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Biomedical subjects

W C Lin

Publications and source records attributed to W C Lin.

At least 109 records · Page 6Linked to original sources
PubMed1995

Effect of intracerebroventricular and intrahypothalamic administrations of picrotoxin on basal and stimulated gastric acid secretion.

The effects of intracerebroventricular and intrahypothalamic injections of picrotoxin, a GABA antagonist, on gastric acid secretion were studied in perfused stomachs of rats under anesthesia. Injection of picrotoxin into the lateral cerebroventricle inhibited the 2-deoxy-D-glucose-stimulated acid secretion. In experiments of intrahypothalamic injections, picrotoxin produced a significant depression of 2-deoxy-D-glucose-stimulated acid secretion when administered to the ventromedial hypothalamus but not to the lateral hypothalamus. In contrast, picrotoxin produced a definite stimulatory effect on basal acid secretion when injected to the lateral hypothalamus or ventromedial hypothalamus; the stimulatory effect of the injection to the lateral hypothalamus was greater than that of the injection to the ventromedial hypothalamus. These findings indicate that picrotoxin acts centrally, probably hypothalamus, to depress the 2-deoxy-D-glucose-stimulated acid secretion. On the other hand, blockade of GABA activity in the lateral hypothalamus or ventromedial hypothalamus may elicit gastric acid secretion. These results, indicate that central GABAergic mechanism is important in regulating gastric acid secretion in the rat.

Animals↗
PubMed1995

Relaxant effects of berberine on the rat fundus.

The present study examines the relaxant effect of berberine on the longitudinal muscle of the isolated rat gastric fundus. Our experiments show that berberine reduces the tonic contraction more than the phasic contraction induced by carbachol. In addition, the carbachol induced phasic contraction in Ca+2 -free solution was only inhibited at the highest concentration of berberine. Berberine inhibited the slow phase more than the fast phase in KCl-induced contractions. These results suggest that the major mechanism of berberine consists of an inhibition of calcium entry from extracellular in the second phase induced by both carbachol and KCl. Conversely, prevention of the mobilization of store Ca+2 in the phasic contraction phase induced by carbachol and prevention of the calcium entry from extracellular in first phase elicited by high levels of potassium play only a minor role in the dilatory effect of berberine.

Animals↗
PubMed1994

Three-dimensional imaging and image analysis of hippocampal neurons: confocal and digitally enhanced wide field microscopy.

The microscopy of biological specimens has traditionally been a two-dimensional imaging method for analyzing what are in reality three-dimensional (3-D) objects. This has been a major limitation of the application of one of science's most widely used tools. Nowhere has this limitation been more acute than in neurobiology, which is dominated by the necessity of understanding both large- and small-scale 3-D anatomy. Fortunately, recent advances in optical instrumentation and computational methods have provided the means for retrieving the third dimension, making full 3-D microscopic imaging possible. Optical designs have concentrated on the confocal imaging mode while computational methods have made 3-D imaging possible with wide field microscopes using deconvolution methods. This work presents a brief review of these methods, especially as applied to neurobiology, and data using both approaches. Specimens several hundred micrometers thick can be sampled allowing essentially intact neurons to be imaged. These neurons or selected components can be contrasted with either fluorescent, absorption, or reflection stains. Image analysis in 3-D is as important as visualization in 3-D. Automated methods of cell counting and analysis by nuclear detection as well as tracing of individual neurons are presented.

Animals↗
PubMed1994

Expression of interleukin 2 receptors on human carcinoma cell lines and tumor growth inhibition by interleukin 2.

We have previously shown that human squamous cell carcinomas (SCC) express the interleukin 2 receptor (IL2R)-alpha and -beta chains, and that the ligand, IL2, directly inhibits growth of the tumor in vitro and in vivo in the tumor xenograft-nude mice model. We now show that the alpha and beta chains of IL2R are expressed on a variety of human carcinoma cell lines and on normal human keratinocytes in early-stage cultures. While all carcinoma cells in a population expressed IL2R-alpha and -beta proteins, in keratinocytes obtained from different normal donors, variable proportions of cells were positive, as measured by flow cytometry. The carcinoma lines and 2/5 keratinocyte lines studied were also found to contain transcripts for the IL2R-gamma chain detectable by combined reverse transcription-PCR (RT-PCR) and hybridization with the specific cDNA probe. Incubation of the gastric (HR) or renal cell carcinoma (RCC) cell lines, but not of other IL2R+ carcinoma cell lines or normal keratinocytes, in the presence of IL2 resulted in dose-dependent inhibition of tumor cell growth. Monoclonal antibodies (MAbs) specific for IL2R-beta chain completely reversed this growth inhibitory effect of IL2. The ligand, IL2, also down-regulated surface expression of its own receptor and of intercellular adhesion molecule-I (ICAM-I) or class I major histocompatibility complex (MHC) antigens on IL2R+ tumor cells. All carcinoma cells studied incubated in the presence of IL2 exhibited significantly increased sensitivity to growth-inhibitory effects of other cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha or transforming growth factor (TGF)-beta. IL2 inhibited growth of the HR cells by arresting a significant proportion of tumor cells in the G0/G1 phase of the cell cycle. Thus, IL2 can have direct effects on IL2R+ carcinoma cells, leading to changes in growth or to increases in sensitivity of tumor cells to cytostatic activities of other cytokines.

Antigens, Neoplasm↗
PubMed1994

Signaling via CD7 molecules on human NK cells. Induction of tyrosine phosphorylation and beta 1 integrin-mediated adhesion to fibronectin.

We have previously reported that CD7 expressed on resting human NK cells is a signal-transducing molecule, which upon ligation with mAb induces a rapid increase in cytoplasmic free calcium, secretion of IFN-gamma, and augmented NK activity against K562 targets. We now demonstrate that Ab-mediated clustering of CD7 molecules on NK cells results in enhanced phosphorylation on tyrosine residues of intracellular proteins of 60, 70, 80, 97, and 120 kDa. In the presence of genistein, a specific inhibitor of protein tyrosine kinase, the enhanced level of tyrosine phosphorylation was blocked, indicating that CD7 may induce signaling via activation of tyrosine kinases. Cross-linking of CD7 or CD16 molecules with primary and secondary Abs, as well as stimulation of NK cells with phorbol ester (PMA) or with calcium ionophore A23187 also induced beta 1 integrin-mediated adhesion of these cells to fibronectin (FN)-coated plastic surfaces. In contrast, cross-linking of CD2 expressed on the surface of NK cells had no significant effect on NK cell adhesion to FN. This adhesion was not associated with up-regulation of expression of alpha 4 beta 1 or alpha 5 beta 1 FN receptors on NK cells, but it required an intact cytoskeleton. The CD7-induced adhesion to FN was mediated by alpha 4 beta 1 and alpha 5 beta 1 integrins, as it was partially blocked by FN connective segment-1 peptide (EILDVPST), the alpha 4 beta 1-binding domain, as well as by RGD-containing peptides, the alpha 5 beta 1-binding domain, but not by EILEVPST or RGE control peptides. NK cell binding to FN was also partially inhibited by mAb to alpha 4, alpha 5, and beta 1 integrins. The mechanism by which cross-linking of CD7 or CD16 on NK cells induced adhesion to FN appeared to involve both protein tyrosine kinase and protein kinase C, because this adhesion was blocked in the presence of either genistein or a protein kinase C inhibitor, staurosporin. Our data demonstrate that signals transduced via triggering of either CD7 or CD16 molecules are involved in the regulation of the functional activity of beta 1 integrins on NK cells.

Alkaloids↗
PubMed1994

Immunotherapy of liver metastases of human gastric carcinoma with interleukin 2-activated natural killer cells.

To better understand the events occurring during immunotherapy of liver metastases with effector cells, we have developed a clinically relevant animal model in which both effector-tumor cell interactions and survival can be evaluated. A cell line of human gastric carcinoma (HR) metastatic to the liver has been established from a patient's liver biopsy. HR cells (10 x 10(6)) injected intrasplenically metastasize into the liver of immunosuppressed nude mice, with micrometastases detectable histologically by day 4 and macrometastases by day 7. The animals subsequently develop ascites and die between days 30 and 40 after tumor injection. To investigate early metastatic events in the liver, HR cells were transduced with a plasmid containing both the lacZ gene under the control of the CMV promoter and NeoR gene. Transfectants selected for neomycin resistance were lacZ gene positive and stained blue in the presence of a beta-galactosidase substrate, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). These transfectants (HRLZ) remained lacZ gene positive for at least 25 passages in vitro. Injected intrasplenically, an HRLZ clone grew invasively in nude mice and formed liver metastases comparably to parental tumor cells. The number and localization of blue X-Gal-positive tumor cells were followed in liver tissues of animals sacrificed at various times, from 1 h to 28 days postinjection of HRLZ cells. HRLZ cells were seen in liver blood vessels and sinusoids within 1 h after injection, and the progressive growth of micrometastases and macrometastases could be followed with precision by X-Gal staining. On day 3 after injection of HRLZ cells, numerous micrometastases were established containing 12-16 tumor cells. When these 3-day established HRLZ micrometastases were treated by the intrasplenic infusion of interleukin 2 (IL2)-activated human natural killer (NK) cells selected by IL2-induced adherence to plastic (A-NK) and systemic IL2, nearly all liver micrometastases were eliminated within 24 h after a single transfer of A-NK cells (P < 0.001). This xenogeneic model was also used for adoptive immunotherapy of 7-day established liver macrometastases with human A-NK cells injected intrasplenically and exogenous IL2 given i.p. A significant decrease in the number of hepatic metastases and the weight of livers (P < 0.003) in comparison with those of control mice was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Cell cycle regulation of V(D)J recombination-activating protein RAG-2.

The antigen receptors of B and T lymphocytes are encoded in multiple germ-line DNA segments that are joined during lymphocyte development. The recombination-activating proteins RAG-1 and RAG-2 are both essential for this process, termed V(D)J rearrangement. Phosphorylation of the RAG-2 protein at Thr-490 by one or more cyclin-dependent kinases is associated with its rapid degradation. In an immature B-cell line and in normal thymocytes, RAG-2 protein accumulates preferentially in the G0/G1 phases of the cell cycle and declines by at least 20-fold before cells enter S phase. The amount of RAG-2 protein remains low throughout the S, G2, and M phases. The amount of RAG-1 protein shows considerably less fluctuation. The variation in RAG-2 protein is likely to be established, at least in part, by a posttranscriptional mechanism. These observations suggest that V(D)J rearrangement occurs entirely or preferentially within G0/G1.

Animals↗
PubMed1994

Proliferation of hematopoietic cell lines induced by a soluble factor derived from human squamous cell carcinomas of the head and neck.

The supernatant of a cell line of squamous cell carcinoma of the head and neck (SCCHN), PCI-50, was previously shown to induce activation, promote proliferation and increase antitumor cytotoxicity of freshly purified human natural killer (NK) cells and CD4+ T lymphocytes [Arch Otolaryngol Head Neck Surg (1994) in press]. This supernatant was found also to promote the growth of a variety of hematopoietic cell lines, including Jurkat, THP-1, K562, NK-92 or Epstein-Barr-virus-transformed B cell lines. The Jurkat cell line was selected as a reporter cell in an 18-h proliferation assay established to measure the growth-promoting activity of PCI-50 supernatant. The presence of soluble tumor-derived factors able to induce proliferation of Jurkat cells was demonstrated in the supernatant produced by several other SCCHN cell lines but not in that produced by a gastric cancer cell line (HR) or renal cell carcinoma line (5117G8). The growth-promoting PCI-50 supernatant was shown to contain 28 +/- 0.5 pg/ml interleukin-6 (IL-6) in vitro but was negative for interferon gamma, IL-1, IL-2, IL-4, tumor necrosis factor alpha, granulocyte/macrophage-colony-stimulating factor and IL-12. The addition of any of these recombinant cytokines to Jurkat cell cultures did not significantly promote growth, while PCI-50 supernatant was consistently growth-stimulatory. This supernatant neither enhanced intracellular Ca2+ concentration in Jurkat cells nor induced up-regulation of activation antigens on the cell surface, although it supported growth of Jurkat cells in the absence of IL-2. The growth-promoting activity in the PCI-50 supernatant was acid-labile at pH 2 for 4 h, heat-resistant at 96 degrees C for 1 h and sensitive to treatments with trypsin and pepsin. Preincubation of the PCI-50 producer cells with tunicamycin or cyclohexamide reduced the level of growth-promoting activity in the supernatant. A partial purification of this activity was achieved using Amicon filtration, chromatography on concanavalin-A-Sepharose and then a hydroxyapatite column and high-pressure liquid chromatography gel filtration. The partially purified glycoprotein had a molecular mass of 50-70 kDa, as determined by gel filtration.

Carcinoma, Squamous Cell↗
PubMed1994

The role of gastric muscle relaxation in cytoprotection induced by san-huang-xie-xin-tang in rats.

We examined the mechanism of the protective effect of San-huang-xie-xin-tang (SHXT), a traditional oriental drug, on the gastric mucosa. SHXT, given intraduodenally, inhibited the gastric secretion in pylorus-ligated rats. SHXT, given orally 30 min before the administration of aspirin, HCl-aspirin and ethanol, protected the gastric mucosa from these agent-induced gastric lesions, although it had no effect on the gastric lesions induced by indomethacin and water immersion stress. Oral administration of SHXT increased gastric contents (muscle relaxation) when they were measured 30 min after SHXT administration. SHXT also inhibited carbachol-contracted gastric muscle in vitro. Neither increased gastric contents nor inhibited ethanol lesions of SHXT were observed when these were tested 4 h after SHXT administration. Pretreatment with indomethacin which is a prostaglandin synthesis inhibitor, and N-ethylmaleimide which is a sulfhydryl blocker did not influence either the increased gastric contents or inhibited ethanol lesions of SHXT. These results indicate that gastric muscle relaxation plays an important role in the gastric protective mechanisms of SHXT, and the endogenous prostaglandins and the sulfhydryl compounds are not necessary for the action of SHXT.

Administration, Oral↗
PubMed1994

Mechanisms of gastric antisecretory action of pentylenetetrazol in rats.

The present study investigated the mechanisms of gastric antisecretory action of pentylenetetrazol (PTZ) in pylorus-ligated rats. Pretreatment with indomethacin, phentolamine, propranolol, 6-hydroxydopamine, flunarizine and nifedipine, which were reported to antagonize seizures induced by PTZ, did not influence the gastric antisecretory action of PTZ. In contrast, pentobarbital and phenobarbital reverse the action of PTZ. These results suggest that prostaglandins production, adrenoceptors and calcium entry are not involved in the gastric antisecretory action of PTZ, and that the GABAA receptor complex plays an important role in the action induced by PTZ.

Animals↗
PubMed1993

Design and synthesis of RNA miniduplexes via a synthetic linker approach. 2. Generation of covalently closed, double-stranded cyclic HIV-1 TAR RNA analogs with high Tat-binding affinity.

We recently developed an approach which allows rapid generation of short, double-stranded oligonucleotides whereby one end of the duplex was joined and stabilized by a synthetic linker of specific design (miniduplexes)(6). Model miniduplexes based on the HIV-1 TAR RNA hairpin were shown to be thermodynamically stable and good substrates for binding by the HIV-1 Tat protein which normally bind to natural TAR (6). In this study, we have extended our studies to the design, synthesis and analysis of the binding properties of covalently closed, double-stranded, cyclic RNA miniduplexes. A strategy using automated chemical synthesis and T4 RNA ligase-catalyzed cyclization was employed to generate cyclic oligoribonucleotides. When both ends of a shortened, wild-type TAR RNA stem (9 bp) were covalently linked through either nucleotidic loops (4-6 nt) or synthetic linkers (derivatized from hexaethylene glycol), the resulting cyclic TAR RNA analogs were good substrates for binding by both Tat-derived peptide or full-length Tat protein. Interestingly, the cyclic TAR analogs failed to show any binding if the synthetic linker was reduced in length (e.g. derivatized from triethylene glycol), although such linkers are acceptable in the hairpin-shaped miniduplexes series (6). This implies that RNA conformational changes are required for Tat binding and that these changes are restricted in certain cyclic variants. Our findings suggest that covalently-closed nucleic acid miniduplexes may be useful both to study nucleic acid-protein interactions as well as to provide a basis for therapeutic intervention as transcription decoys.

Amino Acid Sequence↗
PubMed1993

Regulation of V(D)J recombination activator protein RAG-2 by phosphorylation.

Antigen receptor genes are assembled by site-specific DNA rearrangement. The recombination activator genes RAG-1 and RAG-2 are essential for this process, termed V(D)J rearrangement. The activity and stability of the RAG-2 protein have now been shown to be regulated by phosphorylation. In fibroblasts RAG-2 was phosphorylated predominantly at two serine residues, one of which affected RAG-2 activity in vivo. The threonine at residue 490 was phosphorylated by p34cdc2 kinase in vitro; phosphorylation at this site in vivo was associated with rapid degradation of RAG-2. Instability was transferred to chimeric proteins by a 90-residue portion of RAG-2. Mutation of the p34cdc2 phosphorylation site of the tumor suppressor protein p53 conferred a similar phenotype, suggesting that this association between phosphorylation and degradation is a general mechanism.

3T3 Cells↗
PubMed1993

Design and synthesis of RNA miniduplexes via a synthetic linker approach.

Double-stranded oligodeoxyribonucleotides or single-stranded oligoribonucleotides with specific secondary structure have been proposed as potential antagonists to target nucleic acid-binding proteins (the sense approach). A major limitation of this strategy is that these derivatives are generally considered to be too large for pharmaceutical applications. We have developed a synthetic linker approach whereby nucleic acid duplexes of a much smaller size (miniduplexes) can be generated directly from a standard oligonucleotide synthesis. In this approach, four synthetic linkers (derivatized respectively from 1,9-nonanediol, triethylene glycol, 1,3-propanediol, and hexaethylene glycol) of different length and hydrophobicity were designed and incorporated into a model RNA molecule based on the TAR stem-loop structure of HIV-1. Their thermal stabilities were evaluated by measuring denaturation profiles (Tm measurements). These linker-derivatized RNA molecules were then assessed for their ability to bind to either a full-length protein (HIV-1 Tat protein) or a short peptide (Tat-derived peptide) through RNA mobility shift assays. Results from this study indicate that such modified miniduplex structures retain full binding activity relative to that of the wild-type sequence (Kd values), while Tm values were increased by 24-31 degrees C compared to an open duplex of the same length. This system provides a new direction in the use of nucleic acid miniduplexes as a novel class of oligonucleotide analogues for both fundamental research and possible therapeutic applications.

Amino Acid Sequence↗
PubMed1993

Transfer of interleukin 2 receptor genes into squamous cell carcinoma. Modification of tumor cell growth.

OBJECTIVE: Human squamous cell carcinomas of the head and neck (SCCHN) have been shown to express interleukin 2 receptor (IL-2R), and binding of the ligand, IL-2, to the receptor results in tumor growth inhibition in vitro or in vivo in an SCCHN xenograft model in nude mice. To optimize growth inhibitory effects of IL-2, expression of the alpha or gamma chains of IL-2R in SCCHN was experimentally modified by transfection of tumor cells with the respective IL-2R genes or the lacZ gene as control. DESIGN: Using plasmid vectors containing the IL-2R alpha chain gene under the control of a cytomegalovirus promoter or the IL-2R gamma chain gene under the control of a Rous sarcoma virus promoter, the IL-2R genes were transferred by lipofection into SCCHN cell lines. Stable transfectants were selected, cloned by limiting dilution, and clones were compared with the parental cell lines for their sensitivity to the growth-inhibitory effect of IL-2. RESULTS: Transfer of the IL-2R alpha chain gene into SCCHN cells resulted in significant upregulation of expression of the IL-2R alpha chain on tumor cell surface but not in increased tumor growth inhibition by IL-2. In contrast, SCCHN IL-2R gamma transfectants, which expressed IL-2R gamma chain transcripts as confirmed in RNase protection assays, were significantly inhibited in growth and were sensitive to lower concentrations of IL-2 than the parental cell lines. CONCLUSIONS: Genetic modification of IL-2R expression on IL-2R-positive tumor cells in culture significantly alters their proliferative response to IL-2. These observations open a way for developing new strategies for therapy of SCCHN based on direct interactions of IL-2 with its receptor on tumor cells.

Carcinoma, Squamous Cell↗
PubMed1993

Complementation of two related tumour cell classes during experimental metastasis tagged with different histochemical marker genes.

Intercellular complementation during tumour development and metastasis was analysed for two different oncogene (ras or sis) transformants of Balb/c 3T3 cells, tagged with different histochemical marker genes (lacZ or ALP to generate LZEJ or APSI cells, respectively), by localising them after their co-injection with specific double-staining protocols. This model evaluates whether limited progression of each tumour class can be facilitated reciprocally during co-localisation and co-growth in nude mice by taking advantage of the sensitivity of the histochemical marker genes for localising them. After intravenous co-injection of equal numbers of both cells to analyse experimental metastasis, most foci transiently established in the lung for several hours were comprised of only one cell class. However, a significant fraction of foci contained both cell types, as identified in double-stained whole-lung tissues and in lung sections. Evidence was obtained that LZEJ cells increase the survivability and subsequent growth of APSI-containing micrometastases during co-localisation in lung, when compared to APSI cells injected alone. Conversely, APSI cells facilitate expansion of LZEJ cells from micrometastatic foci into overt-metastatic nodules in the lung. These analyses reveal reciprocity during experimental metastasis by two related tumour cell classes derived from the same parental cell.

3T3 Cells↗
PubMed1993

A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination.

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.

Animals↗
PubMed1992

Receptors for interleukin 2 on human squamous cell carcinoma cell lines and tumor in situ.

Several human head and neck squamous carcinoma cell lines were found to bind 125I-labeled or fluorescein-labeled interleukin 2 (IL-2). This binding was inhibited by an excess of cold ligand, IL-2, and by anti-p55 and anti-p70 monoclonal antibodies to the alpha and beta chains, respectively, of the IL-2 receptor (IL-2R). A small number (300/cell) of high-affinity IL-2R (2 x 10(-12) M) and a larger number (> 13,000/cells) of intermediate-affinity IL-2R (3 x 10(-10) M) were present on these tumor cells. By affinity cross-linking, tumor cells were shown to bind 125I-IL-2 to a M(r) 66,000 and 55,000 doublet peptide. The alpha and beta chains of the IL-2R also were detected on the surface of cultured tumor cells using the relevant monoclonal antibodies and flow cytometry. Immunoperoxidase staining with anti-p70 monoclonal antibody confirmed the expression of IL-2R on squamous cell carcinomas of the head and neck in situ. The presence of transcripts for p55/IL-2R-alpha and p70/IL-2R-beta in PCI-1 cells was confirmed by the polymerase chain reaction followed by hybridization to the IL-2R-alpha complementary DNA probe or IL-2R-beta complementary DNA probe, respectively. Our observations demonstrate that intermediate-affinity and high-affinity IL-2Rs are expressed on some human squamous cell carcinomas of the head and neck and that the receptors are functional, because growth of these tumor cell lines can be directly inhibited by exogenously supplied IL-2. The presence of IL-2R on human solid tumors could be important to consider, in addition to immunomodulatory effects of IL-2, in developing optimal therapeutic strategies for the administration of IL-2 to patients with cancer.

Antibodies, Monoclonal↗