Biomedical subjects

J J Lin

Publications and source records attributed to J J Lin.

At least 91 records · Page 5Linked to original sources

PubMed1996

Molecular cloning and characterization of a novel mouse macrophage gene that encodes a nuclear protein comprising polyglutamine repeats and interspersing histidines.

Simple tandem repeats of the trinucleotide sequence CAG encode homopolymeric stretches of glutamine. Although polyglutamine has been identified in diverse proteins, it is present predominantly in transcription factors. We observed that oncogene-immortalized mouse macrophages express several genes that contain a CAG repeat motif. Therefore, we attempted to clone a novel gene that contains a CAG repeat and is associated with cytokine activation of macrophages. Screening of a mouse macrophage cDNA library with a probe comprising 12 consecutive CAG triplets identified at least one unique clone. The cDNA encodes a protein (named GRP-1 or glutamine repeat protein-1) with 171 amino acids, a calculated molecular mass of 21.6 kDa, and a predicted pI of 10.67. Greater than two-thirds of GRP-1 are only two amino acids, namely glutamine (50%) and histidine (18%). There are four polyglutamine motifs interspersed with histidine-rich regions. There is also a putative nuclear localization signal flanked by sites for possible serine phosphorylation. GRP-1 mRNA was expressed constitutively in some macrophage cell lines and B and T cell lines. Interferon-gamma or lipopolysaccharide augmented GRP-1 mRNA expression in the mouse macrophage cell line ANA-1. Western blot analyses using an antipeptide serum revealed that GRP-1 was localized in the nucleus of ANA-1 macrophages and transfected 3T3 fibroblasts. Overexpression of GRP-1 decreased Sp1-driven chloramphenicol acetyltransferase gene expression in transient cotransfection experiments. Because polyglutamine motifs can cause protein oligomerization and can function as transcriptional activation domains, we suggest that GRP-1 may be a transcription factor associated with interferon-gamma- or lipopolysaccharide-induced activation of macrophages.

Amino Acid Sequence↗

PubMed1996

A PCR-based DNA fingerprinting technique: AFLP for molecular typing of bacteria.

Amplified restriction fragment polymorphism (AFLP) is a PCR-based DNA fingerprinting technique. In AFLP analysis, bacterial genomic DNA is digested with restriction enzymes, ligated to adapters, and a subset of DNA fragments are amplified using primers containing 16 adapter defined sequences with one additional arbitrary nucleotide. Polymorphisms of different Escherichia coli strains or Agrobacterium tumefaciens strains were demonstrated as distinct, unique bands in a denaturing sequencing gel using AFLP. The polymorphisms detected between BL21 and BL21F'IQ and between DH5 alpha and DH5 alpha F'IQ strains indicated that AFLP is able to resolve differences in F' episomal DNA (approximately 100 kb).

Agrobacterium tumefaciens↗

PubMed1996

The melanoma differentiation associated gene mda-7 suppresses cancer cell growth.

Cancer is a disease characterized by defects in growth control, and tumor cells often display abnormal patterns of cellular differentiation. The combination of recombinant human fibroblast interferon and the antileukemic agent mezerein corrects these abnormalities in cultured human melanoma cells resulting in irreversible growth arrest and terminal differentiation. Subtraction hybridization identifies a melanoma differentiation associated gene (mda-7) with elevated expression in growth arrested and terminally differentiated human melanoma cells. Colony formation decreases when mda-7 is transfected into human tumor cells of diverse origin and with multiple genetic defects. In contrast, the effects of mda-7 on growth and colony formation in transient transfection assays with normal cells, including human mammary epithelial, human skin fibroblast, and rat embryo fibroblast, is quantitatively less than that found with cancer cells. Tumor cells expressing elevated mda-7 display suppression in monolayer growth and anchorage independence. Infection with a recombinant type 5 adenovirus expressing antisense mda-7 eliminates mda-7 suppression of the in vitro growth and transformed phenotype. The ability of mda-7 to suppress growth in cancer cells not expressing or containing defects in both the retinoblastoma (RB) and p53 genes indicates a lack of involvement of these critical tumor suppressor elements in mediating mda-7-induced growth inhibition. The lack of protein homology of mda-7 with previously described growth suppressing genes and the differential effect of this gene on normal versus cancer cells suggests that mda-7 may represent a new class of cancer growth suppressing genes with antitumor activity.

Animals↗

PubMed1996

Differential displaying of mRNAs from the atrioventricular region of developing chicken hearts at stages 15 and 21.

In an effort to isolate novel genes that may be involved in the development of the cardiac cushions and then the formation of cardiac valves and septa, we utilized the differential mRNA display method in conjunction with the whole-mount in situ hybridization. The total RNAs used to differentially display were prepared from atrioventricular (AV) canal regions of stage 15 and stage 21 chicken hearts because critical events known to be important for the AV valve and septum formation occur during this period of the development. We have successfully obtained 14 potential candidate genes. Three examples, 15H16 (phospholamban), E13 (skeletal alpha-tropomyosin) and 21C (a novel gene), are discussed here. Levels of mRNA expression in developing hearts were determined by Northern blot analysis and their expression patterns were revealed and compared using whole-mount in situ hybridization. Both phospholamban and skeletal alpha-tropomyosin messages in the myocardium of the AV canal region showed significant decrease during this period of the development. The 21C differential display product detects a novel 9.5 Kb message whose expression is cardiac-specific at early stages of development. The expression level of the 21C gene appeared to be increased from stage 15 to stages 21 and 25 as determined by both Northern blot analysis and in situ hybridization. From these data, we demonstrate that the differential display method together with the whole-mount in situ hybridization could be an effective means for the isolation of novel and differentially expressed genes.

Animals↗

PubMed1996

Overexpression of microfilament-stabilizing human caldesmon fragment, CaD39, affects cell attachment, spreading, and cytokinesis.

Previous studies have demonstrated that overexpression of the carboxyl-terminal fragment, CaD39, of human fibroblast caldesmon in Chinese hamster ovary cells protected endogenous tropomyosin from turnover and stabilized actin microfilament bundles [Warren et al., 1994: J. Cell Biol. 125:359-368]. To assess the consequences of having CaD39-stabilized microfilaments in living cell, we characterized the motile behaviors of stable CaD39-expressing lines. We here found that CaD39-expressing cells adhered faster to plastic, glass, fibronectin-coated glass, and collagen-coated glass than control cells. Moreover, the CaD39-expressing cells also exhibited enhanced spreading immediately after attachment. Despite these differences, overexpression of CaD39 had little effect on the velocity of intracellular granule movement, or the velocity and persistence of cellular translocation. However, CaD39-expressing cells were more elongate and encompassed less area than non-expressing cells during migration in a wound-healing assay. In interphase cells, the expressed CaD39 fragments were found associated with tropomyosin-enriched microfilaments. Like endogenous caldesmon, the CaD39 fragment was also modified at mitosis. Although a significant portion of CaD39 underwent only partial modification, the majority of the CaD39 was released from the microfilaments during mitosis. This is consistent with the finding that the CaD39-induced advantage for attachment and spreading was lost during mitosis. In CaD39-expressing cells, an incomplete release of the CaD39 from microfilaments at mitosis was found which may be responsible for the increase in the frequency of multinuclear cells in CaD39-expressing lines.

Actin Cytoskeleton↗

PubMed1996

Growth hormone and insulin-like growth factor-I and mesangial matrix in uremic rats.

Combined growth hormone (GH) and insulin-like growth factor-I (IGF-I) therapy has been advocated for clinical use to minimize the diabetogenic effect of GH and enhance their anabolic effects. However, GH has been shown to accelerate the development of glomerular sclerosis in experimental animals and IGF-I mediates the renal effects of GH. The purpose of this study was therefore to examine morphometrically the effects of GH (1 mg intraperitoneally three times a week), IGF-I (50 micrograms/kg body weight subcutaneously twice a day), and combined GH/IGF-I treatments in vivo on mesangial matrix at 3-20 days after 5/6 nephrectomy in 140- to 150-g rats. There were no significant changes in growth and renal function after GH and/or IGF-I treatment. The effects of GH and IGF-I on glomerular size were additive, which were more prominent in juxtamedullary glomeruli. GH induced proportional increases in mesangial area (MA) and glomerular area (GA), whereas IGF-I induced a similar increase in GA without a corresponding change in MA. When compared with GH treatment alone, combined GH/ IGF-I treatment resulted in a lesser degree of mesangial expansion despite an enhanced glomerular size. While additional studies are needed to examine the long-term effects of these findings, our results suggest a potentially beneficial effect of combined GH/IGF-I therapy during uremia.

Animals↗

PubMed1996

Down regulation of bcl-2 by p53 in nasopharyngeal carcinoma and lack of detection of its specific t(14;18) chromosomal translocation in fixed tissues.

High levels of bcl-2 protein have been found in a wide variety of human cancers. Since p53 gene inactivation occurs in over half of human cancers, it is possible that loss of p53-mediated repression of bcl-2 gene expression accounts, at least in part, for the frequent abnormalities in bcl-2 protein production seen in tumours. By using immunohistochemical methods, we have analysed thirty-three nasopharyngeal carcinomas for p53 and bcl-2 expression. We found an inverse correlation between the expression of these two proteins (P < 0.001). Moreover, we utilized universal oligonucleotide primers of a region 5' to the bcl-2 MBR and at the 3' end of JH segments to initiate a DNA polymerase chain reaction that amplified these bcl-2-JH junctures. Of the twelve nasopharyngeal carcinomas expressing bcl-2, none showed a t(14;18) chromosome translocation. These findings may indicate potential mechanisms by which bcl-2 regulates apoptosis.

Base Sequence↗

PubMed1996

A high molecular mass non-muscle tropomyosin isoform stimulates retrograde organelle transport.

Although non-muscle tropomyosins (TM) have been implicated in various cellular functions, such as stabilization of actin filaments and possibly regulation of organelle transport, their physiological role is still poorly understood. We have probed the role of a high molecular mass isoform of human fibroblast TM, hTM3, in regulating organelle transport by microinjecting an excess amount of bacterially-expressed protein into normal rat kidney (NRK) epithelial cells. The microinjection induced the dramatic retrograde translocation of organelles into the perinuclear area. Microinjection of hTM5, a low molecular mass isoform had no effect on organelle distribution. Fluorescent staining indicated that hTM3 injection stimulated the retrograde movement of both mitochondria and lysosomes. Moreover, both myosin I and cytoplasmic dynein were found to redistribute with the translocated organelles to the perinuclear area, indicating that these organelles were able to move along both microtubules and actin filaments. The involvement of microtubules was further suggested by the partial inhibition of hTM3-induced organelle movement by the microtubule-depolymerizing drug nocodazole. Our results, along with previous genetic and antibody microinjection studies, suggest that hTM3 may be involved in the regulation of organelle transport.

Actins↗

PubMed1996

Plasminogen activator inhibitor-1 and peritoneal transport in peritoneal dialysis patients.

Plasminogen activator inhibitor-1 (PAI-1) is involved in the accumulation of extracellular matrix in various tissues. Since peritoneal interstitium is one of the resistance sites for transport during peritoneal dialysis (PD), the peritoneal PAI-1 level may have a significant effect on peritoneal transport. It was the purpose of this study to examine the associations between plasma or dialysate PAI-1 levels and the variables of peritoneal transport during peritoneal equilibration tests (PET) in 25 stable, adult PD patients. The dialysate PAI-1 levels increased with dwell time. Hematocrit was a positive predictor of plasma PAI-1 concentration and the change of dialysate PAI-1 amount scaled by body surface area (delta DPAI/ BSA). The peritoneal clearance of PAI-1 was higher than expected for its molecular size, suggesting a local production of PAI-1 in peritoneal tissue. The mass transfer coefficient of creatinine scaled by BSA (MTCcr/BSA) was a positive predictor of delta D PAI/BSA, suggesting the diffusion of plasma PAI-1 into the peritoneal cavity also accounted for part of the increased dialysate PAI-1 levels during PET. delta DPAI/BSA was a positive predictor of MTC of urea (ur) MTCwr/BSA and MTCcr/BSA, parameters for diffusive transport. Our findings are consistent with the hypothesis that peritoneal PAI-1 level has a significant effect on peritoneal transport during PD.

Adult↗

PubMed1995

Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression.

Cultured human melanoma cells lose proliferative capacity and terminally differentiate after treatment with the combination of recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ). Subtraction hybridization of cDNA libraries prepared from actively proliferating human H0-1 melanoma cells from cDNA libraries produced from H0-1 cells treated with IFN-beta + MEZ identifies a novel melanoma differentiation-associated (mda) cDNA, mda-7, that displays elevated expression in differentiation inducer-treated H0-1 cells. mda-7 encodes a novel protein of 206 amino acids with a predicted size of 23.8 kDa. The level of mda-7 mRNA is elevated in actively proliferating normal human melanocytes versus primary and metastatic human melanomas. In the Matrigel-assisted melanoma progression model, mda-7 expression decreases in early vertical growth phase primary human melanoma cells selected for autonomous or enhanced tumor formation in nude mice. Treatment of human melanomas with IFN-beta + MEZ, and to a lesser extent with MEZ, results in growth suppression and induced or enhanced mda-7 expression. Immunoprecipitation analyses using peptide-derived rabbit polyclonal antibodies detect increases in mda-7 protein, and a higher molecular weight protein of approximately 90 to 100 kDa, in MEZ and IFN-beta + MEZ treated H0-1 cells. mda-7 is a highly conserved gene with an homologous sequence in the genome of yeast. Transfection of mda-7 expression constructs into H0-1 and C8161 human melanoma cells reduces growth and inhibits colony formation. These results confirm that mda-7 has antiproliferative properties in human melanoma cells and in this context may contribute to terminal cell differentiation. The mda-7 gene may also function as a negative regulator of melanoma progression.

Amino Acid Sequence↗

PubMed1995

SCH 51344 inhibits ras transformation by a novel mechanism.

A pyrazolo-quinoline compound, 6-methoxy-4-[2-[(2-hydroxyethoxyl)-ethyl]amino]-3-methyl-1M-pyrazo lo [3,4-b]quinoline (SCH 51344), was identified based on its ability to derepress human smooth muscle alpha-actin promoter activity in ras-transformed cells. In this study, we show that SCH 51344 reverts several key aspects of ras transformation, such as morphological changes, actin filament organization, and anchorage-independent growth, and also inhibits Val-12 Ras-induced maturation of Xenopus oocytes. SCH 51344 is also a potent inhibitor of the anchorage-independent growth of human tumor lines known to contain multiple genetic alterations in addition to activated ras genes. We have sought to determine whether SCH 51344 disrupts the signaling pathway that activates mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) in normal and ras-transformed fibroblast cells. NIH 3T3 cells transformed by different oncogenes, which have products that participate at different steps of the Ras signaling pathway, were tested in a soft-agar colony formation assay to determine which step of the pathway is inhibited by SCH 51344. Our results indicate that SCH 51344 inhibits the ability of v-abl, v-mos, H-ras, v-raf, and mutant active MAP kinase kinase-transformed NIH 3T3 cells to grow in soft agar. Only v-fos-transformed cells were found to be resistant to the treatment of SCH 51344. SCH 51344 treatment had very little effect, if any, on the activation of MAP kinase kinase, MAP kinase, and p90RSK activity in response to growth factor stimulation. Treatment of ras-transformed cells with SCH 51344 led to stimulation of serum response factor DNA binding activity and activation of serum response element-dependent gene transcription, accounting for its ability to activate alpha-actin promoter activity in ras-transformed cells. Our results indicate that SCH 51344 inhibits ras transformation by a novel mechanism and acts at a point either downstream or parallel to extracellular signal-regulated kinase-dependent Ras signaling pathway.

3T3 Cells↗

PubMed1995

Alignment of caldesmon on the actin-tropomyosin filaments.

We have reported previously that each smooth-muscle caldesmon binds predominantly to a region within residues 142-227 of tropomyosin, but a weaker binding site also exists at the N-terminal region of tropomyosin [Watson, Kuhn, Novy, Lin and Mak (1990) J. Biol. Chem. 265, 18860-18866]. In view of recent evidence for the presence of tropomyosin-binding sites at both the N- and C-terminal domains of caldesmon, we have studied the binding of the N- and C-terminal fragments of human fibroblast caldesmon expressed in Escherichia coli to tropomyosin and its CNBr fragments. The N-terminal fragment, CaD40 (residues 1-152), binds tropomyosin, but the interaction is mostly abolished in the presence of actin. CaD40 binds strongly to Cn1B(142-281) of tropomyosin, but weakly to Cn1A(11-127). The C-terminal fragment, CaD39, which corresponds to residues 443-736 of gizzard caldesmon, binds tropomyosin, and the interaction is enhanced by actin. CaD39 binds to both Cn1A(11-127) and Cn1B(142-281) of tropomyosin. Our results suggest that the N-terminal domain of caldesmon interacts with the C-terminal half of one tropomyosin molecule, whereas the C-terminal domain binds to both N- and C-terminal regions of the adjacent tropomyosin molecule along the actin filament. In addition, the binding of the N-terminal domain of caldesmon to the actin-tropomyosin filament is weak, which may allow this domain to project off the thin filament to interact with myosin.

Actins↗

PubMed1995

An in vitro assay for Saccharomyces telomerase requires EST1.

Telomerase activity was demonstrated in cell-free extracts from S. cerevisiae through the use of a PCR-based assay. As expected, this activity was eliminated by RNase or phenol treatment of the extract and was dependent on dGTP and dTTP. Telomerase was not detected in extracts prepared from cells grown for approximately 30 or more cell divisions in the absence of the EST1 product, Est1p. TLC1 RNA, which determines the sequence of telomeric DNA in vivo, was present in normal amounts in est1 delta cells. Moreover, TLC1 RNA specifically precipitated with epitope-tagged Est1p. These data indicate that Est1p is either a subunit of yeast telomerase or an accessory protein associated with telomerase that is essential in vitro for its activity.

Base Sequence↗

PubMed1995

Expression of kappa opioid receptors in human and monkey lymphocytes.

mRNA encoding the kappa opioid receptor gene sequence was identified and isolated from various human lymphocytic cells: CEM x174 (a hybrid of T and B origin) cells, Jurkat-T4 cells, human peripheral blood mononuclear cells (PBMC) and purified CD4+ cells. Analyzing the cDNA sequences of RNA transcripts spanning the putative second extracellular loop, which has reported dynorphin specificity, and the seventh transmembrane domain revealed a 100% and 95% homology in amino acid sequence to corresponding kappa opioid receptor sequences in human placenta and rat brain, respectively. Expression of a similar kappa opioid receptor sequence could be detected in normal monkey PBMC but not in monkey PBMC in which the CD4+/CD8+ cell ratio (or CD4+ cell number) was significantly reduced due to prolonged SIV infection. These findings suggest that human and monkey lymphocytes constitutively express kappa opioid receptor mRNA.

Amino Acid Sequence↗

PubMed1995

Rapid diagnosis of tuberculous meningitis by polymerase chain reaction assay of cerebrospinal fluid.

A polymerase chain reaction (PCR) method for the rapid diagnosis of tuberculous meningitis (TBM) was used to study prospectively 47 cerebrospinal fluid (CSF) samples from 45 patients. Twenty CSF samples were from patients with clinically suspected TBM and another 27 samples came from patients without clinically suspected TBM. Mycobacterial DNA was detected in 15 CSF samples (14 from patients with clinically suspected TBM and 1 from a patient not suspected of having TBM). Of the PCR-positive samples, 4 were also positive for mycobacterial culture. However, 32 PCR-negative samples were all culture-negative. All samples were negative for the acid-fast bacillus by direct smear. The single PCR-positive patient in the clinically unsuspected TBM group was initially diagnosed as suffering from aseptic meningitis on the basis of his clinical features. The mycobacterial culture of his CSF specimen was also positive and a revised diagnosis of an aseptic type of TBM was made. The estimations of specificity and sensitivity in this study were 100% and 70% respectively. The results showed that using a PCR to detect mycobacterial DNA in CSF for the early diagnosis of TBM is not only a rapid but also an accurate method.

Base Sequence↗

PubMed1995

Rapid optimization of electroporation conditions for plant cells, protoplasts, and pollen.

The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.

Electroporation↗

PubMed1995

Recurrence of immunoglobulin A-kappa crystalline deposition disease after kidney transplantation.

Cases of immunoglobulin A heavy chain and kappa light chain deposition disease are rare and their clinical presentations vary. We report one patient with histopathologic and clinical findings of a microangiopathic glomerulonephritis due to immunoglobulin A-kappa deposition. Ultrastructural studies revealed highly ordered deposits in the capillary lumen, mesangium, and basement membrane. The disease recurred at 2.5 years after a cadaveric kidney transplantation. Pulse steroid therapy was repeatedly effective in retarding further progression of renal deterioration in this patient.

Female↗

PubMed1995

Forced expression of chimeric human fibroblast tropomyosin mutants affects cytokinesis.

Human fibroblasts generate at least eight tropomyosin (TM) isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) from four distinct genes, and we have previously demonstrated that bacterially produced chimera hTM5/3 exhibits an unusually high affinity for actin filaments and a loss of the salt dependence typical for TM-actin binding (Novy, R.E., J. R. Sellers, L.-F. Liu, and J.J.-C. Lin, 1993. Cell Motil. & Cytoskeleton. 26: 248-261). To examine the functional consequences of expressing this mutant TM isoform in vivo, we have transfected CHO cells with the full-length cDNA for hTM5/3 and compared them to cells transfected with hTM3 and hTM5. Immunofluorescence microscopy reveals that stably transfected CHO cells incorporate force-expressed hTM3 and hTM5 into stress fibers with no significant effect on general cell morphology, microfilament organization or cytokinesis. In stable lines expressing hTM5/3, however, cell division is slow and sometimes incomplete. The doubling time and the incidence of multinucleate cells in the stable hTM5/3 lines roughly parallel expression levels. A closely related chimeric isoform hTM5/2, which differs only in the internal, alternatively spliced exon also produces defects in cytokinesis, suggesting that normal TM function may involve coordination between the amino and carboxy terminal regions. This coordination may be prevented in the chimeric mutants. As bacterially produced hTM5/3 and hTM5/2 can displace hTM3 and hTM5 from actin filaments in vitro, it is likely that CHO-expressed hTM5/3 and hTM5/2 can displace endogenous TMs to act dominantly in vivo. These results support a role for nonmuscle TM isoforms in the fine tuning of microfilament organization during cytokinesis. Additionally, we find that overexpression of TM does not stabilize endogenous microfilaments, rather, the hTM-expressing cells are actually more sensitive to cytochalasin B. This suggests that regulation of microfilament integrity in vivo requires stabilizing factors other than, or in addition to, TM.

Actin Cytoskeleton↗