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Biomedical subjects

C H Lee

Publications and source records attributed to C H Lee.

At least 19 recordsLinked to original sources
PubMed1992

Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family.

High efficiency transient transfection of Cos-7 cells was previously used to establish the functional coupling between G alpha q/G alpha 11 and phospholipase C beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the functional coupling between other guanine nucleotide-binding regulatory protein (G-protein) alpha subunits and phospholipases and to study which G alpha subunits mediate the activation of phospholipase C by the alpha 1-adrenergic receptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they could activate endogenous phospholipases in Cos-7 cells in the presence of AIFn. The synergistic increase in inositol phosphate release in Cos-7 cells after they were cotransfected with cDNAs encoding G alpha subunits and phospholipase C beta 1 indicates that both G alpha 16 and G alpha 14 can activate phospholipase C beta 1. The activation of phospholipase C beta 1 was restricted to members of the Gq subfamily of alpha subunits. They activated phospholipase C beta 1 but not phospholipase C gamma 1, gamma 2, or phospholipase C delta 3. The cotransfection of Cos-7 cells with cDNAs encoding three different alpha 1-adrenergic receptors and G alpha q or G alpha 11 leads to an increase in norepinephrine-dependent inositol phosphate release. This indicates that G alpha q or G alpha 11 can mediate the activation of phospholipase C by all three subtypes of alpha 1-adrenergic receptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the activation of phospholipase C by the alpha 1-adrenergic receptors. The alpha 1 B subtype receptor gave a ligand-mediated synergistic response in the cells cotransfected with either G alpha 14 or G alpha 16. However, the alpha 1 C receptor responded in cells cotransfected with G alpha 14 but not G alpha 16, and the alpha 1 A receptor showed little synergistic response in cells transfected with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C receptors to activate phospholipase C through G alpha q and G alpha 11 was also demonstrated in a cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes.

The relative specificities of members of the G alpha q family of GTP-binding proteins were tested for their ability to activate different phosphoinositide-specific phospholipase C (PI-PLC) beta isozymes. Cos-7 cells were transfected with cDNA corresponding to G alpha q, G alpha 11, G alpha 14, and G alpha 16. Most of the recombinant protein was bound to the cell membrane and these membranes were washed to elute endogenous PI-PLC activity. The membrane preparation was reconstituted with purified preparations of the PI-PLC beta isozymes and guanosine 5'-O-thiotriphosphate (GTP gamma S)-stimulated enzyme activity was measured. All four proteins of the G alpha q family were found to stimulate PI-PLC beta 1, with G alpha q and G alpha 11 being most efficient. On the other hand, G alpha 16 was found to most effectively activate PI-PLC beta 2, while G alpha q, G alpha 11, and G alpha 14 showed less stimulation. Specific anti- G alpha 16 antibody blocked the stimulation of both PI-PLC beta 1 and PI-PLC beta 2 in the enriched membrane fraction. We conclude that there is specificity in the interaction of different members of the Gq family with different PI-PLC beta effectors. This specificity may be important in generating tissue- or receptor-specific responses in vivo.

Amino Acid Sequence↗
PubMed1992

An extremely stable inorganic pyrophosphatase purified from the cytosol of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius strain 7.

A highly active inorganic pyrophosphatase was purified to electrophoretical homogeneity from the cytosol of Sulfolobus acidocaldarius strain 7, an extremely thermoacidophilic archaebacterium. The enzyme has an apparent molecular mass of 80 kDa as estimated by gel permeation chromatography, and showed a 21-kDa polypeptide on SDS-PAGE, suggesting that the archaebacterial enzyme is similar to most of the eubacterial pyrophosphatases rather than eukaryotic ones. The pI = 5.1. The enzyme showed relatively high content of Pro and low content of Ser plus Thr. The optimal pH was 6.5 (at 56 degrees C). From the Arrhenius plot an activation energy of 11.2 kcal/mol was obtained between 37-95 degrees C. The specific activity was 617 mumol Pi release min-1 mg-1 at 56 degrees C. The S. acidocaldarius pyrophosphatase was extremely stable. Complete activity remained after incubation at 100 degrees C for 10 min. No dissociation into subunit or unfolding of polypeptide chain occurred in the presence of 8 M urea. Experiments using guanidine-HCl suggested that the transition between a native tetrameric state and an unfolded state is completely reversible, and essentially independent of any additional factors such as divalent metal cation or dithiothreitol.

Amino Acids↗
PubMed1992

Li+ increases accumulation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in cholinergically stimulated brain cortex slices in guinea pig, mouse and rat. The increases require inositol supplementation in mouse and rat but not in guinea pig.

Li+, beginning at a concentration as low as 1 mM, produced a time- and dose-dependent increase in accumulation of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 in acetylcholine (ACh)-stimulated guinea-pig brain cortex slices prelabelled with [3H]inositol and containing 1 mM-inositol in the final incubation period. Similar results were obtained by mass measurement of samples incubated with 10 mM-Li+ by using a receptor-binding assay, although the percentage stimulation of Ins(1,4,5)P3 accumulation by Li+ was somewhat less by this assay. The increase in accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 by Li+ was absolutely dependent on the presence of ACh. In the absence of added inositol, 1-5 mM-Li+ produced smaller increases in Ins(1,4,5)P3, but the Li(+)-dependent increase in Ins(1,3,4,5)P4 was not as affected by inositol omission. In previous studies with cholinergically stimulated rat and mouse brain cortex slices, Li+ inhibited accumulation of Ins(1,4,5)P3 in rat and inhibited Ins(1,3,4,5)P4 accumulation in rat and mouse [Batty & Nahorski (1987) Biochem. J. 247, 797-800; Whitworth & Kendall (1988) J. Neurochem. 51, 258-265]. We found that Li+ inhibited both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation in these species, but we could reverse this inhibition by adding 10-30 mM-inositol; we then observed a Li(+)-induced increase in Ins(1,4,5)P3 and Ins(1,3,4,5)P4. The species differences observed in the absence of supplemented inositol were explained by the fact that a much higher concentration of inositol was required to bring the Li(+)-elevated levels of CDP-diacylglycerol (CDPDG) down to baseline in the rat and mouse. These data suggest that inositol is more rate-limiting for phosphatidylinositol synthesis in the presence of Li+ in rat and mouse, which can account for the previous reports of inhibition of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation by this ion in these species. Thus, in all species examined. Li+ could be shown to increase accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in cholinergically stimulated brain cortex slices if the slices were supplemented with sufficient inositol to bring the Li(+)-elevated level of CDPDG down to near baseline, as seen in the absence of Li+. In guinea-pig brain cortex slices, increases in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation could then be seen at Li+ concentrations as low as 1 mM, which falls within the therapeutic range of plasma concentrations in the treatment of manic-depressive disorders. These observations may have therapeutic implications.

Animals↗
PubMed1992

Activation of phospholipase C by the alpha subunits of the Gq and G11 proteins in transfected Cos-7 cells.

High efficiency transient transfection was used to introduce cDNA corresponding to various G protein alpha subunits into Cos-7 cells. The proteins that were subsequently synthesized were detected with specific G protein alpha subunit antipeptide antiserum and were localized in the membrane fraction of the cell. Cells that were prelabeled with the [3H]inositol and transfected with G alpha q and G alpha 11 cDNA showed marked increases in formation of [3H]inositol phosphates after stimulation with aluminum fluoride. Co-transfection with cDNAs corresponding to phosphoinositide specific phospholipase C beta 1 (PI-PLC beta 1) and to G alpha q or G alpha 11 resulted in even higher levels of inositol phosphate formation. The introduction of mutations that convert residue glutamine 209 to leucine in G alpha q and G alpha 11 resulted in persistent activation of PI-PLC and high steady state levels of inositol phosphates. On the other hand, transfection with a variety of other G alpha subunit cDNAs, i.e. G alpha Z, G alpha OA, G alpha OB, transducin, and the glutamine 205 to leucine mutants of G alpha Z and of G alpha OA did not increase inositol phosphate formation. To further test the specificity of G protein activation of PI-PLC, a cell-free system was prepared by using washed membranes of transiently transfected cells and purified PI-PLC beta 1. Membranes derived from G alpha q and G alpha 11, but not G alpha OA transfected cells, showed guanosine 5-O-thiotriphosphate (GTP gamma S)-stimulated PIP2 hydrolysis. The activity seen in the system reconstituted with membranes derived from G alpha 11-transfected cells was blocked by preincubation with specific G alpha 11 antipeptide antibodies. All of these results are consistent with the conclusion that G alpha q and G alpha 11 cDNA encode proteins that in the presence of GTP gamma S specifically activate PI-PLC.

Amino Acid Sequence↗
PubMed1992

Diagnosis of chronic hepatitis C after liver transplantation by the detection of viral sequences with polymerase chain reaction.

Chronic hepatitis frequently occurs after liver transplantation. The role of hepatitis C virus infection in patients after liver transplantation is unknown, although antibodies to HCV are detected in some of these cases. The use of polymerase chain reaction techniques for the detection of hepatitis C virus RNA should improve sensitivity and specificity, particularly in these immunosuppressed patients. Our goal was to further clarify the role of hepatitis C virus infection in chronic hepatitis occurring after liver transplantation. Patients with chronic hepatitis of uncertain origin after transplantation were identified. Serum samples taken at the time of the most recent liver biopsy that showed chronic hepatitis were tested for anti-hepatitis C virus using enzyme-linked immunoassay and supplemented by recombinant immunoblot assay (recombinant immunoblot assay I and recombinant immunoblot assay II). The samples were also tested for the presence of hepatitis C virus RNA using polymerase chain reaction. Of the 25 patients with chronic hepatitis, 15 (60%) had hepatitis C virus RNA present. Only seven (47%) of these 15 patients had anti-hepatitis C virus detected. Hepatitis C virus is a major cause of chronic hepatitis occurring after liver transplantation. The magnitude of hepatitis C virus infection will be underestimated if only currently available assays for anti-hepatitis C virus are used.

Base Sequence↗
PubMed1992

Hepatitis C virus does not cause nonalcoholic steatohepatitis.

The pathogenesis of nonalcoholic steatohepatitis (NASH) remains poorly understood. Since inflammation and fatty changes are associated with hepatitis C (HCV) infection, we have tested the role of HCV in the genesis of NASH. Five consecutive cases of classic NASH were tested by Abbott anti-c100-3 EIA and polymerase chain reaction (PCR) to detect HCV-RNA. All serum specimens were negative for anti-c100-3 (or anti-HCV EIA) and HCV PCR. Based on this study, we conclude that HCV does not play a causative or contributing role in the pathogenesis of NASH.

Adult↗
PubMed1992

Uptake, distribution and fate of bacterial lipopolysaccharides in monocytes and macrophages: an ultrastructural and functional correlation.

Bacterial lipopolysaccharides (LPS), which are important components of the cell wall of gram-negative bacteria, induce a number of host responses both beneficial and harmful. The present review elucidates the uptake, distribution and functions of LPS in mononuclear phagocytes in an attempt to gain an insight into the mechanisms which control the pathogenesis of LPS mediated septic shock. The unique feature of LPS bilayer structure, the tagged LPS and antibodies to LPS provide means for studying binding, uptake, fate and subcellular distribution of LPS in tissues and cells. LPS bind to monocytes and macrophages by specific interaction via receptors such as scavenger receptors, CD14 and CD18 and by non-specific interactions, and enter the cells via receptor-mediated endocytosis, absorptive pinocytosis, phagocytosis, and diffusion. The ingested LPS are localized in pinocytic vesicles, phagocytic vacuoles, cytoplasm, mitochondria, rough endoplasmic reticulum, Golgi apparatus, and nucleus. The interactions of LPS with monocytes and macrophages trigger a broad spectrum of cellular responses, including production of important bioactive factors or mediators, such as IL-1, TNF, interferons, prostaglandins, and macrophage-derived growth factor, which are implicated in the pathogenesis of septic shock and wound healing. However, there is no conclusive evidence indicating that production of the mediators can only be induced through specific interactions.

Animals↗
PubMed1992

Repression of the hepatitis B virus enhancer by a cellular factor.

The enhancer element of hepatitis B virus (HBV) regulates the transcription of all HBV mRNA, including the pregenomic mRNA used during replication. This pregenomic mRNA is transcribed from the core gene promoter which is located 500 bp downstream from the HBV enhancer element. To examine the effect of the HBV enhancer on the activity of the core gene promoter, we constructed various plasmids containing different combinations of HBV enhancer and core gene promoter sequences regulating the expression of the chloramphenicol acetyltransferase gene. When the HBV enhancer was positioned immediately adjacent to the core gene promoter, the enhancer increased the activity of the core gene promoter nearly 30-fold. In contrast, the HBV enhancer only modestly stimulated the core gene promoter (less than threefold) at its native position in the HBV genome. This weak HBV enhancer activity was due to a DNA sequence located between the enhancer and the core gene promoter and not due to the increased distance between the enhancer and the core gene promoter. Competition experiments demonstrated that a trans-acting factor(s) bound this sequence and repressed the enhancer. This DNA sequence contains the C/EBP, AP-1 and NF-1 regulatory sites. No inhibition of enhancer activity was observed when only the AP-1 and C/EBP sites were present. Repression of the HBV enhancer was not detected when the NF-1 site was disrupted, indicating that the NF-1 site was involved in the suppression of the HBV enhancer.

Base Sequence↗
PubMed1992

Cell-mediated immunity in cervical intraepithelial neoplasia.

Various immune parameters, i.e., peripheral T and B cell distribution, T cell subpopulation, interleukin 2 (IL-2) activity and in vitro lymphocyte responses to phytohemagglutinin (PHA) and concanavalin A (Con A), were investigated in 24 pretreated patients with cervical intraepithelial neoplasia (CIN) and in 35 normal controls. Of the measured parameters, lymphocyte response to PHA was significantly lower in the CIN than control group (P less than 0.05). The values of all other measured parameters were similar in both groups.

B-Lymphocytes↗
PubMed1992

Lithium enhances accumulation of [3H]inositol radioactivity and mass of second messenger inositol 1,4,5-trisphosphate in monkey cerebral cortex slices.

We previously reported that lithium, in the presence of acetylcholine, increased accumulations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in brain cortex slices from the guinea pig, rabbit, rat, and mouse. In the mouse and rat, the Li(+)-induced increases required supplementation of the medium with inositol. This probably relates to the following facts: (a) Brain cortices of the mouse and rat contain in vivo concentrations of inositol half of that of the guinea pig. (b) Incubated rat brain cortex slices are depleted of inositol by 80%. (c) The slices require 10 mM inositol supplementation to restore in vivo concentrations. We now show that in monkey brain cortex slices, therapeutic concentrations of Li+ increase accumulation of inositol 1,4,5-trisphosphate. The inositol 1,3,4,5-tetrakisphosphate level is not increased. Neither inositol nor an agonist is required. The same effects are seen whether inositol 1,4,5-trisphosphate is quantified by the [3H]inositol prelabeling technique or by mass assay, although mass includes a pool of inositol 1,4,5-trisphosphate that is metabolically inactive. Thus, in a therapeutically relevant model for humans, Li+ increases inositol 1,4,5-trisphosphate levels in brain cortex slices, as was previously seen in lower mammals at non-rate-limiting concentrations of inositol.

Acetylcholine↗
PubMed1992

An enzyme-linked immunosorbent assay for enumeration of Pneumocystis carinii in vitro and in vivo.

An enzyme-linked immunosorbent assay (ELISA) to quantitate Pneumocystis carinii organisms from culture supernatant and rat lung has been developed. A polyclonal antibody specific to P. carinii was produced in Sprague-Dawley rats by allowing P. carinii-infected animals to recover from infection. This antibody reacted strongly to P. carinii proteins of 50 to 55 kDa and weakly to those of 33 and 116 kDa. The ELISA used this convalescent-phase antibody to quantitate the number of P. carinii organisms in lung homogenates of infected rats and supernatants from infected tissue cultures which were used to screen drugs for P. carinii. The results of the ELISA were compared with those of direct microscopic counting of organisms, and the two methods were highly correlated (r > 0.9). Thus, the ELISA can be used as an alternative method for the quantitation of P. carinii organisms, and it is superior to the conventional microscopic method because it is easier to perform and less labor-intensive.

Animals↗
PubMed1992

Identification of hepatitis C viruses with a nonconserved sequence of the 5' untranslated region.

The nucleotide sequence of the 5' untranslated region of hepatitis C virus (HCV) has been shown to be conserved. In contrast, we have detected more sequence variation in this region in several HCV isolates than hitherto expected. The nucleotide sequences of the 5' untranslated regions of these isolates differ significantly from that of the prototype but are very similar to each other. We believe that these isolates belong to the same type of HCV. Among 240 HCV RNA polymerase chain reaction-positive specimens that we examined, 7 belong to this type. The results suggest that the HCV variants that we detected represent a different type of HCV and that they are responsible for approximately 3% of HCV infections in patients that we have examined.

Base Sequence↗
PubMed1992

Relation of left ventricular isovolumic relaxation time and incoordination to transmitral Doppler filling patterns.

OBJECTIVE: To investigate factors during isovolumic relaxation that determine Doppler filling patterns in patients with left ventricular disease, and thus to identify the underlying mechanisms. DESIGN: 85 patients (50 ischaemic heart disease, 35 left ventricular hypertrophy due to aortic stenosis) and 26 controls were studied with Doppler and M mode echocardiography and phonocardiography. 16 patients underwent two studies on separate occasions, to find whether changes in isovolumic relaxation time were reflected by a change in the Doppler A/E ratio. SETTING: A tertiary cardiac referral centre. SUBJECTS: Patients referred for assessment of coronary artery disease or aortic stenosis with left ventricular hypertrophy. MAIN OUTCOMES MEASURES: Doppler filling velocities during early (E wave) and late (A wave) diastole and the A/E ratio, acceleration of the E wave, digitised M mode indices of incoordinate relaxation (change in cavity dimension before mitral valve opening and time from minimum dimension to mitral valve opening), isovolumic relaxation time, M mode measures of diastolic function after mitral valve opening (peak rate of posterior wall thinning and peak rate of dimension increase), and left ventricular end diastolic pressure. RESULTS: A/E correlated with age in normal subjects (r = 0.74), to a lesser extent in left ventricular hypertrophy (r = 0.41), but not significantly in ischaemic heart disease. In all patients, isovolumic relaxation time was significantly and negatively correlated with the acceleration of the E wave, showing its fundamental relation to the force responsible for early diastolic filling (r = -0.71 for left ventricular hypertrophy, and -0.74 for ischaemic heart disease, p value < 0.01). In left ventricular hypertrophy and those ischaemic patients without left ventricular dilatation A/E was correlated both with isovolumic relaxation time (r = 0.68 and 0.60 respectively), and with incoordinate relaxation (r = 0.65 and 0.61). In those ischaemic patients with left ventricular dilatation, the influence of incoordination was lost and isovolumic relaxation time became the dominant influence upon A/E (r = 0.82). Weak correlations of end diastolic pressure and RR interval with A/E, became insignificant once isovolumic relaxation time had been taken into account. Isovolumic relaxation time and incoordination together accounted for over 50% of the variance in the A/E ratio in our patients. Isovolumic relaxation time and the A/E ratio were linearly related. Patients with a short isovolumic relaxation time had evidence of considerable diastolic abnormalities, despite a normal Doppler A/E ratio. In the 16 patients who had two echocardiographic studies, changes in the duration of isovolumic relaxation were accompanied by a change in the Doppler A/E ratio. The relation between these two variables, derived from the group as a whole was similar. CONCLUSIONS: The main factors influencing the A/E ratio in patients with left ventricular disease are two distinct properties of isovolumic relaxation--namely the duration and the extent of incoordinate wall motion. Filling pressure and RR interval are not significant independent determinants, but act only through an effect upon isovolumic relaxation time. Age is an important influence in normal people, but this effect is attenuated in left ventricular hypertrophy and lost in ischaemic ventricular disease.

Adult↗
PubMed1992

Breast conservation therapy in patients with mammographically undetected breast cancer.

The authors reviewed their experience with 542 patients with breast cancer who were treated with conservative surgery and radiation therapy (CSRT) and analyzed the outcome in those patients whose tumors could not be detected with mammography. Fifty-five of the patients (10.1%) had a palpable, pathologically confirmed breast carcinoma and a negative preoperative mammogram. Routine follow-up included annual mammography and physical examination. The local recurrence, 5-year actuarial survival, and 5-year disease-free survival rates for these 55 patients did not differ significantly from those for patients with positive mammograms. There were six cases of local breast recurrence in this subgroup. Four of five cases were visible on mammograms (one patient did not undergo mammography at the time of recurrence); two of the cases were detected with mammography alone following physical examination with negative results. The authors conclude that patients with palpable but mammographically occult early-stage breast cancer are suitable candidates for CSRT and that mammography is a mandatory part of follow-up of conservatively treated patients.

Actuarial Analysis↗
PubMed1992

Molecular characterization of a mouse Y chromosomal repetitive sequence that detects transcripts in the testis.

145SC5 is a Y chromosomal repetitive sequence isolated from a BALB/c mouse and is unique in that it detects poly(A)-containing transcripts in the testis. We determined nucleotide sequences of 145SC5 and a related cDNA clone designated PC11 and compared their sequences to that of pYMT2/B, another related cDNA clone. 145SC5 and PC11 are almost identical (97%), while PC11 and pYMT2/B share 84% identity. In situ hybridization and Southern blot analysis with XXSxr male mice demonstrated that 145SC5-related sequences were distributed over the entire length of the Y chromosome including the short arm. Two types of Y chromosome are present in inbred mouse strains: the Mus musculus musculus type and M.m. domesticus type. Among 26 inbred strains surveyed, 145SC5 detected polymorphims only in the M.m. domesticus Y chromosome.

Amino Acid Sequence↗