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C Lin

Publications and source records attributed to C Lin.

At least 217 records · Page 12Linked to original sources
PubMed1995

Tissue-specific expression of two aldose reductase-like genes in mice: abundant expression of mouse vas deferens protein and fibroblast growth factor-regulated protein in the adrenal gland.

Aldose reductase (AR), the first enzyme in the polyol pathway, has been implicated in the pathogenesis of diabetic complications, although its physiological role is unclear. In mice, besides AR, two AR-like proteins, mouse vas deferens protein (MVDP) and fibroblast growth factor-regulated protein (FR-1), have been reported recently. Tissue-specific expression of these two genes was examined using the RNase protection assay method. Contrary to previous reports, MVDP was detected in a variety of tissues besides the vas deferens. High levels of MVDP mRNA were found in the adrenal glands, and low levels of expression were detected in eye, intestine, seminal vesicle, kidney, liver, testis and lung. The major gene expression pattern for FR-1 was slightly different from that of MVDP, with the highest levels of mRNA detected in testis, heart, adrenal gland, and ovary; less was found in the lung and it was barely detectable in eye, intestine, liver and seminal vesicle tissue. Mouse embryos, as early as 10.5 days post coitum, expressed both genes, although the levels of expression were different. Human AR mRNA was found in human vas deferens, although not at the high level found in mice. The localization of both MVDP and FR-1 transcripts in the adrenal cortex by in situ hybridization led to the speculation that these two AR-like proteins could be related to hormone production.

Adrenal Glands↗
PubMed1995

Heteroligomers of type-I and type-III inositol trisphosphate receptors in WB rat liver epithelial cells.

We have previously shown that a 222-kDa polypeptide co-immunoprecipitates together with the type-I myoinositol 1,4,5-trisphosphate receptor (IP3R) in WB rat liver epithelial cell extracts, when the immunoprecipitation is carried out with a type-I isoform specific antibody (Joseph, S. K. (1994) J. Biol. Chem. 269, 5673-5679). Utilizing isoform-specific antibodies raised to unique sequences within the COOH-terminal region of IP3 receptors, we now report that the co-immunoprecipitating 222-kDa polypeptide is the type-III IP3R isoform and that type-III IP3R antibodies (Abs) can co-immunoprecipitate the type-I IP3R isoform. Co-immunoprecipitation of IP3R isoforms was not due to cross-reactivity of the antibodies for the following reasons: (a) on immunoblots the type-III antibodies did not cross-react with type-I IP3R and vice versa; (b) inclusion of the COOH-terminal type-III peptide had no effect on the ability of type-I IP3R Ab to co-immunoprecipitate the type-III IP3R but blocked the ability of type-III IP3R Ab to coimmunoprecipitate the type-I isoform; and (c) crude hepatocyte lysates contain undetectable amounts of type-III IP3R, and immunoprecipitation with type-III IP3R Ab does not co-immunoprecipitate any other isoforms. However, type-I and type-II IP3R isoforms were co-immunoprecipitated by their respective antibodies in hepatocyte lysates. Sucrose density gradient analysis of WB cell lysates indicated that the co-immunoprecipitating fraction is exclusively located at the density expected for tetrameric receptors, suggesting that co-immunoprecipitation was not a reflection of the nonspecific aggregation of IP3R isoforms. Phosphorylation of either type-I or type-III immunoprecipitates by protein kinase A indicated that only the type-I IP3R could be phosphorylated in vitro. Fractionation of WB cell membranes and immunofluorescence studies showed that the type-I and type-III isoforms have very similar sub-cellular localizations. We conclude that the WB cell contains both type-I and type-III IP3R isoforms and that a proportion of these receptors exist as heterotetramers.

Amino Acid Sequence↗
PubMed1995

Malignant transformation of human fibroblast strain MSU-1.1 by v-fes requires an additional genetic change.

To determine whether the v-fes oncogene can malignantly transform human fibroblasts that have acquired an infinite life span and are partially growth-factor-independent, we transfected cell strain MSU-1.1 with a plasmid containing the v-fes oncogene and a bacterial histidinol dehydrogenase gene. Of the 60 independent histidinol-resistant clones isolated and assayed for v-fes expression using a fes-specific monoclonal antibody, 6 were found to express the v-fes protein at a detectable level. When progeny cells from these 6 clones were further characterized, 3 of the 6 clonal populations exhibited a significant increase in the ability to form medium-sized colonies in agarose, but none were tumorigenic in athymic mice. However, when the 6 populations were propagated for many generations, the same 3 populations acquired the ability to form very large colonies in agarose ( > or = 100 microM in diameter) at a frequency of 2% to 17%, and formed malignant tumors in athymic mice. This suggests that an additional genetic change required for malignant transformation had been spontaneously acquired in 3 of the v-fes -transformed cell strains. To determine whether the change or changes were the equivalent of an activated sis or ras proto-oncogene, we transfected the v-fes oncogene into derivative strains of MSU-1.1 that express a transfected v-sis, c-H-ras or c-N-ras oncogene, but that do not form tumors, and assayed the v-fes-expressing transfectants for tumorigenicity. The results showed that when complemented either by a ras oncogene expressed at a somewhat enhanced level or by the v-sis oncogene, v-fes can supply the additional change required for malignant transformation.

Animals↗
PubMed1995

Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene.

We report the cloning of a novel gene (E5-1) encoded on chromosome 1 which has substantial nucleotide and amino-acid sequence similarity to the S182 gene on chromosome 14q24.3. Mutations, including three new missense mutations in the S182 gene, are associated with the AD3 subtype of early-onset familial Alzheimer's disease (AD). Both the E5-1 and the S182 proteins are predicted to be integral membrane proteins with seven membrane-spanning domains, and a large exposed loop between the sixth and seventh transmembrane domains. Analysis of the nucleotide sequence of the open reading frame (ORF) of the E5-1 gene led to the discovery of two missense substitutions at conserved amino-acid residues in affected members of pedigrees with a form of familial AD that has a later age of onset than the AD3 subtype (50-70 years versus 30-60 years for AD3). These observations imply that the E5-1 gene on chromosome 1 and the S182 gene on chromosome 14q24.3 are members of a family of genes (presenilins) with related functions, and indicates that mutations in conserved residues of E5-1 could also play a role in the genesis of AD. Our results also indicate that still other AD susceptibility genes exist.

Alzheimer Disease↗
PubMed1995

Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.

Arabidopsis↗
PubMed1995

Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1.

The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.

Arabidopsis↗
PubMed1995

The hepatitis C virus NS3 serine proteinase and NS4A cofactor: establishment of a cell-free trans-processing assay.

The hepatitis C virus RNA genome encodes a long polyprotein that is proteolytically processed into at least 10 products. The order of these cleavage products in the polyprotein is NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. A serine proteinase domain located in the N-terminal one-third of nonstructural protein NS3 mediates cleavage at four downstream sites (the 3/4A, 4A/4B, 4B/5A, and 5A/5B sites). In addition to the proteinase catalytic domain, the NS4A protein is required for processing at the 4B/5A site but not at the 5A/5B site. These cleavage events are likely to be essential for virus replication, making the serine proteinase an attractive antiviral target. Here we describe an in vitro assay where the NS3-4A polyprotein, NS3, the serine proteinase domain (the N-terminal 181 residues of NS3), and the NS4A cofactor were produced by cell-free translation and tested for trans-processing of radiolabeled substrates. Polyprotein substrates, NS4A-4B or truncated NS5A-5B, were cleaved in trans by all forms of the proteinase, whereas NS4A was also required for NS4B-5A processing. Proteolysis was abolished by substitution mutations previously shown to inactivate the proteinase or block cleavage at specific sites in vivo. Furthermore, N-terminal sequence analysis established that cleavage in vitro occurred at the authentic 4A/4B site. Translation in the presence of microsomal membranes enhanced processing for some, but not all, proteinase-substrate combinations. Trans-processing was both time and temperature dependent and was eliminated by treatment with a variety of detergents above their critical micelle concentrations. Among many common proteinase inhibitors tested, only high (millimolar) concentrations of serine proteinase inhibitors tosyllysyl chloromethyl ketone and 4-(2-aminoethyl)benzenesulfonyl fluoride inactivated the NS3 proteinase. This in vitro assay should facilitate purification and further characterization of the viral serine proteinase and identification of molecules which selectively inhibit its activity.

Animals↗
PubMed1995

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Amino Acid Sequence↗
PubMed1995

Determination of a cyclic guanine monophosphate phosphodiesterase inhibitor (SCH 51866) in rat serum using capillary zone electrophoresis.

A capillary zone electrophoretic (CZE) assay was developed for the determination of cis-5,6a,7,8,9,9a-hexahydro-2-[4-(trifluoromethyl)phenylmethyl]-5-methyl - cyclopent[4,5]imidazo[2,1-b]purin-4(3H)-one, SCH 51866 (I), a cyclic guanine monophosphate phosphodiesterase inhibitor, in rat serum using acetonitrile deproteination as a clean-up step before injection. The calibration curve was linear over a serum concentration range of 0.5 to 10 micrograms/ml serum with a correlation coefficient (r) > 0.99. The limit of quantitation (LOQ) was established at 0.5 micrograms/ml. Fifty microliters of serum were used for analysis, which allowed serial bleeding (8 samples) from a single rat to characterize the pharmacokinetic profile of I after either oral or intravenous drug administration. In traditional pharmacokinetic and toxicokinetic studies in rodents, one animal provides only one serum sample since 1 to 2 ml of sample volume is required for chromatographic analysis, resulting in the use of a large number of animals per study. This assay yields a significant reduction in the use of animals, hence providing a large reduction in resources and time in drug discovery and development.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease.

Some cases of Alzheimer's disease are inherited as an autosomal dominant trait. Genetic linkage studies have mapped a locus (AD3) associated with susceptibility to a very aggressive form of Alzheimer's disease to chromosome 14q24.3. We have defined a minimal cosegregating region containing the AD3 gene, and isolated at least 19 different transcripts encoded within this region. One of these transcripts (S182) corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. Five different missense mutations have been found that cosegregate with early-onset familial Alzheimer's disease. Because these changes occurred in conserved domains of this gene, and are not present in normal controls, they are likely to be causative of AD3.

Alzheimer Disease↗
PubMed1995

Regulated basal, inducible, and tissue-specific human erythropoietin gene expression in transgenic mice requires multiple cis DNA sequences.

Erythropoietin (Epo) gene expression in kidney and liver is inducible by anemia. To localize the sequences necessary for regulated expression of the Epo gene, we constructed transgenic mice containing five human Epo gene constructs and examined Epo expression under basal conditions and with anemia. Mice containing the Epo gene with 0.3 kb of 5' flanking sequence, 0.7 kb of 3' flanking sequence, and either all introns or only intron I alone were polycythemic, had Epo expression in various tissues (including non-Epo-producing tissues), and induction only in liver. In contrast, mice containing the Epo gene with 8.5 kb of 3' flanking sequence and either 9.5 or 22 kb of 5' flanking sequence had basal expression at low levels in appropriate tissues and were less likely to be markedly polycythemic. Mice with the smaller of these two constructs had induction only in the liver, whereas those with the larger construct had induction in the kidney and liver. These studies indicate that sequences sufficient for induction in the liver are located in close proximity to the Epo gene, including the immediate 5' and 3' flanking sequence and the first intron. They also indicate that sequences required for induction in the kidney are located more than 9.5 kb 5' to the gene. Furthermore, comparison of these and prior transgenic studies suggest that sequences that limit the basal expression of the Epo gene are located downstream of the gene. We conclude that multiple cis DNA sequences are required for regulated Epo gene expression.

Anemia↗
PubMed1995

Subcellular organization of calcium signalling in hepatocytes and the intact liver.

Hepatocytes respond to inositol 1,4,5-trisphosphate (InsP3)-linked agonists with frequency-modulated oscillations in the intracellular free calcium concentration ([Ca2+]i), that occur as waves propagating from a specific origin within each cell. The subcellular distribution and functional organization of InsP3-sensitive Ca2+ pools has been investigated, in both intact and permeabilized cells, by fluorescence imaging of dyes which can be used to monitor luminal Ca2+ content and InsP3-activated ion permeability in a spatially resolved manner. The Ca2+ stores behave as a luminally continuous system distributed throughout the cytoplasm. The structure of the stores, an important determinant of their function, is controlled by the cytoskeleton and can be modulated in a guanine nucleotide-dependent manner. The nuclear matrix is devoid of Ca2+ stores, but Ca2+ waves in the intact cell propagate through this compartment. The organization of [Ca2+]i signals has also been investigated in the perfused liver. Frequency-modulated [Ca2+]i oscillations are still observed at the single cell level, with similar properties to those in the isolated hepatocyte. The [Ca2+]i oscillations propagate between cells in the intact liver, leading to the synchronization of [Ca2+]i signals across part or all of each hepatic lobule.

Animals↗
PubMed1995

Osmotic dilution stimulates axonal outgrowth by making axons more sensitive to tension.

Mechanical tension is a potent stimulator of axonal growth rate, which is also stimulated by osmotic dilution. We wished to determine the relationship, if any, between osmotic stimulation and tensile regulation of axonal growth. We used calibrated glass needles to apply constant force to elongate axons of cultured chick sensory neurons. We find that a neurite being pulled at a constant force will grow 50-300% faster following a 50% dilution of inorganic ions in the culture medium. That is, osmotic dilution appears to cause axons to increase their sensitivity to applied tensions. Experimental interventions suggest that this effect is not mediated by dilution of extracellular calcium, or to osmotic stimulation of adenylate cyclase, or to osmotic stimulation of mechanosensitive ion channels. Rather, experiments measuring the static tension normally borne by neurites suggest a direct mechanical effect on the cytoskeletal proteins of the neurite shaft. Our results are consistent with a formal thermodynamic model for axonal growth in which removing a compressive load on axonal microtubules promotes their assembly, thus promoting axonal elongation.

Adenylyl Cyclases↗
PubMed1995

Defining topological similarities among ion transport proteins with anti-amiloride antibodies.

The structural features of amiloride binding sites on amiloride-sensitive transport proteins have received limited characterization. An antibody that recognizes limited regions of amiloride and can mimic, in binding specificity, certain amiloride-sensitive transport proteins was used as a model to elucidate potential amino acid residue relationships that might define putative amiloride contact sites. Analysis of the structure of this antibody has allowed us to identify sequence relationships among several Na+ selective transport proteins. A structure-based relational database was employed to re-examine sequence homologies among these ion transport proteins. A search of the protein sequence databank identified representative amino acid tracts among amiloride sensitive proteins involving planar residues that might be involved in interacting with amiloride. Computer models of sites within transmembrane domains of NHE1 and NHE2 isoforms of the Na+/H+ exchanger reflective of these planar tracts indicate that amiloride probably spans two helices for interaction with the Na+/H+ exchanger. Structural analysis of this monoclonal anti-amiloride antibody appears to mimic some of the salient features of amiloride binding sites on these proteins.

Amiloride↗
PubMed1995

Mutations throughout an Arabidopsis blue-light photoreceptor impair blue-light-responsive anthocyanin accumulation and inhibition of hypocotyl elongation.

This paper reports the characterization of novel mutations within the Arabidopsis thaliana HY4 gene, which has previously been shown to encode a protein (CRY1) with characteristics of a blue-light photoreceptor. Several point mutations were identified within the amino-terminal domain of CRY1--this region of CRY1 has high homology to photolyase and is likely to be involved in blue-light-mediated electron transfer. Mutations were found within the region of homology to the known chromophore binding domains of photolyase. Point mutations within the 200 amino acid carboxy-terminal extension distinguishing CRY1 from photolyase, likewise disrupt function of the protein. CRY1 was originally defined as the photoreceptor responsible for blue-light-mediated inhibition of hypocotyl elongation and we now report that anthocyanin accumulation in germinating seedlings is an additional phenotype under the control of this photoreceptor--this is shown to be mediated in part by modulation of mRNA levels of chalcone synthase, one of the anthocyanin biosynthetic enzymes. The effect of the novel mutations on both inhibition of hypocotyl elongation and anthocyanin biosynthesis have been evaluated, and it is demonstrated that mutations with less severe effects on hypocotyl elongation show a similarly reduced effect on anthocyanin biosynthesis. These results are consistent with the cryptochrome photoreceptor mediating multiple regulatory pathways by the same primary mode of action.

Amino Acid Sequence↗
PubMed1995

Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 5' non-coding region.

A method is described for identifying different genotypes of hepatitis C virus (HCV) by restriction endonuclease cleavage of sequences amplified by PCR from the 5' non-coding region. Using the enzymes HaeIII-RsaI and HinfI-MvaI, followed by cleavage with BstU1 or ScrFI, it was possible to identify and distinguish HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5 and 6. The method was used to investigate the prevalence of these genotypes in 723 blood donors in 15 countries, the largest survey to date, and one which covered a wide range of geographical regions (Europe, America, Africa and Asia). These results, combined with a review of the existing literature, indicate the existence of several distinct regional patterns of HCV genotype distribution, and provide the framework for future detailed epidemiological investigations of HCV transmission.

Blood Donors↗