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Biomedical subjects

C T Lin

Publications and source records attributed to C T Lin.

At least 19 recordsLinked to original sources
PubMed1998

BCECF in single cultured cells: inhomogeneous distribution but homogeneous response.

Using confocal laser scanning microscopy with a dual-wavelength laser system, the behaviour of BCECF [(2',7'-bis-2-carboxyethyl)-5-(and-6)carboxyfluorescein] was investigated in a variety of cell lines. Selection of a small area for monitoring allowed discrimination between various intracellular organelles, whose identity was established by vital staining. It was found that, after loading the cells with BCECF, both the nucleus and the mitochondria showed a higher level of fluorescence than the cytoplasm. Calibration of the pH-sensitivity of these fluorescence signals using the nigericin method yielded identical curves, as did exposure of the cells to NH4Cl. These studies suggest that BCECF, despite its inhomogeneous intracellular distribution, reports the pH of only one cellular compartment, the cytosol.

Ammonium Chloride↗
PubMed1997

A method for generating subtractive cDNA libraries retaining clones containing repetitive elements.

Here we describe a two-stepped photobiotin-based procedure to enrich a target (canine retinal) cDNA library for tissue specific clones without removing those containing repetitive ( SINE ) elements, despite the presence of these elements in the driver population. In a first hybridization excess SINE elements were hybridized to a driver (canine cerebellar) cDNA. In a second hybridization target cDNA was added to this reaction. The resulting cDNA library was enriched for retinal specific clones, but contained the same ratio of clones with SINE elements found in the unsubtracted library.

Animals↗
PubMed1997

A cruciform-dumbbell model for inverted dimer formation mediated by inverted repeats.

Small inverted repeats (small palindromes) on plasmids have been shown to mediate a recombinational rearrangement event in Escherichia coli leading to the formation of inverted dimers (giant palindromes). This recombinational rearrangement event is efficient and independent of RecA and RecBCD. In this report, we propose a cruciform-dumbbell model to explain the inverted dimer formation mediated by inverted repeats. In this model, the inverted repeats promote the formation of a DNA cruciform which is processed by an endonuclease into a linear DNA with two hairpin loops at its ends. Upon DNA replication, this linear dumbbell-like DNA is then converted to the inverted dimer. In support of this model, linear dumbbell DNA molecules with unidirectional origin of DNA replication (ColE1 ori ) have been constructed and shown to transform E.coli efficiently resulting in the formation of the inverted dimer. The ability of linear dumbbell DNA to transform E.coli suggests that the terminal loops may be important in bypassing the requirement of DNA supercoiling for initiation of replication of the ColE1 ori.

DNA↗
PubMed1997

Regulation of the long terminal repeat in visna virus by a transcription factor related to the AML/PEBP2/CBF superfamily.

The long terminal repeats of maedi visna virus strain 1514 contain a consensus AP-1 binding site which has been shown to be important in controlling virus transcription. However, this consensus site is absent in strain EV-1. Here, we have compared the ability of oligonucleotides corresponding to LTR sequences from EV-1 with those from 1514 to bind transcription factors in competitive gel retardation assays and activate reporter gene expression. The experiments demonstrated no observable binding of AP-1 to the EV-1-derived sequences and significant differences in the abilities of the 1514 and EV-1 sequences to activate transcription. However, both viral sequences interacted with a second, previously undetected, transcription factor. This factor gave specific gel shifts which were competed by an oligonucleotide containing the consensus sequence for the AML/PEBP2/CBF family of transcriptional factors, but not by control AP-1 or OCT-1 oligonucleotides. The factor was therefore denoted AML (vis). A second AML (vis) site, noted upstream of the TATA box proximal AP-1 site, gave single shifts which were competed by the downstream AML (vis) oligonucleotide. Both sites were functional in transfection assays. In gel shift retardation assays, polyclonal antisera directed against known runt domain proteins were able to supershift part of the AML (vis) binding activity in nuclear extracts from physiologically relevant cell types. The results thus suggest that the AML (vis) binding factor belongs to the AML/PEBP2/CBF family of transcription factors and may be important in controlling virus replication in these and other strains of ruminant lentiviruses.

Animals↗
PubMed1997

Co-localization of endogenous and exogenous p53 proteins in nasopharyngeal carcinoma cells.

Recently, we have established nine nasopharyngeal carcinoma (NPC) cell lines in which only one cell line showed the p53 mutation. For investigation of the p53 mutation in this line, immunostaining using anti-p53 antibody was applied and showed the presence of p53 protein in the cytoplasm but not in the nucleus. Single strand conformation polymorphism analysis of the p53 gene showed one normal and one additional DNA band. Cloning and sequencing of PCR-amplified DNA showed an AGA (arginine) to ACA (threonine) heterozygous point mutation at codon 280. Transfection of the p53 DNA binding sequence and chloramphenicol acetyltransferase assay revealed loss of transcriptional activation function of endogenous p53 protein. Co-localization of the endogenous and the transfected exogenous p53 protein by polyclonal antibodies to anti-p53 protein revealed strong exogenous p53 staining in the transfected nuclei and weak staining of endogenous p53 protein in the cytoplasm. We concluded that (a) a heterozygous point mutation at codon 280 was identified in the NPC-TW 06 cell line; (b) the point mutation may cause the stagnation of mutant p53 protein in the cytoplasm, and loss of its transcriptional activation function; (c) endogenous and exogenous p53 protein can be co-localized at the same time in the transfected cells; and (d) 280 mutant p53 protein in NPC cells does not cause a decrease or increase in sensitivity to chemotherapy.

Biopsy↗
PubMed1997

The mechanism of Epstein-Barr virus infection in nasopharyngeal carcinoma cells.

To investigate the relationship between Epstein-Barr virus (EBV) and nasopharyngeal carcinoma (NPC) cells, we examined the pathway of EBV infection in NPC cell lines. We used immunolocalization to investigate the EBV receptor (C3d-R) and polymeric immunoglobulin receptor [secretory component (SC) protein]. We incubated IgA anti-EBV and EBV particles with NPC cells and observed the EBV DNA signal by in situ polymerase chain reaction hybridization and polymerase chain reaction plus Southern blotting. We also colocalized SC protein and EBV RNA in NPC biopsy specimens. Results showed that: 1) NPC cells did not express the EBV receptor but did express SC protein in each line; 2) SC protein was also expressed in some tumor cells but not in untransformed squamous metaplastic epithelia in NPC biopsy specimens; 3) EBV could infect NPC cells through an EBV-IgA and SC complex and retained an EBV viral genome in their nuclei; SC expression could be down-regulated by EBV proteins; and 4) in biopsy specimens, a fraction of tumor cells showed SC protein expression; only a portion of tumor cells contained EBV, and of these cells only a few expressed SC protein. These findings indicate that EBV cannot infect untransformed nasopharyngeal squamous metaplastic epithelia but can enter NPC cells through IgA-mediated endocytosis.

Carcinoma↗
PubMed1997

Phrenic nerve transfer in the repair of brachial plexus injuries: an animal model.

UNLABELLED: Ten young mongrel dogs underwent unilateral denervation of the brachial plexus. In six dogs, a 2-cm segment of phrenic nerve autograft was sutured to either the resected musculocutaneous nerve or the radial nerve. A hemoclip was applied to either musculocutaneous or radial nerve in the control groups. Five months postoperatively, the grafted musculocutaneous nerve demonstrated less fibrous tissue and less muscle atrophy of the biceps when compared to the control group with clipped nerve. In the group with the grafted radial nerve, the electromyographic findings of multiphasic action potential and muscle contraction from electric stimulation suggested reinnervation of the radial nerve. IN CONCLUSION: phrenic nerve transfer may be used to repair specific damages to nerve trunk with histological, electromyographic and clinical recovery.

Animals↗
PubMed1997

Clonal versus polyclonal Epstein-Barr virus infection in nasopharyngeal carcinoma cell lines.

In most nasopharyngeal carcinoma (NPC) biopsy specimens, the Epstein-Barr virus (EBV), particularly in the terminal repeat region genomic structure, reveals a clonal pattern. To evaluate this phenomenon in vitro, we infected EBV-negative NPC cell lines, which express secretory component (SC) protein on their cell surface, with EBV particles. The viral particles were obtained either from a subcloned single cell or from the original B95-8 cell line. EBV infection was performed by incubating IgA anti-EBV and EBV particles with NPC cells and confirmed by direct in situ PCR hybridization. Southern blot analysis of EBV terminal repeat in EBV-infected NPC cell lines was performed using a Xhol 1.9-kb DNA fragment from the right terminus of the EBV genome as a probe. We found that all four NPC cell lines (ie, NPC-TWO1; 03, 04, and 06) expressed SC protein on their surfaces and could be infected by EBV through the EBV IgA-SC complex. Southern blot analysis in the single cell-subcloned B95-8 cell line showed a clonal EBV terminal repeat with a higher molecular size; whereas the original B95-8 line revealed the polyclonal EBV DNA pattern. A similar clonal EBV genomic pattern with lower molecular size was seen in all EBV-infected NPC cell lines. For comparison, six NPC biopsy specimens were also examined; of these, five showed a single band, and the remaining showed one major band and several lower molecular-sized bands. The EBV genomic DNA in the infected cells was shown to be an episomal form. We conclude, therefore, that a single (clonal) form of EBV genome can be obtained from a mixed population of epithelial tumor cells, even when they are infected by multiple virions with single or multiple form(s) of the EBV genomic pattern.

Animals↗
PubMed1996

A system for the analysis of seat support surfaces using surface shape control and simultaneous measurement of applied pressures.

A system for the design and analysis of seat support and buttock tissue interfaces has been developed. It has the ability to control the seating surface shape while measuring the pressure applied to the buttocks by the surface. Pressures are measured over an 11 x 12 rectangular array of support elements using silicon pressure sensors mounted in a swiveling head atop each support element. Control of surface shape is mediated by selective linear translation of the support elements along their respective vertical axes. Closed-loop control of the system allows for the dynamic formulation of a support surface on the basis of programmable criteria. The system is intended to function as a research tool to facilitate the study of the relationships between support surface shape and interface pressure, and support surface shape and soft tissue distortion. The purpose of this paper is to present the system instrumentation and the rationale behind its design and development. The paper also presents the results of several tests to evaluate the accuracy and performance of the system. This evaluation included a pilot study on 10 able-bodied subjects. The results of these system evaluations indicate that the system is capable of making repeatable and precise measurements of pressure and surface element position and can formulate support surface shapes that satisfy specified optimization criteria.

Back↗
PubMed1996

Canine infectious cyclic thrombocytopenia found in Taiwan.

Here were report the first canine infectious cyclic thrombocytopenia (CICT) found in Taiwan. Platelet-specific inclusions were detected in the blood smear of a military working dog. To identify the etiologic agent, the patient's blood was transmitted to three six-month-old German Shepherd dogs. The Ehrlichia platys-like inclusions were observed six to eight days after inoculation. Indirect fluorescent antibody test showed that the serum from the patient reacted specifically with the microorganisms within the platelets. Typical hematologic manifestations of E. platys infection, cyclic parasitemia and concomitant thrombocytopenia, were observed in these dogs. The prevalence of CICT in north Taiwan was also studied, and the incidence was 8.9% (4 out of 45) in civilian dogs and 97.1% (34 out of 35) in dogs from a heavily tick infested kennel.

Animals↗
PubMed1995

Lateral mobility of Fc gamma RIIa is reduced by protein kinase C activation.

The lateral mobility of membrane proteins can reflect the extent of various protein-protein interactions. Using the fluorescence recovery after photobleaching technique, we have studied the lateral mobility of human Fc gamma RIIa and some Fc gamma RIIa mutants expressed in either P388D1 cells, a mouse macrophage-like cell line, or in Chinese hamster ovary (CHO) cells [1]. After treatment with phorbol myristate acetate (PMA), only the Fc gamma RIIa molecules capable of mediating rapid endocytosis of immune complexes exhibited a reduced lateral diffusion coefficient with respect to untreated controls. Wild type Fc gamma RIIa expressed in CHO cells, and nonfunctional Fc gamma RIIa mutants expressed in P388D1 cells did not show any differences upon PMA treatment. This finding suggests that protein kinase C activation evokes additional protein-protein interactions with the cytoplasmic domain of functional Fc gamma RIIa, which reduced receptor lateral mobility. The identity of these putative interacting proteins and the nature of the interactions remain to be elucidated.

Animals↗
PubMed1995

A movable and regulable inactivation function within the central region of a temperature-sensitive p53 mutant.

p53 is the most frequently mutated gene in human cancer. Naturally occurring mutations of p53 are mainly located within a region containing residues 100-300 and are predominantly of missense type, resulting in loss of the protein's DNA binding activity. Here we show that this type of mutation also represses the p53 N-terminal activation domain. The repression activity is localized in the central region of mutant p53 containing residues 101-318. Interestingly, the central region of a temperature-sensitive mutant p53N247I possesses a movable and regulable inactivation function. It represses other activities present on the same polypeptide chain without strict regard to the configuration of that polypeptide only at the nonpermissive temperature (37 degrees C) and not at the permissive temperature (30 degrees C). Furthermore, this mutant p53 region exhibits no other activity, and its function is independent of endogenous p53 status.

Animals↗
PubMed1995

The Ca2+ dependence of human Fc gamma receptor-initiated phagocytosis.

Differing roles for [Ca2+]i transients in Fc gamma R-mediated phagocytosis have been suggested based on the observations that antibody-opsonized erythrocyte phagocytosis by human neutrophils shows a [Ca2+]i dependence, while that by murine macrophages appears [Ca2+]i-independent. To explore whether this difference might reflect different receptor isoforms or different cell types, we studied the [Ca2+]i dependence of receptor-initiated phagocytosis by human Fc gamma RIIa and a panel of Fc gamma RIIa cytoplasmic domain mutants expressed in murine P388D1 cells and by human Fc gamma R endogenously expressed on human neutrophils and monocytes. Wild-type and point mutants of huFc gamma RIIa stably transfected into murine P388D1 cells have different capacities to initiate a [Ca2+]i transient, which are closely correlated with quantitative phagocytosis (r = 0.94, p < 0.0001). Phagocytosis both by huFc gamma RIIa in P388D1 cells and by huFc gamma RIIa endogenously expressed on neutrophils and blood monocytes shows [Ca2+]i dependence. Phagocytosis of antibody-opsonized erythrocytes by neutrophils demonstrated greater susceptibility to [Ca2+]i quenching compared with Fc gamma RIIa-specific internalization with E-IV.3, suggesting that the phagocytosis activating property of Fc gamma RIIIb in neutrophils also engages a [Ca2+]i-dependent element. In contrast, phagocytosis by human Fc gamma RIa, endogenously expressed on blood monocytes, is [Ca2+]i-independent. Despite the importance of a consensus tyrosine activation motif for both receptors, Fc gamma RIa and Fc gamma RIIa engage at least some distinct signaling elements to initiate phagocytosis. The recognition that both of the phagocytic receptors on murine macrophages and human Fc gamma RIa associate with the Fc epsilon RI gamma-chain, which contains a tyrosine activation motif distinct from that in the Fc gamma RIIa cytoplasmic domain, suggests that [Ca2+]i-independent phagocytosis is a property associated with the utilization of gamma-chains by Fc gamma R.

Amino Acid Sequence↗
PubMed1995

PLS analysis of distance matrices to detect nonlinear relationships between biological potency and molecular properties.

Although the statistical method of partial least squares (PLS) is widely used for the analysis of the relationship between molecular properties and biological potency, it is recognized that PLS detects only linear relationships. We tested two types of properties: simulated univariate data and electrostatic molecular field as a function of Hammett sigma constants. In both cases we compared relationships in which the function is linear, asymptotic, or rises to an optimum and then falls. We found that PLS analysis of the matrix of the distances between every pair of compounds detects all three types of relationships with the same quality of cross-validation. The successful application of the method requires that the distance matrices be constructed such that each contains only information about one property (for example, the electrostatic field around the functional group of interest). Carbo and Hodgkin similarities perform less well than distances.

Models, Chemical↗
PubMed1995

Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method.

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 degrees C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant kd calculated for the monomer at 85 degrees C was 0.029 min-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 degrees C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 degrees C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.

Base Sequence↗