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Biomedical subjects

J L Chen

Publications and source records attributed to J L Chen.

At least 91 records · Page 5Linked to original sources
PubMed1997

Inositol triphosphate receptors in sea urchin sperm.

Inositol 1,4,5-triphosphate (Ins(1,4,5)P3) is a second messenger that regulates Ca2+ channels in many important cell signalling pathways. In sea urchin sperm the outer investment of the egg triggers the acrosome reaction (AR) that involves Ins(1,4,5)P3 production and the opening of two Ca2+ channels. Here we have sought to identify a high-affinity Ins(1,4,5)P3 receptor in Strongylocentrotus purpuratus sperm. An Ins(1,4,5)P3 binding component was affinity-purified 12-fold from sperm extracts. It displayed similar characteristics to the Ins(1,4,5)P3 receptor from other sources: pH-dependent high affinity for Ins(1,4,5)P3 (KD = 261 nM), a tau1/2 of association and dissociation of 50 and 40 s, respectively, specificity (IC50 > 5 microM for Ins(1)P1, Ins(1,4)P2 and Ins(1,3,4,5)P4), and pharmacological sensitivity (10 and 100 microg heparin/ml inhibited 75% and 100% binding respectively). An antibody against the carboxy-terminal of the type I Ins(1,4,5)P3 receptor of somatic cells recognised a plasma membrane component in the sperm head and less intensely in the flagella. This antibody also recognised a 240 kDa band from isolated head plasma membranes, and weakly in flagellar membrane. This IP3 receptor-like protein may mediate the sustained uptake of Ca2+ through the second Ca2+ channel opened during the AR.

Animals↗
PubMed1997

International differences in approaches to 131I therapy for Graves' disease: case selection and restrictions recommended to patients in Japan, Korea, and China.

Members of the American Thyroid Association (ATA), European Thyroid Association (ETA), Japan Thyroid Association (JTA), Korean Thyroid Association (KTA), and Chinese Thyroid Association (CTA) were surveyed independently through an identical questionnaire on their management of Graves' disease. One of the major purposes of the survey was to determine how expert thyroidologists in different regions of the world use three different therapies available for a typical Graves' patient as well as for clinical variations provided. In this report, we summarized, contrasted, and interpreted the results of the surveys in three Asian countries by focusing on therapeutic preference of radioiodine. For the index patient with hyperthyroidism due to Graves' disease, radioiodine was the therapy of choice for 69% of ATA respondents but only 22%, 22%, 11%, and 11% of ETA, CTA, JTA, and KTA respondents, respectively. The goal of radioiodine therapy in Asian countries was to restore the euthyroid state. For the case of recurrence after surgery, there was consensus on choosing radioiodine among all countries surveyed. To interpret the reluctance to advocate radioiodine therapy for a typical Graves' patient in Japan, a new survey concerning the current trends in radioiodine therapy in Japan was conducted among clinical members of the JTA. The phobia of radiation and stringent safety rules for radioactive pharmaceuticals were the two major reasons found. Nevertheless, the percentage of JTA respondents who aim for the euthyroid of hypothyroid state by radioiodine therapy for typical Graves' significantly increased compared with respondents to the former survey in 1988. Moreover, a considerable number of JTA members believe that more radioiodine therapy should be applied for achievement of the rapid improvement of hyperthyroidism, for convenience, and for medical cost benefits.

Adult↗
PubMed1997

Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data [Harris ME et al., 1994 EMBO J 13:3953-3963]. However, several substructures of that model were poorly constrained by the available data. In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex. Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards. Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure. The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set. The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA. The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.

Affinity Labels↗
PubMed1997

Display of disparate transcription phenotype by the phosphorylation negative P protein mutants of vesicular stomatitis virus, Indiana serotype, expressed in E. coli and eucaryotic cells.

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo. We have recently shown that the P protein of VSV, New Jersey serotype (PNJ), expressed in E. coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII). In the present work, we are studying the role of phosphorylation in the activation of the P protein of Indiana serotype (PIND), which is highly nonhomologous in amino acid sequence yet structurally similar to its New Jersey counterpart. Despite the fact that E. coli-expressed PIND required phosphorylation by CKII for activation, the phosphorylation negative P protein mutants generated by altering the phosphate acceptors S and T to alanine, surprisingly, showed transcription activity similar to wild-type in vitro. Alteration of S and T residues to phenylalanine, similarly, supported substantial transcription activity (approx. 60% of wild-type), whereas substitution with arginine residue abrogated transcription (approx. 5% of wild-type). In contrast, the same mutants, when expressed in eucaryotic cells, exhibited greatly reduced transcription activity in vitro. This disparate display of transcription phenotype by the PIND mutants expressed in bacteria and eucaryotic cells suggests that these mutants are unique in assuming different secondary structure or conformation when synthesized in two different cellular milieu. The findings that, unless phosphorylated by CKII, the bacterially expressed unphosphorylated (P0) form of PIND, as well as the phosphorylation negative mutants expressed in eucaryotic cells, demonstrates transcription negative phenotype indicate that, like PNJ, phosphorylation of PIND is essential for its activity.

Animals↗
PubMed1996

Molecular cloning and analysis of two subunits of the human TFIID complex: hTAFII130 and hTAFII100.

Transcription factor TFIID is a multiprotein complex composed of the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). TFIID plays an essential role in mediating transcriptional activation by gene-specific activators. Numerous transcriptional activators have been characterized from mammalian cells; however, molecular analysis of the components of mammalian TFIID has been incomplete. Here we describe isolation of cDNAs encoding two TAF subunits of the human transcription factor TFIID. The first cDNA is predicted to encode the C-terminal 947 residues of the 130-kDa human TAF subunit, hTAFII130. The second cDNA encodes the C-terminal 801 residues of the 100-kDa subunit, hTAFII100. Recombinant TAFs expressed in human cells by transient transfections are capable of associating with the endogenous TAFs and TBP to form a TFIID complex in vivo. Protein binding experiments demonstrate that hTAFII130, like its Drosophila homolog dTAFII110, interacts with the glutamine-rich activation domains of the human transcription factor Sp1. Furthermore, hTAFII130 shows reduced binding to the Sp1 mutants with impaired ability to activate transcription, suggesting a role for hTAFII130 as a direct coactivator target for Sp1.

Amino Acid Sequence↗
PubMed1996

Pfmrk, a MO15-related protein kinase from Plasmodium falciparum. Gene cloning, sequence, stage-specific expression and chromosome localization.

Cyclin-dependent kinases (Cdks) play a central role in the regulation of the eukaryotic cell cycle. A novel gene encoding a Cdk-like protein, Pfmrk, has been isolated from the human malaria parasite Plasmodium falciparum. The gene has no introns and comprises an open reading frame encoding a protein of 324 amino acids with a predicted molecular mass of 38 kDa. Database searches revealed a striking similarity to the Cdk subfamily with the highest similarity to human MO15 (Cdk7). The overall sequence of Pfmrk shares 62% similarity and 46% identity with human MO15, in comparison to the 49-58% similarity and 34-43% identity with other human Cdks. Pfmrk contains two unique inserts: one consisting of 5 amino acids just before the cyclin-binding motif and the other composed of 13 amino acids within the T-loop equivalent region. Southern blots of genomic DNA digests and chromosomal separations showed that Pfmrk is a single-copy gene conserved between several parasite strains and is located on chromosome 10. A 2500-nucleotide transcript of this gene is expressed predominantly in the sexual blood stages (gametocytes), suggesting that Pfmrk may be involved in sexual stage development.

Amino Acid Sequence↗
PubMed1996

Identification of the promoter of the mouse obese gene.

Primer extension and RACE (rapid amplification of cDNA ends) assays were used to identify and sequence the 5' terminus of mouse ob mRNA. This sequence was used to obtain a recombinant bacteriophage containing the first exon of the encoding gene. DNA sequence analysis of the region immediately upstream of the first exon of the mouse ob gene revealed DNA sequences corresponding to presumptive cis-regulatory elements. A canonical TATA box was observed 30-34 base pairs upstream from the start site of transcription and a putative binding site for members of the C/EBP family of transcription factors was identified immediately upstream from the TATA box. Nuclear extracts prepared from primary adipocytes contained a DNA binding activity capable of avid and specific interaction with the putative C/EBP response element; antibodies to C/EBP alpha neutralized the DNA binding activity present in adipocyte nuclear extracts. When linked to a firefly luciferase reporter and transfected into primary adipocytes, the presumptive promoter of the mouse ob gene facilitated luciferase expression. When transfected into HepG2 cells, which lack C/EBP alpha, the mouse ob promoter was only weakly active. Supplementation of C/EBP alpha by cotransfection with a C/EBP alpha expression vector markedly stimulated luciferase expression. Finally, an ob promoter variant mutated at the C/EBP response element was inactive in both primary adipocytes and HepG2 cells. These observations provide evidence for identification of a functional promoter capable of directing expression of the mouse ob gene.

Adipose Tissue↗
PubMed1996

Expression and localization of DNA topoisomerase II during rat spermatogenesis.

The potential role(s) of DNA topoisomerase II (topo II) during chromatin changes that characterize different stages of spermatogenesis was investigated in the rat by an analysis of the expression and localization of topo II mRNA and protein in individual spermatogenic cells. Expression of topo II was restricted to spermatogonia, spermatocytes, and round and early-elongating spermatids. Two protein bands of 177 and 170 kDa were detected in immunoblots of spermatocytes and round spermatids, while bands of 148 and 142 kDa were prominent in preparations of elongating spermatids. Topo II levels and distribution patterns, as observed by immuno-fluorescent microscopy, exhibited cell type-specific variations. Differences in topo II staining patterns were also apparent when nuclear matrices of spermatogenic cells were prepared with different extraction conditions. In addition to its possible function as a structural component, topo II, associated with nuclear matrix preparations from spermatogenic cells, possessed catalytic activity. These observations indicate that both the 177 and 170 kDa and the 148 and 142 kDa forms of topo II share similar structural and functional properties. Topo II beta mRNA was transcribed in rat spermatogenic cells at 6.2 kb. Relative levels of topo II beta mRNA were high in spermatogonia and spermatocytes, and decreased in both round and early-elongating spermatids. Changes in topo II expression levels and localization patterns represent distinct stage-specific markers for the maturation of spermatogenic cells, and are consistent with the involvement of topo II in mediating DNA modifications and chromatin changes during spermatogenesis.

Animals↗
PubMed1996

Rapid distribution of intraventricularly administered sucrose into cerebrospinal fluid cisterns via subarachnoid velae in rat.

The intracranial distribution of [14C]sucrose, an extracellular marker infused for 30 s into one lateral ventricle, was determined by autoradiography of frozen-dried brain sections. Within 3.5 min [14C]sucrose appeared in: (i) the third ventricle, including optic, infundibular and mammillary recesses; (ii) the aqueduct of Sylvius; (iii) the velum interpositum, a part of the subarachnoid space that runs along the roof of the third ventricle and contains many blood vessels; (iv) the mesencephalic and fourth ventricles; and (v) the superior medullary velum, a highly vascular extension of the subarachnoid space that terminates at the walls of the mesencephalic and fourth ventricles. Within 5 min, radioactivity was present in the interpeduncular, ambient and quadrigeminal cisterns, which encircle the midbrain. By 10 min, approximately 11% of the radioactivity had passed into the subarachnoid space via a previously undescribed flow pathway that included the velum interpositum and superior medullary velum. At many places along the ventricular system, [14C]sucrose appeared to move from cerebrospinal fluid into the adjacent tissue by simple diffusion, as reported previously (Blasberg R. G. et al. (1974) J. Pharmac. exp. Ther. 195, 73-83; Levin V. A. et al. (1970) Am. J. Physiol. 219, 1528-1533; Patlak C. and Fenstermacher J.D. (1975) Am. J. Physiol. 229, 877-884; Rosenberg G. A. and Kyner W.T. (1980) Brain Res. 193, 56-66; Rosenberg G. A. et al. (1986) Am. J. Physiol 251, F485-F489). Little sucrose was, however, taken up by: (i) circumventricular organs such as the subfornical organ; (ii) medullary and cerebellar tissue next to the lateral recesses; and (iii) the superior and inferior colliculi and cerebral peduncles. For the latter two groups of structures, entry from cerebrospinal fluid was apparently blocked by a thick, multilayered glia limitans. Although [14C]sucrose was virtually absent from the rest of the subarachnoid system after 1 h, it remained in the perivascular spaces and/or walls of pial arteries and arterioles for more than 3 h. Certain transport proteins, protease inhibitors, growth factors and other neurobiologically active materials are present in cerebrospinal fluid, and their distribution to the brain and its blood vessels may be important. The present work shows, in the rat, that the flow of cerebrospinal fluid and the disposition of its constituents is fairly complex and differs among regions. Flow was rapid throughout the ventricular system and into various subarachnoid velae and cisterns, but was surprisingly slow and slight over the cerebral and cerebellar cortices. The cerebrospinal fluid-to-tissue flux of material was relatively free at many interfaces, but was greatly restricted at others, the latter indicating that the old concept of a "cerebrospinal fluid-brain barrier" may hold at such places. Finally, radiolabeled sucrose was retained longer within the walls and perivascular spaces of pial arteries and arterioles than in other subarachnoid tissues; one function of the cerebrospinal fluid system or "third circulation" may thus be delivering factors and agents to these pial blood vessels.

Animals↗
PubMed1996

Use of computed tomography scanning before and after sitting in an emergency department.

RATIONALE AND OBJECTIVES: Before a computed tomography (CT) scanner was installed in the emergency department, this hospital had no dedicated CT scanner for patients in the emergency department, and transporting these patients to the CT scanners in the radiology department took approximately 8-10 min each way. We sought to determine whether the presence of a CT scanner within the emergency department would lead to an increase in the number of emergent cranial CT examinations and a decrease in the diagnostic yield of these examinations. METHODS: More than 8,000 records of cranial CT examinations were reviewed for the 12 months before and the 12 months after the installation of the CT scanner in the emergency department. A positive case was defined as one that showed acute abnormality such as ischemia, hemorrhage, edema, or mass effect. RESULTS: Our results show a 30.1% increase in the number of CT scans of the head ordered by physicians in the emergency department after the installation of a CT scanner (p < .0001). This is compared with the background 1.8% increase in the total number of emergency department visits. However, in both periods, 12% of the total number of head scans done were positive. CONCLUSION: The convenience of having a CT scanner in the emergency department leads to greater use of CT examinations. However, the increase in the number of emergent CT studies is justified because the number of positive CT examinations increases proportionately.

Emergency Service, Hospital↗
PubMed1996

Two new naphthoquinones with antiviral activity from Rhinacanthus nasutus.

Two new naphthoquinones, rhinacanthin-C (1) and rhinacanthin-D (2), exhibit inhibitory activity against cytomegalovirus (CMV), with EC50 values of 0.02 and 0.22 microgram/mL, respectively, against human CMV. They were isolated from the medicinal plant Rhinacanthus nasutus (Acanthaceae). The structures of the compounds were determined by analysis of their spectroscopic data, in particular, 2D NMR.

Antiviral Agents↗
PubMed1996

The role of megakaryocyte growth and development factor in terminal stages of thrombopoiesis.

Thrombopoietin (TPO), the ligand for the c-Mpl cytokine receptor, is a recently identified cytokine with potent effects on platelet production. The receptor-binding portion of c-Mpl ligand is encompassed in another molecule known as megakaryocyte growth and development factor, or MGDF. Although it is clear that the administration of TPO or MGDF to animals dramatically increases the platelet count, the specific stage(s) of thrombopoiesis during which these molecules are principally active have not been unambiguously determined. Pharmacology studies administering MGDF at doses ranging from 0.1 to 630 micrograms/kg/d to mice revealed a biphasic response in platelet production. Administration of the drug at concentrations from 6 to 60 micrograms/kg/d resulted in platelet counts 5-fold above normal. However, doses > 60 micrograms/kg/d resulted in less-than-optimal platelet production. This phenomenon was investigated in vitro. Using an established culture system for the generation of human megakaryocytes and platelets, MGDF was shown to be optimally and equivalently active in the generation of mature megakaryocytes at concentrations from 10 to 1000 ng/ml. However, the cytokine was not required for proplatelet formation and in fact was inhibitory to that process in a dose-dependent manner. When MGDF was added to human megakaryocytes at concentrations of 200 ng/ml or greater, proplatelet formation was inhibited to 30% of control values. MGDF-mediated inhibition was specific, since the addition of the truncated form of the c-Mpl receptor reversed the inhibition in a dose-dependent manner. Other recombinant factors, interleukin-6, interleukin-11 and erythropoietin had no significant positive or negative effects in this human proplatelet assay. Together, these data suggest that although TPO and MGDF promote the full spectrum of megakaryocyte growth and development, they are not necessary for proplatelet formation, and may in part regulate platelet shedding by their absence.

Animals↗
PubMed1996

Pharmacokinetics and intraocular pressure lowering effect of timolol preparations in rabbit eyes.

Two timolol preparations, a gel and an eyedrop with a thickening agent, and one commercial eyedrop without a thickening agent, were studied in rabbits. After topical administration of these three preparations in rabbits, aqueous humor was withdrawn and the proteins removed from the samples by precipitation with acetonitrile. Timolol concentrations were determined directly by an HPLC method. The HPLC mobile phase was composed of methanol and 5 mM d-camphorsulfonic acid (in 1% acetic acid) with a ratio of 49:51 (v/v). A reversed phase C18 column was used to separate samples with a flow rate of 0.8 mL/min and a UV detector set at 284 nm. A two-compartment pharmacokinetic model was used to fit the aqueous humor level for determining the drainage (kd) and absorption rate constants (ka) in the precorneal area as well as the elimination rate constant (ke) of timolol in aqueous humor. For ka +kd, the eyedrop without a thickening agent had the highest value (0.160 min-1), followed by the eyedrop with a thickening agent (0.030 min-1), and the gel had the lowest value (0.009 min-1). It suggests that the gel has a longer retention time in eyes to improve ocular bioavailability and decrease side effects. The AUC0 approximately infinity for the aqueous humor profile with time coordinates were 4142, 2974, and 1604 micrograms min/mL, for the gel, the eyedrop with a thickening agent, and the eyedrop without a thickening agent, respectively. In another study, timolol preparations were also topically administered in alpha-chymotrypsin-induced glaucoma rabbits for determining the lowering effect on intraocular pressure (IOP). The durations of depressing IOP for the gel, the eyedrop with a thickening agent, and the eyedrop without a thickening agent were 24, 14 and 10 hrs, respectively. Thus, the gel preparation has a longer duration and a higher ocular bioavailability which might be further developed in the treatment of open-angle glaucoma.

Absorption↗
PubMed1996

Chronic TPA treatment inhibits expression of proximal tubule-specific properties by LLC-PK1 cells.

Confluent LLC-PK1 cell populations expressed progressively proximal tubule-specific properties, including gamma-glutamyl transpeptidase activity, sodium hexose symport activity, alkaline phosphatase activity, and villin protein. This was paralleled by an increase in villin protein manifested at the single cell level. Chronic treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited expression of proximal tubule-specific properties at the levels of enzyme activity, protein content, and mRNA content. Inhibition occurred in all cells of the population. TPA treatment induced a decrease in total protein kinase C (PKC)-alpha protein content and a change in subcellular localization from predominantly soluble to predominantly particulate. PKC-epsilon protein content was unchanged by TPA treatment. PKC-epsilon was localized in both soluble and particulate fractions of control cells but was localized predominantly in particulate fractions after TPA treatment. PKC-delta was barely detectable in control cells, but content was markedly increased by TPA. These results suggest that TPA-induced inhibition of expression of proximal tubule-specific properties is mediated through modulation of content and/or subcellular localization of one or more PKC isozymes, likely PKC-alpha.

Alkaline Phosphatase↗
PubMed1995

Coactivator and promoter-selective properties of RNA polymerase I TAFs.

Human ribosomal RNA synthesis by RNA polymerase I requires the activator UBF and the promoter selectivity factor SL1, which consists of the TATA binding protein (TBP) and three associated subunits, TAFI110, TAFI63, and TAFI48. Here it is shown that both TAFI110 and TAFI63 contact the promoter, whereas TAFI48 serves as a target for interaction with UBF and is required to form a transcriptionally active SL1 complex responsive to UBF in vitro. TAFI48 also alters the ability of TBP to interact with TATA box elements, and the resulting complex fails to support transcription by RNA polymerase II. Thus, TAFI48 may function both as a target to mediate UBF activation and as a class-specific promoter selectivity factor.

DNA-Binding Proteins↗