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Biomedical subjects

J J Lin

Publications and source records attributed to J J Lin.

At least 127 records · Page 7Linked to original sources
PubMed1994

The variant mRNA isoform of human metastasis gene (CD44V) detected in the cell lines of human hepatocellular carcinoma.

The interaction of the cell surface receptor CD44 molecular with its ligands (addressin, extracellular matrix etc.,) plays an important role in fulfilling the lymphocyte homing and immune reaction. Recently alternatively spliced products of CD44 gene are found to be involved in tumor metastasis as well. Our report found that CD44 prototype RNA (CD44S) was present in all five tumor cell lines. Isoform CD44 RNA (CD44V) was recognized in three metastasized hepatocellular carcinoma cell lines, J5, HCC36, HEP3B. In addition, the J5 CD44 RNA isoform expressed two distinct transcripts which are of the same size as MDA-231 breast tumor cell line. The MDA-231 CD44 RNA variant (CD44V) has been confirmed to contain metastasis domain 4 and 5. It is implicated that the alternative RNA splicing may also play a major role in hepatocellular carcinoma metastasis.

Base Sequence↗
PubMed1994

The isolation of differentially expressed genes in fibroblast growth factor stimulated BC3H1 cells by subtractive hybridization.

We have developed a subtractive hybridization procedure based on the hybridization of a single-stranded phagemid cDNA library (target) to biotinylated RNA (driver). We have applied this method to fibroblast growth factor (FGF) induced-uninduced mouse brain tumor-derived muscle-like cell. BC3Hl cDNA libraries. After hybridization to a C(o)t value of 1000, cDNAs common to the target and driver populations were subtracted up to 231-fold, whereas several highly induced genes were enriched from 2-15-fold. Interestingly, moderately induced genes (e.g., the 12-fold-induced nur/77 gene) were subtracted even at a low C(o)t value of 50. Therefore, at every C(o)t tested, subtractive hybridization tended to equalize the uninduced and moderately induced common sequences within target populations regardless of the abundance of the gene species. These observations suggest that subtractive hybridization should only be used for identifying target genes that are either uniquely expressed or highly induced.

Animals↗
PubMed1993

The 3' flanking region of the human erythropoietin-encoding gene contains nitrogen-regulatory/oxygen-sensing consensus sequences and tissue-specific transcriptional regulatory elements.

We have reported the identification of a classical canonical CAAT box, TATA boxes and other transcriptional regulatory elements in the 5' flanking region of the human erythropoietin (hEp)-encoding gene [Lee-Huang et al., Gene 128 (1993) 227-236]. These elements were not found in the hEp genomic clones reported by others. Our genomic clone extends in both directions beyond any reported clones, by 3.9 kb on the 5' side and by 1.8 kb on the 3' side. Many important regulatory elements are found in these extended flanking regions. We report here the genomic structure of the extended 3' flanking region of hEp. This region contains the following regulatory elements: nitrogen-regulatory/oxygen-sensing consensus sequences, 5'-TTTTGCA and 5'-CCCTGCA; tissue-specific regulatory elements, including binding sites for A-activator, 5'-GTGGTGCAA; for DBP, 5'-TGATTTTGT; for HNF, 5'-T(A/G)TTTGT; and for C/EBP, 5'-T(T/G) (T/G)TGCAAT; a lymphokine-responsive element, 5'-GTGAAACCCC (Rev), as well as binding sites for AP and Sp1. In addition, the nucleotide (nt) sequence in this region is rich in inverted repeats (palindromes) that allow the formation of hairpin loops. A total of 14 potential stem loops with a maximum loop size of 20 nt are found. The identification of these regulatory elements in hEp should provide further insight into the tissue-specific and inducible expression of hEp. Such knowledge should be useful in the clinical modulation of erythropoiesis under physiologic and pathologic conditions.

Base Sequence↗
PubMed1993

Partial antinociceptive cross-tolerance to intracerebroventricular beta-endorphin in mice tolerant to systemic morphine.

The effects of subcutaneous morphine pellet-implantation on antinociception induced by intracerebroventricular (i.c.v.) administration of beta-endorphin or morphine and intrathecal (i.t.) administration of morphine, [D-Pen2,D-Pen5]enkephalin (DPDPE), [D-Ala2,NMePhe4,Gly5-ol]enkephalin (DAMGO), serotonin or norepinephrine were studied in male ICR mice. The tail-flick and hot-plate responses were used for antinociceptive tests. The ED50 values for i.c.v. administered morphine for antinociception in morphine pellet-implanted mice were increased from 3.3- and 2.2-fold at 0 h to 14.2- and 19.0-fold at 4 h and declined to 4.8- and 3.0-fold at 8 h after pellet removal in the tail-flick and hot-plate tests, respectively. On the other hand, the ED50 values for i.c.v. administered beta-endorphin for antinociception were only slightly increased (1.7- to 5.1-fold increases) throughout the same time course. The inhibition of the tail-flick response induced by i.t. injection of morphine, DPDPE and serotonin, but not norepinephrine or DAMGO, was attenuated in morphine pellet-implanted mice. These findings are consistent with previous studies indicating that different neuronal mechanisms are involved in morphine- and beta-endorphin-induced antinociception.

Analgesics↗
PubMed1993

The human erythropoietin-encoding gene contains a CAAT box, TATA boxes and other transcriptional regulatory elements in its 5' flanking region.

We have reported the cloning and expression of a human erythropoietin (hEp)-encoding cDNA [Lee-Huang, Proc. Natl. Acad. Sci. USA 81 (1984) 2708-2712]. Using this hEp cDNA as a probe, we isolated a 9.3-kb BamHI genomic Ep clone from a human leukocyte library soon thereafter. The size and restriction map of this clone is in agreement with restriction analysis of human genomic DNA probed with the hEp cDNA, demonstrating that this clone is representative of the single hEp gene. This clone is unique in that it extends beyond any reported hEp genomic clone by 3.9 kb on the 5' side and by 1.8 kb on the 3' side. The promoter function of the newly described 5' flanking region has been demonstrated by the expression of biologically active hEp in transfected cells. We find that, despite reports to the contrary, hEp does contain classic canonical TATA boxes and a CAAT box. The 5'-flanking region also contains cytokine-responsive consensus sequences, tissue-specific and metal-responsive elements, CRE and GRE sites, and binding sites for transcription factors, including AP1, NF-kappa beta and Sp1. These regulatory elements have not been found in the hEp genomic clones thus far reported. The identification of these elements and their precise localization in hEp should be useful in studying the regulation of hEp expression, as well as in gene therapy and physiologic modulation of this hormone.

Activating Transcription Factor 2↗
PubMed1993

Expression of smooth muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins.

The cDNA encoding the beta-tropomyosin isoform of chicken smooth muscle (CSM beta) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated beta-tropomyosin (rCSM beta) and a mutant (rCSM beta-7) with a 7-residue deletion at its amino-terminus. Furthermore, the cDNA coding for human fibroblast tropomyosin isoform 3 (hTM3) was also used to produce unacetylated hTM3 (called PEThTM3). All of bacterially-made tropomyosins were high alpha-helical in structure as judged by CD analysis and resistant to heat denaturation. Both the rCSM beta and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14 x 10(6) and 2.78 x 10(6) M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSM beta) appeared to have a comparable actin-binding affinity to that of gel-purified CSM beta homodimer (1.25 x 10(6) M-1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28 x 10(7) M-1). The amino-terminal deletion mutant rCSM beta-7 failed to bind to F-actin. Effects of gizzard caldesmon on the actin binding of these bacterially made tropomyosins were also examined. Under the binding condition containing 0.5 mM MgCl2 and 30 mM KCl, caldesmon greatly enhanced the binding of rCSM beta to F-actin. However, under the same condition, there was a slight enhancement in the actin-binding for gel-purified CSM beta or PEThTM3 (1.2-1.6-fold stimulation) and no enhancement for native gizzard tropomyosin. Neither the presence of caldesmon nor native gizzard tropomyosin induced detectable binding of the amino-terminal deletion mutant rCSM beta-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSM beta in the actin binding and the caldesmon enhancement.

Actins↗
PubMed1993

Telomeric DNA binding proteins.

The physical ends of eukaryotic chromosomes form a specialized nucleoprotein complex composed of DNA and DNA binding proteins. This nucleoprotein complex, termed the telomere, is essential for chromosome stability. In most organisms, the DNA portion of the nucleo-protein complex consists of simple tandem DNA repeats with one strand guanine rich. The protein portion of the complex is less well understood. The experiments presented in two recent papers represent different stages in the characterization of the telomeric DNA binding proteins. The first paper presents a structure-function study of the Oxytricha telomeric DNA binding proteins and the second paper shows the identification and initial characterization of a telomeric DNA binding activity from Xenopus laevis. These two reports provided valuable information in understanding the structure and function of telomeres.

Animals↗
PubMed1993

Human fibroblast tropomyosin isoforms: characterization of cDNA clones and analysis of tropomyosin isoform expression in human tissues and in normal and transformed cells.

A tropomyosin-specific oligonucleotide probe (REN29) designed to hybridize to all known human tropomyosin isoforms was used to study tropomyosin mRNA levels in normal and transformed human cells. At least four different sizes of RNAs were detected in normal human fibroblast KD cells by Northern blot analysis. The major bands of 1.1 kb RNA for hTM1 and 3.0 kb RNA for hTM4 were decreased substantially in various transformed cell lines. One of the minor RNA bands (2.0 kb for hTM2 and hTM3) appeared to be absent in a human pancreatic carcinoma cell line. The level of the other minor RNA band (2.5 kb for hTM5) was found to be unchanged or slightly decreased in transformed cells. This differential expression of tropomyosin isoforms at the RNA level was not totally in agreement with the difference in the protein amounts found in normal and transformed cells, suggesting that translational control may also play an important role in the expression of some tropomyosin isoforms. The REN29 probe was further used to screen lambda gt10 and lambda gt11 cDNA libraries, which were constructed from poly(A)+ RNAs of human fibroblast cell lines HuT-14 and WI-38, respectively. In addition to cDNA clones encoding known isoforms, we obtained three classes of new cDNA clones that encode two low M(r) isoforms (hTM5a and hTM5b), and a high M(r) isoform (hTMsm alpha). Sequence comparison revealed that hTM5a and hTM5b are alternatively spliced products derived from the same gene that encodes hTM2 and hTM3. Northern blot analysis and amino acid sequence comparison suggested that the hTMsm alpha represents a smooth muscle tropomyosin which is also expressed in human fibroblasts. The exon specific for, and common to, hTM5a and hTM5b was found to be highly expressed in small intestine. However, there was no detectable expression of this exon in stomach and skeletal muscle. The difference in tissue-specific expression suggests that different isoforms may perform distinct functions in different tissues.

Amino Acid Sequence↗
PubMed1993

In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants.

At least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full-length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5, PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F-actin was stronger than the PEThTM3 isoform. However, analysis of actin-binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N-terminal fragment of hTM5 fused to the C-terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N-terminal fragment of hTM3 fused to the C-terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F-actin than their parental tropomyosins. A bacterially made C-terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin-binding of these bacterially expressed tropomyosins. However, PETCaD39's enhancement of binding to F-actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low M(r) isoform PEThTM4 appeared to be able to amplify the actin-activated HMM ATPase activity by 4.7 fold, while the high M(r) isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low M(r) isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell.

Actins↗
PubMed1993

Electron microscopic images suggest both ends of caldesmon interact with actin filaments.

An improved rotary shadowing technique enabled us to visualize chicken gizzard caldesmon (CaD) and its complexes with one or two covalently linked calmodulin (CaM) molecules by electron microscopy. Using a monoclonal antibody against an epitope in the N-terminal region of CaD (anti-N), we can now identify the end of the molecule that is involved in binding to another protein molecule. Thus in the 1:1 complex of CaD and CaM, the CaM molecule was almost always associated with the C-terminus of CaD, indicating preferential CaM-binding to the C-terminal region. We have also studied binding of CaD to filamentous actin (F-actin), using an EM technique that avoids spraying or freeze drying and thereby preserves the structure of F-actin. Only one end of CaD appeared to bind to F-actin, leaving the rest of the molecule projecting away from the filament. While the majority of anti-N bound at the free end of CaD, some antibody molecules were found on F-actin. These findings suggest that either end of CaD can bind to F-actin. Experiments using a monoclonal antibody against the C-terminus of CaD (anti-C) supported this idea. When the native thin filaments that contain endogenous CaD were incubated with anti-N, almost all the bound antibodies were found on the filaments, indicating that the N-terminal regions of CaD interact with actin, and that the binding affinity of the N-terminal region of CaD for actin is higher in vivo than that in vitro, either because the properties of CaD have been altered during purification, or because of the presence of some other component(s) associated with the native filaments.

Actins↗
PubMed1993

Forced expression and assembly of rat cardiac troponin T isoforms in cultured muscle and nonmuscle cells.

Cardiac troponin T (cTnT), a tropomyosin (TM)-binding subunit of the troponin complex, undergoes a developmentally regulated isoform switch from embryonic form to adult form in the rat heart. To investigate the in vivo assembly of cTnT isoforms, we transiently transfected cDNA clones of either rat cTnT isoform into nonmuscle CHO cells and chick embryo myogenic (CEM) cells. As determined by Western blotting, both isoforms can be expressed in CHO and CEM cells. The expressed proteins had the same mobility as native rat cTnT proteins on SDS polyacrylamide gels and were recognized by anti-TnT antibodies. Conventional and confocal microscopy of transfected cells, double-labelled with antibodies against cTnT and against TM, revealed that neither isoform appears to associate with the nonmuscle TM in CHO cells, although both are able to colocalize with muscle TM-containing microfilament bundles in the myogenic CEM cells. There was no appreciable cTnT isoform-related difference in association with TM, suggesting that the functional significance of isoform variability in rat cTnT does not correspond to an assembly advantage for the maturing cardiac thin filament. To help determine whether cTnT nonassembly in CHO environment is primarily due to the nonmuscle nature of the endogenous TM, or if it involves the absence of other factors specific to muscle, we have isolated several stably-transfected clones of skeletal beta TM-expressing CHO cells which incorporate this muscle TM onto stress fibres. When either isoform of cTnT was transiently expressed in these beta TM-CHO cells, the strictly filamentous beta TM staining pattern was no longer observed. Instead, beta TM codistributed with cTnT in brightly staining aggregates not associated with the intact stress fibres. This suggests that both isoforms of cTnT are interacting with the beta TM in the nonmuscle environment and that other muscle-specific proteins may indeed be required for stable assembly of cTnT onto microfilaments. It also suggests that the interaction between cTnT and muscle TM is stronger than that between muscle TM and nonmuscle microfilaments.

Actin Cytoskeleton↗
PubMed1993

IgG and IgA classes of anti-neutrophil cytoplasmic autoantibodies in a 13-year-old girl with recurrent Henoch-Schonlein purpura.

We describe a 13-year-old girl with recurrent Henoch-Schonlein purpura whose symptoms were precipitated by upper respiratory tract infections. Her serum was positive for both IgG and IgA classes of anti-neutrophil cytoplasmic autoantibodies by immunofluorescence. The titers of both autoantibodies correlated with disease activity. The immunopathology underlying these findings is discussed.

Adolescent↗
PubMed1992

Complete nucleotide sequence and structural organization of rat cardiac troponin T gene. A single gene generates embryonic and adult isoforms via developmentally regulated alternative splicing.

We have previously demonstrated that rat cardiac troponin T (TnT) is expressed as two different isoforms during development, the larger, more acidic embryonic isoform and the smaller, more basic adult isoform, which appear to be generated from a common transcript of the cardiac TnT gene by alternative RNA splicing. In this study, Southern blot analysis confirmed the existence of a single copy of cardiac TnT gene in the rat genome. For investigation of the molecular mechanism of isoform switch and the control of this gene expression in myocardial development, several overlapping genomic clones were isolated from a rat genomic library. Complete nucleotide sequences were determined from these genomic clones and revealed a 19,186 base-pair DNA fragment containing 16 exons of rat cardiac TnT gene. Its DNA sequence and exon organization appeared to differ from that of the rat fast skeletal muscle TnT gene or chicken cardiac TnT gene. Comparison of genomic and cDNA clones also confirmed that the cardiac TnT isoform switching was due to the inclusion or exclusion of exon 4 during RNA processing. Sequence analysis allowed us to further identify the other alternatively spliced exon containing only nine nucleotides in size (exon 12). The inclusion and complete or partial exclusion of this exon may be responsible for generating three classes of mRNAs detected by our cDNA clones. The functional significance of this variation in TnT isoforms remained unknown, but its splicing pattern did not appear to link to the developmental changes. The 5' upstream structure was very similar to that in chicken cardiac TnT gene but differed from that in the rat fast skeletal muscle TnT gene, suggesting a similar regulatory mechanism for mammalian and avian cardiac TnT expression.

Alternative Splicing↗
PubMed1992

Active site of (A)BC excinuclease. I. Evidence for 5' incision by UvrC through a catalytic site involving Asp399, Asp438, Asp466, and His538 residues.

(A)BC excinuclease of Escherichia coli removes damaged nucleotides from DNA by hydrolyzing the 8th phosphodiester bond 5' and the 15th phosphodiester bond 3' to the modified base. The activity results from the ordered action of UvrA, UvrB, and UvrC proteins. The role of UvrA is to help assemble the UvrB.DNA complex, and it is not involved in the actual incision reactions which are carried out by UvrB and UvrC. To investigate the role of UvrC in the nuclease activity a subset of His, Asp, and Glu residues in the C-terminal half of the protein were mutagenized in vitro. The effect of these mutations on UV resistance in vivo and incision activity in vitro were investigated. Mutations, H538F, D399A, D438A, and D466A conferred extreme UV sensitivity. Enzyme reconstituted with these mutant proteins carried out normal 3' incision but was completely defective in 5' incision activity. Our data suggest that UvrC makes the 5' incision by employing a mechanism whereby the three carboxylates acting in concert with H538 and a Mg2+ ion facilitate nucleophilic attack by an active site water molecule.

Adenosine Triphosphatases↗
PubMed1992

Active site of (A)BC excinuclease. II. Binding, bending, and catalysis mutants of UvrB reveal a direct role in 3' and an indirect role in 5' incision.

UvrB plays a central role in (A)BC excinuclease. To study its role in the incision reactions, conserved His and Asp residues in this subunit were mutagenized. All His and the majority of Asp mutants behaved like wild-type protein in vivo and in vitro. However, three mutants, D337A, D478A, and D510A, either completely or partially abolished UvrB activity. All three mutant proteins associate with UvrA normally but D337A and D510A were unable to bind to DNA specifically. The UvrB-D478A mutant bound to DNA specifically but failed to denature and kink the DNA. However, UvrB-D478A was efficiently loaded onto DNA preincised at the 3' site and promoted near-normal incision by UvrC at the 5' site. We propose that D478 is involved in bending DNA and catalysis of the 3' incision and that the 3' incision precedes the 5' incision. UvrB which is missing the carboxyl-terminal 43 amino acids binds to, and kinks DNA but is unable to make the 3' incision suggesting that it is missing a residue involved in catalysis. This residue was identified to be E639 by site-specific mutagenesis.

Adenosine Triphosphatases↗
PubMed1992

Calponin and tropomyosin interactions.

The interaction between chicken gizzard calponin and tropomyosin was examined using viscosity, light scattering, electron microscopy and affinity chromatography. At neutral pH, 10 mM NaCl and in the absence of Mg2+, calponin induced tropomyosin filaments to form paracrystals thus decreasing the viscosity while increasing dramatically the light scattering of the tropomyosin solution. Electron micrographs of the uranyl acetate stained calponin-tropomyosin complex showed the presence of spindle shaped paracrystals with regular striation patterns and repeating units of about 400 A. Under similar conditions, smooth muscle caldesmon also induced tropomyosin to form paracrystals. To localize the calponin-binding site on tropomyosin, binding of fragments of tropomyosin, generated by chemical and mutational means, to a calponin-affinity column was studied. The COOH-terminal tropomyosin fragment Cn1B(142-281) and the NH2-terminal fragment CSM-beta(1/8/12-227) bound to a calponin-affinity column with an affinity similar to that of intact tropomyosin; while the NH2-terminal fragment, Cn1A(11-127), did not bind, indicating that the calponin-binding site(s) resides within residues 142-227 of tropomyosin. To determine the involvement in calponin binding of the area around Cys-190 of tropomyosin, fragments with cleavage sites near or at Cys-190 were used. Thus, while fragments Cy2(190-284) and CSM-beta(1/8/12-200) bound weakly to the calponin-affinity column, fragment Cy1(1-189) did not. These results demonstrate that calponin binds to tropomyosin between residues 142 and 227, and that the integrity of the region around Cys-190 of tropomyosin is important for strong interaction between the two proteins.

Animals↗
PubMed1992

Enhanced degradation of the ferritin repressor protein during induction of ferritin messenger RNA translation.

Induction of ferritin synthesis in cultured cells by heme or iron is accompanied by degradation of the ferritin repressor protein (FRP). Intermediates in the degradative pathway apparently include FRP covalently linked in larger aggregates. The effect of iron on FRP degradation is enhanced by porphyrin precursors but is decreased by inhibitors of porphyrin synthesis, which implies that heme is an active agent. These results suggest that translational induction in this system may be caused by enhanced repressor degradation. While unique among translational regulatory systems, this process is common to a variety of other biosynthetic control mechanisms.

5-Aminolevulinate Synthetase↗