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Biomedical subjects

C Lin

Publications and source records attributed to C Lin.

At least 181 records · Page 10Linked to original sources
PubMed1997

Identification of an amiloride binding domain within the alpha-subunit of the epithelial Na+ channel.

Limited information is available regarding domains within the epithelial Na+ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278-283) within the extracellular domain of alpharENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant alpharENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type alpharENaC was inhibited by amiloride with a Ki of 169 nM. Deletion of the entire WYRFHY tract (alpharENaC Delta278-283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (Ki = 26.5 microM). Similar results were obtained when either alpharENaC or alpharENaC Delta278-283 were co-expressed with wt beta- and gammarENaC (Ki values of 155 nM and 22.8 microM, respectively). Moreover, alpharENaC H282D was insensitive to submicromolar concentrations of amiloride (Ki = 6.52 microM), whereas alpharENaC H282R was inhibited by amiloride with a Ki of 29 nM. These mutations do not alter ENaC Na+:K+ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of alpharENaC and mutations preceding the second membrane-spanning domains of alpha-, beta-, and gammarENaC alters amiloride's Ki, suggest that selected regions of the extracellular loop of alpharENaC may be in close proximity to residues within the channel pore.

Actins↗
PubMed1997

Analysis of the 5' sequence, genomic structure, and alternative splicing of the presenilin-1 gene (PSEN1) associated with early onset Alzheimer disease.

Mutations in the human presenilin genes (PSEN1 and PSEN2) are associated with early onset familial Alzheimer disease. The presenilin genes encode integral membrane proteins with similar structures, which suggests that they may have closely related, but as yet unknown functions. Analysis of the 5' upstream sequence and the structure of the PSEN1 gene reveals that the 5' sequence contains multiple putative transcription regulatory elements including clusters of STAT elements involved in transcriptional activation in response to signal transduction. The first four exons contain untranslated sequences, with Exons 1 and 2 representing alternate initial transcription sites. The function of these alternate initial exons is unclear. Exon 4 bears the first ATG sequence. The last 12 bp of Exon 4 is used as an alternative splice donor site. Exon 9 is alternately spliced in leukocytes, but not in most other tissues. Splicing of Exon 9 is predicted to cause significant structural changes to the protein. The majority of transcripts expressed in most tissues are polyadenylated 1127 bp from the TAG stop codon in Exon 13. A small proportion of transcripts contain the same 5'UTR and ORF but are polyadenylated 4435 bp from the stop codon. The longer polyadenylated transcripts contain three additional palindromes and at least one additional stem-loop structure with stabilities greater than -16 kcal/mol.

Alternative Splicing↗
PubMed1997

Cloning and characterization of the Drosophila presenilin homologue.

Mutations in two genes, PS1 and PS2, coding for the presenilins, have been linked to the early onset form of familial Alzheimer's disease (AD). Here we report the identification of a Drosophila melanogaster homologue of human PS genes, Dps, which maps to band 77B-C on chromosome 3 and is expressed at multiple developmental stages. The predicted amino acid sequence of the Dps product is 53% identical to human presenilins, with the greatest similarity in the putative transmembrane domains, the hydrophobic domains at the beginning and the end of the cytoplasmic TM6-TM7 loop and the C-terminus. Analysis of Dps in a genetically tractable model system such as Drosophila may provide insight into the mechanisms of Alzheimer's disease (AD) necessary for the development of rational therapeutic approaches.

Alzheimer Disease↗
PubMed1997

Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line.

Mutations in a Cl- channel (cystic fibrosis transmembrane conductance regulator or CFTR) are responsible for the cystic fibrosis (CF) phenotype. Increased Na+ transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+ channel open probability (Po). The Xenopus renal epithelial cell line, A6, expresses both cAMP-activated 8-picosiemen (pS) Cl- channels and amiloride-sensitive 4-pS Na+ channels, and provides a model system for examining the interactions of CFTR and epithelial Na+ channels. A6 cells express CFTR mRNA, as demonstrated by reverse transcriptase-polymerase chain reaction and partial sequence analysis. A phosphorothioate antisense oligonucleotide, complementary to the 5' end of the open reading frame of Xenopus CFTR, was used to inhibit functional expression of CFTR in A6 cells. Parallel studies utilized the corresponding sense oligonucleotide as a control. CFTR protein expression was markedly reduced in cells incubated with the antisense oligonucleotide. Incubation of A6 cells with the antisense oligonucleotide led to inhibition of forskolin-activated amiloride-insensitive short circuit current (Isc). After a 30-min exposure to 10 microM forskolin, 8-pS Cl- channel activity was detected in only 1 of 31 (3%) cell-attached patches on cells treated with antisense oligonucleotide, compared to 5 of 19 (26%) patches from control cells. A shift in the single-channel current-voltage relationship derived from antisense-treated cells was also consistent with a reduction in Cl- reabsorption. Both amiloride-sensitive Isc and Na+ channel Po were significantly increased in antisense-treated, forskolin-stimulated A6 cells, when compared with forskolin-stimulated controls. These data suggest that the regulation of Na+ channels by CFTR is not limited to respiratory epithelia and to epithelial cells in culture overexpressing CFTR and epithelial Na+ channels.

Amiloride↗
PubMed1997

Relationship between energetic, ionic, and functional status in the perfused rat heart following thermal injury: a 31P and 23Na NMR study.

To test the hypothesis that energy deficits and intracellular ion derangements may be the cellular basis for intrinsic myocardial dysfunction in rats after burn trauma, we examined ATP metabolism, intracellular pH, sodium, and mechanical performance simultaneously in perfused beating hearts from sham burn or burned rats (43% TBSA, 3 degrees scald burn, resuscitated for 24 hr with lactated Ringer's solution, Parkland formula). Intracellular calcium was also measured in myocytes harvested from parallel groups of sham burn and burn resuscitated rats. Burn trauma caused a 46% decrease in left ventricular developed pressure, a 69% decrease in +dP/dtmax, and a 72% decrease in -dP/dtmax. Intracellular to external standard sodium ratio increased (+58%) from 0.318 +/- 0.027 to 0.500 +/- 0.048 (P < 0.05), and intracellular calcium increased (+67%) from 206 +/- 13 to 445 +/- 37 nM (P < 0.01). Burn hearts exhibited decreased functional response to isoproterenol challenge compared to sham burn controls, but energy metabolism was similar in all hearts, regardless of burn injury. Our data suggest that burn trauma alters intracellular cardiomyocyte calcium and sodium homeostasis, and ionic derangements are not related to either altered intracellular pH or high energy phosphate deficits.

Adrenergic beta-Agonists↗
PubMed1997

Single-dose pharmacokinetics of felbamate in patients with renal dysfunction.

AIMS: The purpose of this study was to evaluate the effects of renal impairment on the single-dose pharmacokinetics of the antiepileptic felbamate. METHODS: Twelve subjects with three levels of renal dysfunction (creatinine clearance > 30-80, > 10-30 or 5-10 m min(-1)) and four controls with normal renal function (creatinine clearance > 80 ml min(-1) were studied). Plasma and urine samples were obtained for 144 h following administration of a single 1200 mg dose. RESULTS: Compared with controls, apparent total body clearance, renal clearance and urinary excretion of felbamate were decreased, and half-life, Cmax and AUC values were increased in subjects with renal dysfunction. The magnitude of these changes was associated with the degree of renal dysfunction. Nonrenal clearance and apparent volume of distribution values were also lower in renal dysfunction subjects, but there was no association between the extent of these changes and degree of renal dysfunction. Renal clearance of felbamate accounted for approximately 30% of apparent total body clearance in the control group and from 9-22% in the renal failure patients. Renal clearance of felbamate was significantly correlated with creatinine clearance (r = 0.75; P< 0.001). CONCLUSIONS: These data suggest that initial dosage and titration of felbamate may require adjustment in patients with renal dysfunction.

Adult↗
PubMed1997

Polynucleotide modulation of the protease, nucleoside triphosphatase, and helicase activities of a hepatitis C virus NS3-NS4A complex isolated from transfected COS cells.

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregated in two somewhat independent domains. The essential machinery of a serine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing in vivo. This may reflect economical packaging of essential viral replicative components, but it could also mean that there is functional interdependence between the two domains. In this study, a full-length NS3-NS4A complex was isolated after expression and autoprocessing in transiently transfected COS cells. The protein was used to examine the effects of polynucleotides on the NTPase, helicase, and protease activities. Unlike the previously reported behavior of a separately expressed NS3 helicase domain, the full NS3-NS4A complex demonstrated optimal NTPase activity between pH 7.5 and 8.5. All three NS3-NS4A activities were modulated by polynucleotides, with poly(U) having the most remarkable effect. These findings suggest that the domains within NS3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this complex HCV replicase component.

Acid Anhydride Hydrolases↗
PubMed1997

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication.

Amino Acid Sequence↗
PubMed1997

The hepatitis C virus NS4A protein: interactions with the NS4B and NS5A proteins.

Hepatitis C virus encodes a large polyprotein precursor that is proteolytically processed into at least 10 distinct products, in the order NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase encoded in the N-terminal 181 residues of the NS3 nonstructural protein is responsible for cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the nonstructural region. NS4A, a 54-residue nonstructural protein which forms a stable complex with the NS3 proteinase, is required as a cofactor for cleavage at the 3/4A and 4B/5A sites and enhances processing at the 4A/4B and 5A/5B sites. Recently reported crystal structures demonstrated that NS4A forms an integral part of the NS3 serine proteinase. In this report, we present evidence that NS4A forms a nonionic-detergent-stable complex with the NS4B5A polyprotein substrate, which may explain the requirement of NS4A for the 4B/5A cleavage. Isoleucine-29 of NS4A, which has been previously shown to be essential for its proteinase cofactor activity and formation of the NS3 complex, was found to be important for the interaction between NS4A and the NS4B5A substrate. In addition, two more hydrophobic residues in the NS4A central region (valine-23 and isoleucine-25) were also shown to be essential for the cofactor activity and for the interaction with either the NS3 proteinase or the NS4B5A polyprotein substrate. Finally, the possible mechanisms by which these viral proteins interact with each other are discussed.

Animals↗
PubMed1997

Rac is required for growth cone function but not neurite assembly.

Recent work has suggested that rac1 and other members of the rho family of small GTP-binding proteins play an important role in the formation of neural processes. We have explored the mechanism of this effect by comparing the spontaneous, growth cone-mediated growth and experimental tension-induced growth of axons in normal PC12 cells and in mutant cells expressing a dominant negative form of rac. PC12 that have been primed by exposure to NGF, but not naive PC12 cells, initiate a microtubule-rich process de novo in response to tension applied to cell body. As in chick sensory neurons, neurite elongation rate is proportional to applied tension above a threshold. Addition of cyclic AMP, which has been shown to rapidly augment NGF-induced neurite outgrowth in PC12, causes a rapid increase in the rate of neurite elongation at a given tension level. Expression of a dominant negative form of rac1 inhibits spontaneous, growth cone-mediated neurite elongation in response to NGF, but does not substantially affect tension-induced neurite elongation. That is, rac-deficient cells show a normal linear relationship between applied tension and elongation rate and the elongations contain a normal density of axial microtubules by immunofluorescent assay. Thus, rac1 is apparently required for the mechanisms that normally generate tension in an elongating neurite, but if this tension is provided from an outside source, then axonal elongation can proceed normally in rac1-deficient cells. We conclude that rac1 is required for the adhesive and motile function of growth cones rather than the assembly of neurites per se.

Animals↗
PubMed1997

[Construction of poliovirus cDNA chimera with (NKNDD)n and (NANP)n].

Oligonucleotides encoding one copy of NKNDD, containing a P.f. B epitope NKND, and two copies of CSP repeated B epitope NANP were synthesized. After self-ligation, NKNDD and (NANP)2 were cloned into the intermediate vector pSKMM. Series of clones with different copies of NKNDD and NANP were identified. (NKNDD)n/MM with three, four, five, eight copies of NKNDD and (NANP)n/MM with four, six copies of NANP were chosen. Then (NKNDD)n and (NANP)n of these recombinants were recovered respectively and finally inserted into the N-Ag I site of poliovirus type I Mahoney strain full-length cDNA, which is contained in the expression plasmid pSV202H+. As using semi-quantitative hybridization in identifying tranformants, the procedure in our experiment for cloning different copies of NKNDD and NANP was greatly simplified.

Animals↗
PubMed1997

[Fully length MDR1 cDNA transfer conferring resistance to adriamycine on sensitive cells GLC].

Human lung cancer leads the mortality of cancers and the chemotherapy is often uneffective because of drug resistance. In order to study the role of mdr-1 gene in resistant lung cancer, the fully length mdr-1 cDNA was transferred into a sensitive lung cancer cell line GLC. The mdr-1 cDNA was constructed in a retroviral vector, pDORneo. The transfection of recombinant plasmid was carried out by lipofectin. Supernatant containing infective viruses derived from a G418 resistant clone of package cell PA317 was used to infect GLC cell which is sensitive to chemotherapeutic agents. After G418 and adriamycine selections, three P-glycoprotein positive clones were isolated and the integration of mdr-1 cDNA was demonstrated by PCR of genomic DNA. The relative resistance of 3 clones to adriamycine as and elevated by 5.4, 6.0 respectively 7.8 times compared with the untransfected cell and the transcription of mdr-1 gene in these transfected cells as obviously enhanced by in situ hybridization. This results suggest that the mdr-1 gene plays a role in increasing drug resistance of human lung cancer.

Adenocarcinoma↗
PubMed1997

[Structure of rubescensin E].

Rubescensin E(I), a new diterpenoid, was isolated from the leaves of Rabdosia rubescens collcted in Jiyuan county, Henan Province On the basis of spectroscopic evidences, the structure of rubescensin E lished as ent-7 beta, 20-epoxy-7 alpha, 14 alpha-dihydroxy-6 alpha, 15 alpha-diacetoxy-16kaurene.

Diterpenes↗
PubMed1997

[Dose distributions of linac-based radiosurgery for pineal region mass].

The doses of linac convergent-beam irradiations to pineal region were measured in an anthropomorphic phantom. The feasibility of a linac accelerator as a radiosurgery tool to treat small sized intracranial masses was evaluated. The influences of some irradiation parameters on the dose distributions were discussed. Linac-based radiosurgery can result in dose distributions similar to those achieved by gamma knife, so it is another safe and effective treatment technique of neurosurgery. The smaller the aperture is and the greater the irradiation arc travel is, the larger the dose gradient will be and the better the dose distribution will be. The maximum target doses are dependent greatly upon the collimater aperture. The appropriate irradiation arc travel and margin isodose were also discussed.

Brain Neoplasms↗
PubMed1997

[Preparation of no-sugar huazhen granules].

This article introduces the preperation, especially its technologies of spraying, drying and making grain of no-sugar Huazhen Granules (composed of Flos lonicerae, Radix Bupleuri, etc.). Ideal grain can be obtained, the capacity of taking can be reduced greatly.

Desiccation↗
PubMed1997

[Evaluation of effectiveness of comprehensive control for diarrhea diseases in rural areas of east Fujian and analysis of its cost-benefit].

An intervention trial with comprehensive prevention and control for diarrhea diseases was carried out in the rural areas of East Fujian, China during 1991 to 1993, focusing on improvement of drinking water, health education, and supervision and direction to villagers to improve their environmental and family health. Results showed the intervention could reduce the incidence of diarrhea diseases by 55.1 percent in average. Economic benefit analysis of the effectiveness of the intervention showed ratios of cost to benefit were 1:5.6, 1:4.9 and 1:6.5 in 1991, 1992 and 1993, respectively, and net benefit amounted to 13,674,000 Chinese yuan. It suggests that effectiveness of this comprehensive control was significant and cost-effective.

China↗
PubMed1996

Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance.

Cold acclimation in plants is associated with the expression of COR (cold-regulated) genes that encode polypeptides of unknown function. It has been widely speculated that products of these genes might have roles in freezing tolerance. Here we provide direct evidence in support of this hypothesis. We show that constitutive expression of COR15a, a cold-regulated gene of Arabidopsis thaliana that encodes a chloroplast-targeted polypeptide, enhances the in vivo freezing tolerance of chloroplasts in nonacclimated plants by almost 2 degrees C, nearly one-third of the increase that occurs upon cold acclimation of wild-type plants. Significantly, constitutive expression of COR15a also affects the in vitro freezing tolerance of protoplasts. At temperatures between -5 and -8 degrees C, the survival of protoplasts isolated from leaves of nonacclimated transgenic plants expressing COR15a was greater than that of protoplasts isolated from leaves of nonacclimated wild-type plants. At temperatures between -2 and -4 degrees C, constitutive expression of COR15a had a slight negative effect on survival. The implications of these data regarding possible modes of COR15a action are discussed.

Journal Article↗