Biomedical subjects
Publications and source records attributed to S Chen.
A 29-year-old man experienced intractable partial seizures as the initial manifestation of neurosyphilis. The diagnosis was made after the onset of dementia 9 months later. Both the epilepsy and dementia resolved with penicillin therapy. Syphilis should be considered in patients with adult-onset focal epilepsy, particularly if there is associated dementia. Treatment may be successful even when the diagnosis is delayed.
The molecular basis of the interaction of DT-diaphorase with a cytotoxic nitrobenzamide CB1954 [5-(aziridin-1-yl)-2, 4-dinitrobenzamide] and five inhibitors was investigated with wild-type DT-diaphorase (human and rat) and five mutants [three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)]. hY155F and hH161Q were generated to evaluate a hypothesis that Tyr155 and His161 participate in the obligatory two-electron transfer reaction of the enzyme. The catalytic properties of hY155F and hH161Q were compared with a naturally occurring mutant, hP187S. Pro187 to Ser mutation disturbs the structure of the central parallel beta-sheet, resulting in a reduction of the binding affinity of the flavin-adenine dinucleotide prosthetic group. With NADH as the electron donor and menadione as the electron acceptor, the k(cat) values for the wild-type human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as 66 +/- 1, 23 +/- 0, 5 +/- 0 and 8 +/- 2 x 10(3) min(-1), respectively. Because hY155F still has significant catalytic activity, the hydroxyl group on Tyr155 may not be as important as proposed. Interestingly, hY155F was found to be 3. 3 times more active than the human wild-type DT-diaphorase in the reduction of CB1954. Computer modeling based on our results suggests that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128. Dicoumarol, Cibacron blue, chrysin, 7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the enzyme with respect to nicotinamide coenzymes. The binding orientations of dicoumarol, flavones, and phenindone in the active site of DT-diaphorase were predicted by results from our inhibitor-binding studies and computer modeling based on published X-ray structures. Our studies generated results that explain why dicoumarol is a potent inhibitor and binds differently from flavones and phenindone in the active site of DT-diaphorase.
An Mn(2+) and Cd(2+) uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd(2+) sensitivity as well as energy-dependent Cd(2+) uptake activity. Both transcription and translation of mntA were induced by Mn(2+) starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd(2+) uptake systems have been identified in L. plantarum: one is a high-affinity Mn(2+) and Cd(2+) uptake system that is expressed in Mn(2+)-starved cells, and the other is a nonsaturable Cd(2+) uptake system that is expressed in Cd(2+)-sufficient cells (Z. Hao, H. R. Reiske, and D. B. Wilson, Appl. Environ. Microbiol. 65:592-99, 1999). MntA was not detected in an Mn(2+)-dependent mutant of L. plantarum which had lost high-affinity Mn(2+) and Cd(2+) uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn(2+) and Cd(2+) transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn(2+)- and Cd(2+)-binding sites of MntA are discussed. A second clone containing a low-affinity Cd(2+) transport system was also isolated.
Members of the NF-kappaB/RelB family of transcription factors play important roles in the regulation of inflammatory and immune responses. RelB, a member of this family, has been characterized as a transcription activator and is involved in the constitutive NF-kappaB activity in lymphoid tissues. However, in a previous study we observed an overexpression of chemokines in RelB-deficient fibroblasts. Here we show that RelB is an important transcription suppressor in fibroblasts which limits the expression of proinflammatory mediators and may exert its function by modulating the stability of IkappaBalpha protein. Fibroblasts from relb(-/-) mice overexpress interleukin-1alpha (IL-1alpha), IL-1beta, and tumor necrosis factor alpha in response to lipopolysaccharide (LPS) stimulation. These cells have an augmented and prolonged LPS-inducible IKK activity and an accelerated degradation which results in a diminished level of IkappaBalpha protein, despite an upregulated IkappaBalpha mRNA expression. Consequently, NF-kappaB activity was augmented and postinduction repression of NF-kappaB activity was impaired in these cells. The increased kappaB-binding activity and cytokine overexpression was suppressed by introducing RelB cDNA or a dominant negative IkappaBalpha into relb(-/-) fibroblasts. Our findings suggest a novel transcription suppression function of RelB in fibroblasts.
Several autoantibodies related to Type 1 diabetes mellitus and their corresponding autoantigens have been previously identified. While peptide antigens are more widely recognized, lipid antigens like sulfatides and gangliosides are also known epitopes for the diabetic humoral immune response. Islet cell antibodies (ICA) in Type 1 diabetes are heterogeneous immunoglobulins directed against selected antigens in the islets of Langerhans. Moreover, ICA may be the best predictive marker of disease in family members of patients with Type 1 diabetes. The aims of this study were: (1) to purify lipids from porcine pancreas that contain ICA epitopes; (2) to characterize these lipid antigens, and (3) to use the purified lipids in an assay to detect antibodies in patients with Type 1 diabetes. A unique family of 4 lysophospholipids, 1 fully characterized as lysophosphatidylmyoinositol, partially inhibited ICA staining, and therefore, were considered to be candidate antigens for an ICA immunoassay. Using a dot blot immunoassay, we detected antibodies directed against these phospholipids in 28 out of 46 (61%) diabetic sera, while detecting only 1 false positive out of 28 nondiabetic sera (3.6%; p < 0.0001 comparing diabetic vs. nondiabetic serum). Therefore, lysophospholipid immunoassay positivity is present in sera of Type 1 diabetic patients. Furthermore, we detected 15 out of 23 ICA-negative diabetic sera (65.2%), showing that our phospholipid immunoassay does not correlate with ICA positivity.
Trilinolein, a triacylglycerol with linoleic acid, was found to inhibit Ca2+ influx in cardiomyocytes. The purpose of this study was to evaluate the effects of trilinolein on ventricular arrhythmia induced by intravenous strophanthidin in guinea pigs anesthetized with intraperitoneal urethane. After strophanthidin- induced ventricular tachycardia, treatment with trilinolein (0.1, 1, 10 and 100 microg/kg) or control (0.04% propylene glycol) did not terminate ventricular tachycardia. However, 1, 10 and 100 microg/kg trilinolein could narrow the width of the QRS complex during ventricular tachycardia. Pretreatment with trilinolein before strophanthidin administration did not prevent the occurrence of ventricular tachycardia; the doses of strophanthidin required to induce arrhythmias (ventricular extrasystole and ventricular tachycardia) were similar in guinea pigs pretreated with trilinolein or control. However, there were fewer ventricular extrasystoles in guinea pigs pretreated with trilinolein than in the control group (ANOVA, p < 0.01). Moreover, the ventricular extrasystoles were fewer in guinea pigs pretreated with higher doses of trilinolein (100 microg/kg, 103 +/- 60; 10 microg/kg, 188 +/- 86) than lower doses of trilinolein (1 microg/kg, 366 +/- 102; 0.1 microg/kg, 436 +/- 145). This study demonstrated that trilinolein was not effective in terminating or preventing strophanthidin-induced ventricular tachycardia. However, trilinolein could improve ventricular depolarization and suppress ventricular extrasystoles.
Doxorubicin is an antineoplastic agent with significant cardiotoxicity. We examined the effects of this agent on the expression of the natriuretic peptide (NP) genes in cultured neonatal rat atrial myocytes. Doxorubicin suppressed NP secretion, steady-state NP mRNA levels, and NP gene promoter activity. In each instance, brain NP (BNP) proved to be more sensitive than atrial NP (ANP) to the inhibitory effects of the drug. ICRF-187 and probucol reversed the inhibition by doxorubicin of ANP mRNA accumulation and ANP gene promoter activity while exerting no effect on BNP mRNA levels or promoter activity. This represents the first identification of the NP genes as targets of doxorubicin toxicity in the myocardial cell. This inhibition operates predominantly at a transcriptional locus and has more potent effects on BNP versus ANP secretion/gene expression. Measurement of BNP secretion/gene expression may provide a sensitive marker of early doxorubicin cardiotoxicity.
The expression of the lutropin receptor (LHR) in granulosa cells is a complex phenomenon under the hormonal control of FSH and estradiol. Using primary cultures of granulosa cells from immature female rats pretreated with diethylstilbestrol (a compound with estrogen-like activity), the role of FSH in LHR induction was studied. Previous studies from our laboratory have shown that FSH or 8-bromo-cAMP addition to these cells causes a marked increase in the rate of transcription of the rat LHR (rLHR) gene. The present studies were undertaken to compare the properties of the rLHR gene in undifferentiated vs. differentiated rat granulosa cells as a means of determining those elements that confer basal activity and cAMP responsiveness. Our studies show that the proximal 155 bp (relative to the translational initiation codon) of the 5'-flanking region of rLHR gene represent a minimal promoter that accounts for the basal expression of this receptor in rat granulosa cells. A major domain located between nucleotides (nt) -90 and -120, with another one possibly being between nt -120 and -155, induced activation of basal transcriptional activity. An inhibitory domain was observed to lie between nt -186 and -1375. Our data further show that multiple elements within the 2.1 kb of the 5'-flanking region of the rLHR gene are involved in the 8-bromo-cAMP-induced expression of the LHR gene. Of these, the three Sp1-binding sites within the proximal portion of the 5'-flanking region appear to be important for both basal as well as cAMP-induced rLHR gene transcription.
Autoimmune Addison's disease and premature ovarian failure are characterized by the presence of organ-specific autoantibodies. The main adrenal and gonadal autoantigens have been identified and cloned, and the relationship between the autoantibodies detected by immunofluorescence techniques and those detected by the new assays using recombinant autoantigens needed to be investigated. We studied 165 patients with Addison's disease: 143 patients had different forms of autoimmune Addison's disease (13 with idiopathic premature ovarian failure) and 22 had nonautoimmune Addison's disease. Adrenal-cortex autoantibodies and steroid-producing cell autoantibodies were measured by the immunofluorescence techniques. Autoantibodies to steroid 21-hydroxylase, 17alpha-hydroxylase, and P450 side chain cleavage enzyme were measured by immunoprecipitation assay using 35S-labeled recombinant proteins. Adrenal-cortex autoantibodies and autoantibodies to 21-hydroxylase were found in 81% of the patients with autoimmune Addison's disease. None of the patients with nonautoimmune Addison's disease had adrenal-cortex autoantibodies or autoantibodies to 21-hydroxylase. A high association between these two markers in patients with different forms of autoimmune Addison's disease and in those with short- or long-standing disease was found. Steroid-producing cells autoantibodies were found in 26% of the patients with autoimmune Addison's disease, and autoantibodies to 17alpha-hydroxylase and/or P450 side chain cleavage enzyme in 36% of the patients. Steroid-producing cells autoantibodies were found in 11/13 (85%) of patients with idiopathic premature ovarian failure associated with autoimmune Addison's disease, and autoantibodies to 17alpha-hydroxylase and/or P450 side chain cleavage were found 12/13 (92%) of patients; the only case negative for all these three markers suffered from Turner's syndrome. Provided that a high standard of immunofluorescence technique is maintained, measurement of adrenal cortex autoantibodies or steroid-producing cells autoantibodies by either immunofluorescence or immunoprecipitation assay is essentially equivalent.
In GH-deficient children showing ectopic posterior pituitary hyperintense signal (EPP), the anatomical details of the pituitary-hypothalamic region and the relationship between the visibility of the pituitary stalk and anterior pituitary function were studied by magnetic resonance imaging (MRI). The absence or presence of the pituitary stalk was recorded by MRI before and after the injection of gadolinium in 25 children with GH deficiency and EPP at the age of 8.7+/-5.0 yr (16 males and 9 females). Patients were classified into 2 groups according to the presence (group 1; n = 14), or the absence (group 2; n = 11) of pituitary stalk visibility after gadolinium injection. Most patients in group 1 (12 of 14) demonstrated isolated GH deficiency, whereas all but 1 patient in group 2 showed multiple anterior pituitary hormone deficiency. The prevalence of a normally sized adenohypophysis was higher in group 1 than in group 2 (50% vs. 9%; P<0.05). Although the EPP was found at the median eminence in all group 2 patients, it was visualized in group 1 at different levels of the pituitary stalk in 60% of cases (8 of 14; at the proximal end of the pituitary stalk, n = 4; in the middle of the pituitary stalk, n = 2; at the distal end of the pituitary stalk, n = 2). This demonstrates that the ectopic posterior pituitary migration abnormality may be complete or partial. In conclusion, although the pathogenesis of GH deficiency with EPP remains unclear, these results suggest that in cases of GH deficiency associated with ectopic posterior pituitary hyperintense signal, patients with no visible pituitary stalk on MRI after gadolinium injection present a more severe form of the disease in childhood associated with multiple anterior pituitary hormone deficiency, whereas visibility of the pituitary stalk is related to isolated GH deficiency. Nevertheless, careful follow-up of these latter patients is necessary, as the natural history of the disease is not established until adulthood.
Estrogen receptor (ER) functions as a ligand-activated transcription factor for estrogen-regulated genes. Because of the critical role of the ER in the proliferation of certain estrogen-dependent cancer cell types such as the mammary tumor, inhibitors of estrogen action at the level of receptor function are of major clinical interest. Here we describe developments of two ribozymes that can selectively degrade the human ER mRNA and inhibit trans-activation of an artificial promoter containing the estrogen response element. Two ribozymes, designated RZ-1 and RZ-2, cleave the human ER alpha mRNA at nucleotide positions +956 and +889, respectively. These cleavage sites lie within the coding sequence for the DNA-binding domain of the receptor protein. Both RZ-1 and RZ-2 were also effective in inhibiting the progression of quiescent MCF-7 breast cancer cells to the S phase of the cell cycle after their exposure to 17beta-estradiol (10(-9) M). These results provide a new avenue for inhibition of estrogen action by selective mRNA degradation with its potential therapeutic application through targeted gene delivery vectors.
In the nematode Caenorhabditis elegans, CED-4 plays a central role in the regulation of programmed cell death. To identify proteins with essential or pleiotropic activities that might also regulate cell death, we used the yeast two-hybrid system to screen for CED-4-binding proteins. We identified MAC-1, a member of the AAA family of ATPases that is similar to Smallminded of Drosophila. Immunoprecipitation studies confirm that MAC-1 interacts with CED-4, and also with Apaf-1, the mammalian homologue of CED-4. Furthermore, MAC-1 can form a multi-protein complex that also includes CED-3 or CED-9. A MAC-1 transgene under the control of a heat shock promoter prevents some natural cell deaths in C. elegans, and this protection is enhanced in a ced-9(n1950sd)/+ genetic background. We observe a similar effect in mammalian cells, where expression of MAC-1 can prevent CED-4 and CED-3 from inducing apoptosis. Finally, mac-1 is an essential gene, since inactivation by RNA-mediated interference causes worms to arrest early in larval development. This arrest is similar to that observed in Smallminded mutants, but is not related to the ability of MAC-1 to bind CED-4, since it still occurs in ced-3 or ced-4 null mutants. These results suggest that MAC-1 identifies a new class of proteins that are essential for development, and which might regulate cell death in specific circumstances.
The performance of mobile telemedical communications links based on the IS-54 and GSM cellular telephone standards (the most widely used commercial systems in North America and Europe, respectively) was studied by computer simulations. A photoplethysmography signal was used to investigate the transmission of medical data over simulated mobile phone channels. Various conditions were simulated in the communications path between a mobile transmitter and receiver, from perfect to distorted conditions. The results showed successful transmission, with bit error rates of better than 10(-7) at the receiver for the IS-54 standard. The performance of the IS-54 standard was superior to that of GSM in terms of minimum path delay variations, especially in built-up (urban) areas.
Explore the source record for details and available documents.
Endocrine disruptors are hormone mimics that modify hormonal action in humans and animals. It is thought that some endocrine disruptors modify estrogen and androgen action in humans and animals by suppressing aromatase activity. Aromatase cytochrome P450 is the key enzyme that converts C19 androgens to aromatic C18 estrogenic steroids. We have developed a novel aromatase inhibitor screening method that allows us to identify antiaromatase activity of various environmental chemicals. The screen was developed by coexpressing the human aromatase and the mouse androgen receptor in yeast cells, which carry the androgen-responsive ss-galactosidase reporter plasmid. Functional expression of aromatase in yeast has been demonstrated using the [3H]-water release assay with intact cells as well as with yeast microsomes. The aromatase activity could be blocked by known aromatase inhibitors such as aminoglutethimide (AG). Yeast-produced androgen receptors were able to transactivate a yeast basal promoter linked to an androgen-responsive element in response to androgens. The resultant triple yeast transformant responded to the treatment of testosterone, androstenedione, or 5 alpha-dihydrotestosterone (5 alpha-DHT). In the absence of the aromatase inhibitor AG, transcriptional activation was observed only for the nonaromatizable androgen 5 alpha-DHT. However, the two aromatizable androgens (testosterone and androstenedione) induced the reporter activity in the presence of AG. Using this yeast-based assay, we confirmed that two flavones, chrysin and alpha-naphtholflavone, are inhibitors of aromatase. Thus, this yeast system allows us to develop a high-throughput screening method, without using radioactive substrate, to identify aromatase inhibitors as well as new ligands (nonaromatizable androgen mimics) for the androgen receptors. In addition, this screening method also allows us to distinguish nonandrogenic aromatase inhibitors from inhibitors with androgenic activity. This yeast screening method will be useful to screen environmental chemicals for their antiaromatase activity and for their interaction with androgen receptor.
Aromatase has been shown to be expressed at a higher level in human breast cancer tissue than in normal breast tissue, by means of enzyme activity measurement, immunocytochemistry, and RT-PCR analysis. Cell culture including MCF-7 breast cancer cells, animal experiments using aromatase-transfected breast cancer cells, and transgenic mouse studies have demonstrated that estrogen production in situ plays a more important role than circulating estrogens in breast tumor promotion. In addition, tumor aromatase is believed to be able to stimulate breast cancer growth through both autocrine and paracrine pathways, as demonstrated by a three-dimensional cell culture study. RT-PCR and gene transcriptional studies have revealed that the aromatase promoter is switched from a glucocorticoid-stimulated promoter, I.4, in normal tissue to cAMP-stimulated promoters, I.3 and II, in cancerous tissue. Recently, we identified and characterized a cAMP-responsive element (CREaro) upstream from promoter I.3 by DNA deletion and mutational analyses. Our results from promoter functional analysis also demonstrated an interaction between the CREaro and the silencer element (S1) that was identified previously in our laboratory. In the presence of cAMP, the positive regulatory CREaro can overcome the action of the silencer on the function of promoter I.3. On the basis of results generated from our own and other laboratories, we propose that, in normal breast adipose stromal cells and fibroblasts, aromatase expression is driven by promoter I.4 (glucocorticoid dependent), and that the action of promoters I.3 and II is suppressed by the silencer negative regulatory element. However, in cancer cells and surrounding adipose stromal cells, the cAMP level increases, and aromatase promoters are switched to cAMP-dependent promoters - I.3 and II. Furthermore, we applied the yeast one-hybrid screening method to search for proteins interacting with the silencer element, S1. The major protein identified was ERRalpha-1; however, SF-1, which is present in the ovary, is not detected in breast cancer tissue. Using a reporter plasmid with the aromatase genomic fragment containing promoter I.3 and S1, in breast cancer SK-BR-3 cells, ERRalpha-1 was found to have a positive regulatory function. It is believed that the silencer element in the human aromatase gene may function differently in different tissues, as a result of distinct expression patterns of transcription factors.
The effects of tetrandrine (Tet) on the contractile function and microvascular permeability in stunned rat myocardium in vivo were studied. Stunned myocardium was induced by 15 (MS(15) group) or 20 (MS(20) group) min of myocardial ischemia plus 60 min of reperfusion. The following was shown. (1) FITC-BSA concentration was 166.0 +/- 7. 9 microg/g myocardium in the control group. The concentrations in ischemic myocardium increased by 35.4 and 45.6% in MS(15) and MS(20) groups respectively (p<0.05). (2) Administration of Tet (64.2 and 96. 3 micromol/kg, I.P.) 20 min before ischemia not only ameliorated the contractile function, but also reduced the FITC-BSA concentrations in ischemic myocardium. At 60 min after reperfusion, the contractile function parameters in Tet-treated groups were significantly superior to those in corresponding stunning groups. FITC-BSA concentrations in Tet-treated groups were lower than those in stunning groups. Then, there was already no significant difference in FITC-BSA concentrations between Tet-treated groups and the control group. The FITC-BSA concentrations at the end of experiments were correlated negatively with dp/dt(max) (r = -0.83, p<0.01). (3) Tet inhibited KCl-induced calcium influx in isolated cardiomyocytes. The results suggest that Tet given before ischemia may be involved in the reduction of microvascular permeability in stunned myocardium, which might be associated with its calcium channel blocking effect.
To determine whether hypoglycemic agents such as sulfonylureas, biguanides and the newly developed insulin sensitizers such as troglitazone, have hypotensive effects in an animal model of non-insulin-dependent diabetes mellitus associated with insulin resistance, male Otsuka Long Evans Tokushima Fatty (OLETF) rats aged 12 weeks were administered following hypoglycemic agents or vehicle by gavage for 26 weeks; glibenclamide (5 mg/kg/day), metformin (100 mg/kg/day) and troglitazone (70 mg/kg/day). The gain in body weight was similar in the different groups. At 36 weeks of age, troglitazone significantly decreased fasting plasma glucose levels when compared to controls. The area under the curve (AUC) for insulin during glucose loading (2 g/kg, i.p.) was 50% lower in the group treated with troglitazone. Serum triglyceride levels in troglitazone-treated rats were also significantly lower than in the glibenclamide-treated group. Plasma membrane GLUT4 protein content was significantly augmented by a factor of 1.48-fold (p<0.02) in the glibenclamide-treated group and tended to be increased 1.32 times by administration of metformin (p=0.06). The systolic blood pressure increased with age in controls and the glibenclamide-treated group. In contrast, treatment with either metformin or troglitazone significantly decreased systolic blood pressure after the age of 29 weeks. Plasma norepinephrine and epinephrine concentrations did not show a significant decrease in the treated group when compared with the control group. These results suggest that metformin and troglitazone, but not glibenclamide, lower blood pressure in an animal model of insulin resistance, providing further evidence of the beneficial effect of insulin sensitizing hypoglycemic agents on blood pressure.