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S Chen

Publications and source records attributed to S Chen.

At least 1,189 records · Page 66Linked to original sources
PubMed1993

Catalytic efficiency of expressed aromatase following site-directed mutagenesis.

Mutant aromatase cytochrome P-450s, expressed in CHO cells after transfection with cDNAs, have been characterized in terms of their catalytic efficiencies. After solubilization from microsomes, specific aromatase P-450 content of wild-type and mutants Pro308Phe, Asp309Asn, Asp309Ala and Phe406Arg was quantitated by a sandwich enzyme-linked immunosorbent assay (ELISA). Microsomal aromatase activity was determined by the 3H-water method using [1 beta-3H]androstenedione as substrate. Estimations of the actual turnover rate (catalytic efficiency) were derived from the combined data. The P-450 content in the mutants varied but was always less than that in the wild type. Hence, the decreases in the Vmax observed in the mutant enzymes did not correlate completely with reductions in catalytic effectiveness. In recent studies on the structure-function relationship of aromatase cytochrome P-450, the observed reduction of enzyme activity in terms of Vmax following site-directed mutagenesis led to the assumption that there was a corresponding loss of catalytic effectiveness. The present study reveals that a lower P-450 content can contribute significantly to decreasing catalytic activity in the mutants. In fact, in mutant Phe406Arg which exhibited virtually no catalytically active aromatase, the specific P-450 content was below the detectable level. Because of its location, the result of this latter mutation could be a major structural perturbation of the heme-binding property. Thus, interpretation of losses and reductions in aromatase activity resulting from single amino-acid replacement should take into account changes in the specific content of aromatase cytochrome P-450.

Androstenedione↗
PubMed1993

Genomic sequences capable of committing mouse and rat fibroblasts to adipogenesis.

The mouse Swiss 3T3-F442A/3T3-C2 cell system is well suited for the isolation of genes involved in commitment to adipogenesis. 3T3-F442A cells convert to adipocytes with high efficiency in response to confluence and insulin. The sister clonal line 3T3-C2 does not respond to these signals, but can convert to adipocytes when transfected with DNA from 3T3-F442A preadipocytes or from human fat. Human fat-tissue biopsy FO46 DNA transfected into 3T3-C2 gave rise to fat foci after two rounds of transfection and selection. A cosmid library of a subclone of secondary transfectant 3T3-C2/FO46-1 was screened for the human repetitive Alu sequence. Five out of eight Alu+ recombinant clones committed 3T3-C2 cells to adipogenesis. The adipose commitment (AC) activity of one cosmid, p18A4, was found to reside in two small, non-identical, subcloned sequences 1.2kb and 2.0kb in length, each separately able to commit 3T3-C2, precrisis mouse and rat fibroblasts and the multipotential C3H10T1/2 cell line to adipogenesis. We conclude that commitment to adipogenesis can be effected in vitro with high efficiency by transfection of specific sequences into a variety of host cells.

3T3 Cells↗
PubMed1993

ATP induces large quaternary rearrangements in a cage-like chaperonin structure.

BACKGROUND: The chaperonins, a family of molecular chaperones, are large oligomeric proteins that bind nonnative intermediates of protein folding. They couple the release and correct folding of their ligands to the binding and hydrolysis of ATP. Chaperonin 60 (cpn60) is a decatetramer (14-mer) of 60 kD subunits. Folding of some ligands also requires the cooperation of cpn10, a heptamer of 10 kD subunits. RESULTS: We have determined the three-dimensional arrangements of subunits in Rhodobacter sphaeroides cpn60 in the nucleotide-free and ATP-bound forms. Negative stain electron microscopy and tilt reconstruction show the cylindrical structure of the decatetramer comprising two rings of seven subunits. The decatetramer consists of two cages joined base-to-base without a continuous central channel. These cages appear to contain bound polypeptide with an asymmetric distribution between the two rings. The two major domains of each subunit are connected on the exterior of the cylinder by a narrower bridge of density that could be a hinge region. Binding of ATP to cpn60 causes a major rearrangement of the protein density, which is reversed upon the hydrolysis of the ATP. Cpn10 binds to only one end of the cpn60 structure and is visible as an additional layer of density forming a cap on one end of the cpn60 cylinder. CONCLUSIONS: The observed rearrangement is consistent with an inward 5-10 degrees rotation of subunits, pivoting about the subunit contacts between the two heptamers, and thus bringing cpn60 domains towards the position occupied by the bound polypeptide. This change could explain the stimulation of ATPase activity by ligands, and the effects of ATP on lowering the affinity of cpn60 for ligands and on triggering the release of folding polypeptides.

Journal Article↗
PubMed1993

Nerve growth factor receptor-containing cholinergic neurons of the basal forebrain project to the thalamic reticular nucleus in the rat.

The origin of nerve growth factor receptor-immunoreactive (NGFr-ir) fibers innervating the thalamic reticular nucleus (Rt) was here investigated in the rat using retrograde tracers in combination with immunocytochemistry. Neurons retrogradely labeled from Rt were scattered ipsilaterally throughout the medial septal nucleus and the other cell groups of the basal forebrain, which contained NGFr-ir cells; 10-20% of these retrogradely labeled neurons were also NGFr-ir. Furthermore, a few retrogradely labeled NGFr-ir cells were detected in the basal forebrain on the contralateral side. Retrograde tracing combined with a double immunocytochemical procedure revealed that all the NGFr-ir neurons labeled from Rt also displayed immunoreactivity for choline acetyltransferase. The present results demonstrate that the NGFr-ir neurons of the basal forebrain which project to Rt are cholinergic. The possible functional implications of these findings are discussed.

Animals↗
PubMed1993

Inhibition of NAD(P)H:quinone acceptor oxidoreductase by flavones: a structure-activity study.

A structure-activity study was carried out to determine the important regions of baicalein and oroxylin A, two flavones isolated from the Chinese herb Scutellariae radix, in inhibiting NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase). This quinone reductase is a vitamin K reductase. It is a target for and has been used as a model enzyme to investigate the mode of action of oral anticoagulants. The two flavones were found to inhibit this quinone reductase in nanomolar ranges. The 5-hydroxyl, 7-hydroxyl, 8-hydroxyl, and 2-phenyl groups of these flavones were found to be important for their inhibition of the enzyme. The inhibition profiles of the flavones on the NADH-menadione reductase activity, the NADH-potassium ferricyanide reductase activity, and the NADH-methyl red reductase activity of this enzyme were different. Therefore, even though the flavones were found to be competitive inhibitors with respect to NADH, they probably did not inhibit the enzyme by binding to the nicotinamide nucleotide binding site. Inhibition kinetic studies which indicated that these compounds bound to different sites than those for dicoumarol and phenindone were performed. These results indicate that these flavones are a new type of inhibitor of NAD(P)H:quinone acceptor oxidoreductase and potentially useful as anticoagulant drugs.

Azo Compounds↗
PubMed1993

Regulation of the activity and phosphorylation of the plasma membrane Ca(2+)-ATPase by protein kinase C in intact human erythrocytes.

Previously the plasma membrane-bound or purified Ca(2+)-translocation ATPase (Ca2+ pump) was found to be activated and phosphorylated by protein kinase C in vitro (K. K. W. Wang et al. 1991, J. Biol. Chem. 266, 9078-9085). We now show that in intact human erythrocytes phorbol-12-myristate 13-acetate (PMA), a known stimulator of protein kinase C, decreases the amplitude of the intracellular calcium ([Ca2+]i) transient induced by 2.5 microM CaCl2 and 10 microM A23187. Since PMA did not affect Ca2+ influx, the decrease in amplitude was most likely due to the stimulation of the Ca2+ pump, the major mechanism of calcium extrusion in these cells. The effect was dose-dependent, the maximum decrease in amplitude (33%) occurring at 1 microM PMA. The depression of the [Ca2+]i transient was further enhanced by the phosphatase inhibitor okadaic acid. It was reversed by the protein kinase C inhibitor staurosporine and could not be mimicked by inactive PMA analogues. In erythrocytes labeled with [32P]orthophosphate, PMA treatment phosphorylated the Ca(2+)-ATPase in a dose-dependent manner. The phosphorylation was inhibited by staurosporine and was slightly enhanced by okadaic acid. Changes in lipid phosphorylation and content were studied under the same conditions in intact cells. The turnover of 32P and lipid phosphate in phosphatidylinositol 4,5-bisphosphate (PIP2) was inhibited by 1 mM adriamycin, concomitant with an increased amplitude of the [Ca2+]i transient. The PIP2 content and its 32P radioactive did not, however, change with PMA stimulation. We conclude that while both protein kinase C and polyphosphoinositides are regulators of Ca(2+)-ATPase activity in the intact human erythrocyte, stimulation of the enzyme activity by PMA is predominantly protein kinase C-mediated.

Calcium↗
PubMed1993

Physiology of muscarinic receptor-operated nonselective cation channels in guinea-pig ileal smooth muscle.

Stimulations of autonomic nerves in smooth muscle often evoke both fast and slow excitatory junction potentials (EJPs), which are thought to involve activations of several distinct types of nonselective cation channels (NSC channels). The ACh-activated NSC channel in guinea-pig ileum (I(ns), ACh) is one probably responsible for the slow EJP and seems to undergo various regulations. This short paper will review the physiology of I(ns),ACh, with particular emphasis on its dynamic interactions with other physiologically important factors such as the membrane potential, [Ca2+]i and pH.

Acetylcholine↗
PubMed1993

Colocalization of neurotransmitters in the deep cerebellar nuclei.

An abundance of glycine and glycine receptor immunoreactivities was found in all three parts of the deep cerebellar nuclei. Glycine immunoreactivity was restricted to small neurons throughout most of the deep cerebellar nuclei except for a few large positive neurons in the ventral part of the fastigial nuclei. In addition, glycine immunoreactivity was found in boutons outlining somata of large glycine negative neurons. Complementary to the glycine positive boutons was an intense glycine receptor immunoreactivity on large deep cerebellar nuclei neurons. Comparisons of immunoreactivities for glycine, GABA and aspartate in consecutive one micron sections revealed that many small neurons colocalized glycine and GABA, while some large neurons in the fastigal region colocalized glycine and aspartate. Ultrastructural investigations revealed glycine receptors on postsynaptic sites of dendrites and somata. Most boutons, which were presynaptic to glycine receptor sites, were filled with small flattened vesicles; however, a small percentage of boutons had round clear or dense core vesicles. Frequently, each bouton apposed multiple active zones on the dendrite or soma. One of these active zones was positive for glycine receptor and another was negative. This study supports: (1) glycine as a neurotransmitter in deep cerebellar nuclei, and (2) glycine and GABA colocalization in the same cell and bouton, but releasing to different receptor sites on the target neuron. Furthermore, the coexistence of glycine with GABA in the same deep cerebellar neuron may play an important role in controlling the conset and duration of signal transmission.

Animals↗
PubMed1993

Variation of haemorheological parameters in renal and pancreatic rejection.

Abnormalities of haemorheology were found in animal and human recipients of kidney and/or pancreas allografts during rejection episodes. Thirteen diabetic canines received solitary pancreatic transplantation and another 13 diabetic and uremic canines underwent combined pancreas and kidney transplantation. Determination of haemorheological parameters was performed before and after operation respectively. During rejection episodes of kidney or pancreas allografts, the values of plasma viscosity, blood reductive viscosity and fibrinogen were significantly higher than those without rejection. On the basis of animal experiments, the determination of haemorheological parameters had been performed on 33 patients (30 receiving renal transplant, 2 pancreatic transplant alone and the remaining 1 combined renal and pancreatic transplant). Consecutive monitoring on these patients showed that a rise in the values of plasma viscosity, blood reductive viscosity and fibrinogen could be demonstrated during rejection episodes. The changes appeared one to three days prior to clinical manifestations and were in accordance with the termination of rejection. Our studies suggest that variation of haemorheological parameters are associated with rejection and the abnormal haemorheology may be an essential factor contributing to graft dysfunction. Moreover, the use of these assays will be beneficial to early diagnosis and better management of rejection in the future.

Adult↗
PubMed1993

Compartmentation of the cerebellar cortex by protein kinase C delta.

Six isozymes of protein kinase C were analysed in the rat cerebellum using immunohistochemistry. The results revealed a non-uniform distribution of protein kinase C delta among Purkinje, basket, and stellate cells. Serial-section mapping of the delta immunoreactivity revealed that (i) the number and intensity of labeled Purkinje cells increased from rostral to caudal while labeled basket-stellate cells decreased caudally; (ii) the majority of Purkinje cells were labeled in the nodulus, flocculus, and paraflocculus while the anterior lobules were mostly negative; and (iii) labeled Purkinje cells formed distinct parasagittal bands in lobules 6-9 of the paravermis and vermis. The banding of protein kinase C delta within subsets of Purkinje cells suggests units of cerebellar circuitry with specific signaling properties through protein phosphorylation. The visual-vestibular regions of the cerebellum contained the highest amount of the isozyme.

Animals↗
PubMed1993

Effects of caloric restriction on rodent drug and carcinogen metabolizing enzymes: implications for mutagenesis and cancer.

Caloric restriction in rodents results in increased longevity and a decreased rate of spontaneous and chemically induced neoplasia. The low rates of spontaneous neoplasia and other pathologies have made calorically restricted rodents attractive for use in chronic bioassays. However, caloric restriction also alters hepatic drug metabolizing enzyme (DME) expression and so may also alter the biotransformation rates of test chemicals. These alterations in DME expression may be divided into two types: (1) those that are the direct result of caloric restriction itself and are detectable from shortly after the restriction is initiated; (2) those which are the result of pathological conditions that are delayed by caloric restriction. These latter alterations do not usually become apparent until late in the life of the organism. In rats, the largest direct effect of caloric restriction on liver DMEs is an apparent de-differentiation of sex-specific enzyme expression. This includes a 40-70% decrease in cytochrome P450 2C11 (CYP2C11) expression in males and a 20-30% reduction of corticosterone sulfotransferase activity in females. Changes in DME activities that occur late in life in calorically restricted rats include a stimulation of CYP2E1-dependent 4-nitrophenol hydroxylase activity and a delay in the disappearance of male-specific enzyme activities in senescent males. It is probable that altered DME expression is associated with altered metabolic activation of chemical carcinogens. For example the relative expression of hepatic CYP2C11 in ad libitum-fed or calorically restricted rats of different ages is closely correlated with the amount of genetic damage in 2-acetylaminofluorene- or aflatoxin B1-pretreated hepatocytes isolated from rats of the same age and caloric intake. This suggests that altered hepatic drug and carcinogen metabolism in calorically restricted rats can influence the carcinogenicity of test chemicals.

Animals↗
PubMed1993

Southern blot and dot blot hybridisation compared to PCR for the detection of human papillomavirus DNA in biopsies of the uterine cervix from women with dysplasia.

The aim of this study was to compare the sensitivity of Southern blot (SB) and dot blot (DB) hybridisation with polymerase chain reaction (PCR) for the detection of HPV in cervical biopsies from samples with differing histology. One hundred and forty seven women with cervical dysplasia had biopsies performed; one sample was analyzed for HPV DNA from types 6/11, 16, and 18 by SB, DB and PCR (L1 consensus primer and type specific probes) while an adjacent sample was examined histologically. The histology of the samples was normal in 40 (27%), squamous metaplasia in 25 (17%), inflammation 2 (1%) HPV infection 24 (17%), cervical intraepithelial neoplasia (CIN) grade I in 11 (7%), CIN II in 18 (12%), CIN III in 22 (15%), while 5 (3%) had invasive cancer. The number of biopsies positive for HPV DNA from types 6/11, 16, and 18, using the different hybridisation methods was 56 (38%) by dot blot, 57 (39%) by Southern blot hybridisation and 66 (45%) by PCR. When the L1 consensus primer was used 100 (68%) specimens were positive by PCR. The sensitivity of SB and DB hybridisation, as compared with PCR (type specific probes 6/11, 16, 18) was greater in biopsies with abnormal histology (histological grades of HPV infection and greater, as a group) (sensitivity of SB 83%, DB 74%) than those with normal and metaplastic change (as a group) (sensitivity of SB 44%, DB 35%) (P < 0.005 for SB and DB) (inflammation excluded from analysis). This study demonstrated that the sensitivity of SB and DB hybridisation, relative to PCR is greater in samples with abnormal histology than in samples with normal histology.

Journal Article↗
PubMed1993

Structure-function studies of human aromatase.

Site-directed mutagenesis experiments have been carried out to determine the structure-function relationship of human aromatase. By sequence comparison, the region in aromatase that corresponds to the distal helix of cytochrome P-450cam has been identified to be Gln-298 to Val-313. Eight aromatase mutants with changes in this region, i.e. C299A, E302L, P308F, D309N, D309A, T310S, T310C, and S312C, have been generated using a mammalian cell stable-expression system. The results from site-directed mutagenesis studies indicate that the region containing Gln-298 to Val-313 is indeed a very important part of the active site of aromatase. The catalytic properties of P308F, D309N, and D309A have been examined in detail and are discussed. Active site-directed labeling is also an important approach to investigate the structure-function relationship of aromatase. HPLC-linked electrospray mass spectrometry is indicated as a useful technique for the characterization of active site-directed probe-modified enzyme. The mass spectral analysis of aromatase suggests that aromatase is glycosylated.

Amino Acid Sequence↗