement in technology and increasing clinical experience, one or more of these minimally invasive techniques may soon challenge surgical resection as the treatment of choice for patients with limited hepatic tumor.

Antineoplastic Agents↗