Biomedical subjects
Publications and source records attributed to M C Chen.
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Fluorescence in situ hybridization (FISH), Southern, and slot blotting were used to detect Epstein-Barr virus (EBV) DNA and RNA sequences in a Burkitt's lymphoma (BL) cell line derived from a North American patient (NAB-2). FISH analysis after hybridization with a BamHI "V" region of EBV showed that NAB-2 cells have EBV genome integrated at a single site on the short arm of chromosome 2(p13). Single hybridization signals were detected at homologous sites on both chromatoids and nuclei. Furthermore, hybridization of intact nuclei without formamide denaturation and heat allowed the detection of single specific viral RNA transcripts visible as "tracks" or "traces." Southern blot analysis confirmed the integration of EBV genome into the host DNA. Quantification of slot blot hybridization revealed that NAB-2 cells have on average one copy of EBV per cell. Virus insertion into chromosomal DNA caused a stable modification site expressed as a distinctive achromatic region adjacent to the band 2p13. The chromatid lesion at the site of EBV integration involving a recombinogenic and fragile site may have contributed to the development of the NAB-2 BL.
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Studying primary cultures of replicating canine oxyntic mucosal cells, we found evidence for modulation of cell growth by endogenous factors. [3H]thymidine incorporation into DNA was rapid with cells cultured in medium free of serum or added growth factors, and growth rates of these cultures were markedly dependent on plating density, indicating mitogenic control by soluble endogenous growth factors. Data indicated that endogenous transforming growth factor-alpha (TGF-alpha) exerted mitogenic control under the following conditions. 1) TGF-alpha was detected in the cultured cells by radioimmunoassay and immunohistochemistry. 2) TGF-alpha-like immunoreactivity and receptor reactivity were present in the medium in concentrations sufficient to exert mitogenic control. 3) Receptors for TGF-alpha and epidermal growth factor (EGF) were present in the cultures. 4) Immunoabsorption by a TGF-alpha-specific antisera reduced [3H]thymidine incorporation. TGF-alpha was localized to parietal cells by immunohistochemistry and cell separation. In contrast, combined [3H]thymidine autoradiography and immunohistochemistry with anti-TGF-alpha did not detect TGF-alpha in dividing cells. We conclude that parietal cell TGF-alpha exerts paracrine control of mucosal cell growth in vitro, and we speculate that this is an important paracrine mechanism in vivo.
To overcome the problem of poor mixing of gastrointestinal tract contents with a diluted iodinated contrast agent in abdominal computed tomography, pure water was adopted as an oral contrast agent. In 25 cases of clinically suspected gastric carcinoma, a subsequent pathological examination revealed six cases of early gastric cancer and 19 cases of advanced gastric cancer. We performed CT of the stomach using the following revised procedure: patients were given 600-1000 mL of water by mouth after an intramuscular dose of Buscopan to distend and immobilize the stomach. Gastric mucosal enhancement and the poorer enhanced submucosal layer were demonstrated by a bolus intravenous injection of iodine-containing contrast medium using an automatic injector synchronized with the CT machine. Based on abnormal gastric wall thickening and the abnormal mural enhancement patterns, an accuracy of 96% was attained in differentiating early gastric cancer from advanced gastric cancer, but the detection rates for extragastric invasion, gastric ulcers and lymphadenopathy were 67%, 43% and 63%, respectively. CT staging of gastric cancer was 72%, and was especially accurate for stages I and IV. Water as oral contrast agent for CT of the GI tract was readily accepted by patients and caused no side effects. Using water as an oral contrast for gastric CT is of great help in staging gastric cancer.
Transcriptional analysis of the promoter of rice psaA-psaB-rps14 operon was performed with Bal 31 deleted mutants in spinach chloroplast extracts. Two functional promoters in this operon (denoted as "-175" and "-129" promoters) were revealed to be utilized by the spinach transcription machinery; however only the "-175" promoter is utilized in vivo. The existence of psaA promoter-specific binding factors in the chloroplast extracts was indicated by competitive gel retardation assay and exonuclease III protection analysis. After ammonium sulfate fractionation of the extracts followed by heparin-agarose column chromatography, the sequence-specific binding factors were characterized to be 66-kDa and 31-kDa polypeptides. Southwestern analysis demonstrated that the two protein factors interact independently with the psaA promoter-containing DNA fragment.
We investigated both sodium-lithium countertransport (Na-Li CT) and ouabain-sensitive sodium transport (Na pump) of erythrocytes in healthy subjects (group A), patients with non-insulin-dependent diabetes (NIDDM) without nephropathy (group B), patients in the proteinuric stage (group C), and those in the renal insufficiency stage (group D). Erythrocytes from all four groups had a similar initial water and ionic content and were loaded with similar degrees of Li and Na for efflux studies. There were no significant differences in erythrocyte Na-Li CT or Na pump among the four groups. However, the maximal rate of Na-Li CT was significantly higher in a group of subjects with essential hypertension when compared with groups A, B and C, consistent with the view that there is a genetic marker for essential hypertension. Ouabain-insensitive Na efflux (Na leak) of erythrocytes was found to be significantly higher in group D than in groups A or B. Also, a significant positive correlation existed between Na leak and urine protein levels of the subjects studied. Our results thus indicate that in contrast with insulin-dependent diabetic patients (IDDM) where an elevated Na-Li CT is observed, with diabetic nephropathy, Na-Li CT in NIDDM is apparently not associated with nephropathy; rather the ouabain-insensitive Na efflux appears to be correlated with the stages of nephropathy in NIDDM. The association suggests that the rate of ouabain-insensitive Na efflux may provide an index for assessing the degree of nephropathy in NIDDM patients.
We have described a a 23-year-old black woman with sickle cell beta-thalassemia who had a urinary tract infection and who was incidentally found to be pancytopenic. Although her anemia was categorized as "normocytic, normochromic" by an electronic particle counter, evaluation of the pancytopenia confirmed unsuspected pernicious anemia. Greater vigilance and a higher index of suspicion are crucial for early diagnosis of pernicious anemia in patients with other known hemoglobinopathies.
The complete nucleotide sequence of the RNA genome of papaya ringspot virus (PRSV) was determined from four overlapping cDNA clones and by direct sequencing of viral RNA. The genomic RNA is 10326 nucleotides in length, excluding the poly(A) tract, and contains one large open reading frame that starts at nucleotide positions 86 to 88 and ends at positions 10118 to 10120, encoding a polyprotein of 3344 amino acids. The highly conserved sequence AAAUAAAANANCUCAACACAACAUA at the 5' end of the RNA of PRSV and those of the other five reported potyviruses shows 80% similarity, suggesting that this region may play a common important role for potyvirus replication. Two cleavage sites of the polyprotein were determined by amino acid sequencing of the N termini of helper component (HC-Pro, amorphous inclusion) and cylindrical inclusion (CI) proteins. Other cleavage sites were predicted by analogy with the other potyviruses. The genetic organization of PRSV is similar to that of the other potyviruses except that the first protein processed from the N terminus of the polyprotein (NT protein) has an M(r) of 63K, 18K to 34K larger than those of the other potyviruses. The cleavage site for liberating the N terminus of the HC-Pro protein was found at the same location down-stream from the consensus sequence FI(V)VRG as that reported for tobacco vein mottling virus. The NT protein of potyviruses is the most variable and may be considered important for identification of individual potyviruses. The most conserved protein of potyviruses appears to be the NIb protein, the putative polymerase for the replication of the potyviral RNA. The genetic organization of PRSV RNA is tentatively proposed to be VPg-5' leader-63K NT-52K HC-Pro-46K-72K CI-6K-48K NIa-59K NIb-35K coat protein-3' non-coding region-poly(A) tract.
Using enzyme-dispersed canine oxyntic mucosal cells, we studied regulation of histamine release from fractions in which mast cells were largely removed by density gradient. Histamine-like immunoreactivity was demonstrated using peroxidase-anti-peroxidase immunohistochemistry. Histamine-containing cells in the small cell elutriator fractions (SCEF) were further separated by albumin step density gradients. Approximately 2.5% of cells in the low density fraction (LDF) contained histamine-like immunoreactivity; this fraction was largely depleted of the more dense mast cells (0.5%). These two fractions were cultured for 48-64 h on a Matrigel substrate. The cell content of histamine and release into the medium were measured by radioenzymatic assay. Gastrin, carbachol, and forskolin increased histamine release from the LDF. The induction of histamine release by gastrin was evident within 5 min and was sustained for at least 60 min. The response to gastrin was dose dependent between concentrations of 10(-11) and 10(-8) M. In contrast, in the mast cell-enriched SCEF, basal release was higher and gastrin was without effect; however, concanavalin A stimulated and epinephrine inhibited histamine release indicating that histamine-release mechanisms were intact in this fraction. Our methods provide a preparation of low density oxyntic mucosal histamine cells that demonstrate gastrin-responsive histamine release; we speculate that enterochromaffin-like cells account for this gastrin response.
Rates of sodium (Na+)-stimulated lithium (Li+) efflux (Na(+)-Li+ countertransport) and ouabain-sensitive Na+ efflux (Na+ pump) were determined in erythrocytes of Chinese normotensive and hypertensive subjects. Near-maximal rate of Na(+)-Li+ countertransport was found to be significantly higher in hypertensive than normotensive subjects. No significant difference was observed for the rate of Na+ pump between them. A second series of study involved normotensive subjects without and with hypertensive parent(s) (group A and B, respectively) and hypertensive subjects (group C). We found that the rate of Na(+)-Li+ countertransport in group A was significantly lower than that of group B and C, while no difference existed between group B and C. No significant difference was observed for the rate of Na+ pump among the three groups. Our results suggested that Na(+)-Li+ countertransport activity could be a genetic marker for essential hypertension in Chinese, similar to that as proposed in Caucasians.
Endometriosis is a common disease with 8-15% occurrence in women during their reproductive period. Involvement of the bowel wall occurs rather frequently and probably presents in 12-34% of patient with pelvic endometriosis. However, it is classically asymptomatic and difficult to find out. From 1977 to 1987, we had six patients of colorectal endometriosis with mainly bowel symptoms. Five of them were located at rectum and sigmoid colon, one at the hepatic flexure of colon. All of the cases developed constipation or diarrhea, and four of them had severe abdominal pain. Four cases developed rectal bleeding. Previous operation for pelvic endometriosis was noted in two cases. The detailed examination included digital examination, endoscopy, barium enema and CT scan. Suspected malignancy was the indication for surgery in 4 cases and one in rectal stenosis. Low diagnostic rate was due to the fact that endometrial tissue rarely infiltrates the mucosa. Therefore, pathology of biopsied specimen often reveals non-conclusive finding to prevent differential diagnosis. However bowel resection offers conclusive diagnosis and chances of cure. In our study, only one suspected endometriosis case received bilateral oophorectomy. The stenotic segment of rectum restored to normal caliber after operation.
Traditional evaluation of aortic stenosis usually requires cardiac catheterization for estimation of aortic valve area by the Gorlin formula. However, it is invasive and has been shown to be inaccurate in the presence of aortic regurgitation or low cardiac output. Evaluation of aortic stenosis by Doppler echocardiography using a continuity equation has been proved to have excellent correlation with cardiac catheterization. It requires hand tracing and computer software for calculation of time velocity integral. Recently, a simplified continuity equation was introduced, which uses the peak velocities instead of time velocity integrals. It saves time and obviates the computer assistance. The purpose of this study is to evaluate its accuracy using the standard continuity equation as the reference standard. Seventy patients with pure or combined aortic stenosis were examined. There was an excellent correlation between these two methods (r = 0.979, 95% confidence interval: 0.978-0.980). The aortic valve area derived from the peak velocity method = 0.018 + 0.949 X aortic valve area derived from the time velocity integral. The difference between the two methods was 0.04 +/- 0.08 cm2 (mean +/- SD) with a 95% confidence interval of -0.12 to 0.20 cm2. Furthermore, the correlation still remained good even in the presence of significant aortic regurgitation or impaired left ventricular function. In conclusion, the simplified continuity equation is a safe, time-saving, and accurate method for the evaluation of aortic stenosis.
The interaction of tetramethylpyrazine, a vasoactive ingredient of a Chinese traditional medicinal plant, with the vascular muscle alpha 1-adrenoceptors was investigated by a direct radioligand binding technique using [3H]prazosin and vascular smooth muscle microsomes isolated from dog aorta and mesenteric artery. Tetramethylpyrazine inhibited the binding of [3H]prazosin to vascular muscle membranes in a concentration-dependent manner at a suboptimal concentration of prazosin. Scatchard analysis of the effect of tetramethylpyrazine on the saturation profile of [3H]prazosin binding to vascular muscle microsomes of either arterial muscle indicated a substantial increase of Kd values (the affinity for prazosin) without a change in Bmax (maximal binding sites for prazosin). Thus, the present results provide supporting evidence that the inhibitory effect of tetramethylpyrazine on the vasoconstriction of dog mesenteric artery induced by phenylephrine in the earlier studies may be, at least, in part due to a direct action at the recognition sites of alpha 1-adrenoceptors. Amiloride and amiloride-related compounds, which shares a common pyrazine ring structure with tetramethylpyrazine and other related derivatives, also inhibits the binding of [3H]prazosin to aortic muscle microsomal membranes. Functional studies of dog saphenous vein also indicated that both tetramethylpyrazine and its ethyl derivatives inhibited the responses induced by phenylephrine and B-HT 920 in the presence and absence of extracellular Ca2+. Together with our earlier findings that amiloride also inhibits [3H]prazosin and [3H]rauwolscine binding to vascular muscle alpha 1- and alpha 2-adrenoceptors, the present radioligand binding study in canine arteries and functional study in saphenous veins suggest that the above compounds containing the pyrazine nucleus indeed interacted at the alpha-adrenoceptor sites.
The effects of two structurally similar pyrazine derivatives, tetramethylpyrazine (TMP) and tetraethylpyrazine (TEP) on the contractile responses of dog saphenous vein to KCl (via membrane depolarization), phenylephrine (PHE, alpha 1-adrenergic agonist), and B-HT 920 (alpha 2-adrenergic agonist) were investigated. The relaxant or inhibitory effect of TMP and TEP was most potent on KCl-induced responses and least potent on PHE-induced responses. Their effect on KCl-induced responses was more prominent at 30 mM KCl than at 100 mM KCl. In Ca(2+)-free medium, PHE and B-HT 920 elicited transient responses, which were also markedly and reversibly inhibited by TMP and TEP. Similar results were also obtained when prostaglandin F2 alpha was used as an agonist. In all four types of contractile responses involving different receptors, the inhibitory effect of TEP was consistently more potent than that of TMP. We conclude that both TMP and TEP behave as a nonselective smooth muscle relaxant having similar and multiple actions including their general interference with the processes involving both Ca2+ entry and intracellular Ca2+ release.
Manganese(II)-N,N'-dipyridoxylethylenediamine-N,N'-diacetate-5,5'-bis (phosphate) (MnDPDP) is a paramagnetic complex designed for use as a hepatobiliary agent. The T1 relaxivity of MnDPDP (2.8 [mmol/L]-1.sec-1 in aqueous solution) was similar to that of gadolinium diethylenetriaminepentaacetic acid (DTPA) (4.5 [mmol/L]-1.sec-1) and gadolinium tetraazocyclodecanetetraacetic acid (DOTA) (3.8 [mmol/L]-1.sec-1). However, in liver tissue the T1 relaxivity of MnDPDP (21.7 [mmol/L]-1.sec-1) was threefold higher than that reported for Gd-DOTA (6.7 [mmol/L]-1.sec-1). Maximum liver T1 relaxation enhancement occurred 30 minutes after injection of MnDPDP, at which time 54MnDPDP biodistribution studies indicated that 13% of total body activity was in the liver. Enhanced (MnDPDP, 50 mumol/kg) MR images showed a fivefold increase in tumor-liver contrast-to-noise ratio over baseline unenhanced images. Results of the authors' acute and subchronic toxicity studies suggest that MnDPDP will be safe at the doses necessary for clinical imaging; at 10 mumol/kg, the safety factor (LD50/effective dose) for MnDPDP is 540, significantly greater than the safety factor of Gd-DTPA (ie, 60-100).
We report methods allowing the culture of rapidly dividing gastric epithelial cells to investigate the regulation of mucosal cell replication. Cells from canine fundic mucosa were dispersed by enzyme treatment, enriched by filtration and elutriation, and cultured on collagen gel in DMEM/F12 medium. After 48 h, greater than 95% of the cells displayed immunoreactivity with antibody to cytokeratin, an epithelial marker. The cells formed confluent monolayers by 72 h with a transmembrane resistance of 1,600 ohm.cm2 when mounted in a Ussing chamber indicating retention of epithelial cell characteristics. Calf serum (0.1-2%) produced a dose-dependent mitogenic effect evident by increases in [3H]-thymidine incorporation into acid-precipitated material and in cell number. After an 18-24-h incubation with [3H]-thymidine, approximately 55% of the cells cultured in 2% serum showed evidence of DNA synthesis by autoradiography and all of the replicating cells were cytokeratin positive. Using comparable culture conditions, a similar proportion of cells incubated for 18-24 h with bromodeoxyuridine displayed nuclear anti-bromodeoxyuridine immunoreactivity, thus indicating that over half of the cells in these cultures synthesized DNA during this period. As with serum, epidermal growth factor and transforming growth factor alpha (TGF alpha) (10 pM to 1 nM), insulin (10 nM to 1 microM) and insulinlike growth factor-I (IGF-I, 1-100 nM) increased [3H]-thymidine uptake. The greater potency of IGF-I, compared to insulin, suggests the presence of IGF-I receptors. We conclude that this culture preparation is composed of fundic mucosal epithelial cells and contains a predominance of dividing epithelial cells. EGF/TGF alpha and IGF-I are potential factors directly regulating proliferation of fundic mucosal cells.
The receptors mediating the physiologic actions of gastrin on acid secretion and growth have thus far not been localized to specific cells or fully characterized. Studies in canine fundic mucosa indicate that gastrin receptors are present on several cell types, including parietal cells and somatostatin cells. There is also increasing evidence for a gastrin-inducible pool of histamine in the fundic mucosa which is presumably stored in histamine-enterochromaffin-like cells. From the vantage point of studies in the canine fundic mucosa, the issue is no longer which cell type has the gastrin receptor but to sort out the mechanisms by which the effects of the gastrin receptors on endocrine/paracrine (histamine and somatostatin) cells and exocrine (parietal) cells are integrated to regulate secretory function and mucosal growth and differentiation.