A highly sensitive and specific multiplex RT-PCR to detect foot-and-mouth disease virus in tissue and food samples.

Three sets of primers to detect foot-and-mouth disease virus (FMDV) using multiplex RT-PCR were designed based on several reference nucleotide sequences, and their reaction conditions were determined. By testing ten-fold serial dilutions of FMDV, the sensitivity of multiplex RT-PCR is 100 times higher than conventional RT-PCR. Meanwhile, its specificity was confirmed compared with other related vesicular disease viruses. Furthermore, 30 field samples from different animals were tested, and the results supported the method's potential applications in routine veterinary quarantine and epidemic surveillance of FMDV.

The complete genome sequence of foot-and-mouth disease virus O/Akesu/58 strain and its some molecular characteristics.

The complete genome of O/Akesu/58 strain of foot-and-mouth disease virus (FMDV) was sequenced. The phylogenetic analysis revealed that it is not closely related to epidemic strains or previous strains compared with reference sequences (the identities of complete VP1 nucleotide sequences range from 77.5 to 84.0%). Its cell-receptor-binding site is a SGD (Ser-Gly-Asp) motif instead of RGD (Arg-Gly-Asp), and 43 bases were deleted in PKs region of the 5'UTR, although deletions were not found in other gene regions.

S-adenosyl-L-homocysteine hydrolase is necessary for aldosterone-induced activity of epithelial Na(+) channels.

The A6 cell line was used to study the role of S-adenosyl-L-homocysteine hydrolase (SAHHase) in the aldosterone-induced activation of the epithelial Na(+) channel (ENaC). Because aldosterone increases methylation of several different molecules, and because this methylation is associated with increased Na(+) reabsorption, we tested the hypothesis that aldosterone increases the expression and activity of SAHHase protein. The rationale for this work is that general methylation may be promoted by activation of SAHHase, the only enzyme known to metabolize SAH, a potent end-product inhibitor of methylation. Although aldosterone increased SAHHase activity, steroid did not affect SAHHase expression. Antisense SAHHase oligonucleotide decreased SAHHase expression and activity. Moreover, this oligonucleotide, as well as a pharmacological inhibitor of SAHHase, decreased aldosterone-induced activity of ENaC via a decrease in ENaC open probability. The kinetics of ENaC in cells treated with antisense plus aldosterone were similar to those reported previously for the channel in the absence of steroid. This is the first report showing that active SAHHase, in part, increases ENaC open probability by reducing the transition rate from open states in response to aldosterone. Thus aldosterone-induced SAHHase activity plays a critical role in shifting ENaC from a gating mode with short open and closed times to one with longer open and closed times.

Contrasting effects of cPLA2 on epithelial Na+ transport.

Activity of the epithelial Na+ channel (ENaC) is the limiting step for discretionary Na+ reabsorption in the cortical collecting duct. Xenopus laevis kidney A6 cells were used to investigate the effects of cytosolic phospholipase A2 (cPLA2) activity on Na+ transport. Application of aristolochic acid, a cPLA2 inhibitor, to the apical membrane of monolayers produced a decrease in apical [3H]arachidonic acid (AA) release and led to an approximate twofold increase in transepithelial Na+ current. Increased current was abolished by the nonmetabolized AA analog 5,8,11,14-eicosatetraynoic acid (ETYA), suggesting that AA, rather than one of its metabolic products, affected current. In single channel studies, ETYA produced a decrease in ENaC open probability. This suggests that cPLA2 is tonically active in A6 cells and that the end effect of liberated AA at the apical membrane is to reduce Na+ transport via actions on ENaC. In contrast, aristolochic acid applied basolaterally inhibited current, and the effect was not reversed by ETYA. Basolateral application of the cyclooxygenase inhibitor ibuprofen also inhibited current. Both effects were reversed by prostaglandin E2 (PGE2). This suggests that cPLA2 activity and free AA, which is metabolized to PGE2, are necessary to support transport. This study supports the fine-tuning of Na+ transport and reabsorption through the regulation of free AA and AA metabolism.

Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway.

Amiloride-sensitive epithelial Na(+) channels (ENaC) are responsible for trans-epithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, gamma) each containing a proline rich region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions with the ubiquitin ligase Neural precursor cell-expressed developmentally down-regulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132, increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important regulator of ENaC function.

The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells.

Rapamycin and FK-506 are immunosuppressive drugs that bind a ubiquitous immunophilin, FKBP12, but immunosuppressive mechanisms and side effects appear to be different. Rapamycin binds renal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na(+) channels in A6 cells. Channel NP(o) was 0.5 +/- 0.08 (n = 6) during the first 5 min but fell close to zero after 20 min. Application of 1 microM rapamycin reactivated Na(+) channels (NP(o) = 0.47 +/- 0.1; n=6), but 1 microM FK-506 did not. Also, GF-109203X, a protein kinase C (PKC) inhibitor, mimicked the rapamycin-induced reactivation in a nonadditive manner. However, rapamycin did not reactivate Na(+) channels if cells were exposed to 1 microM FK-506 before rapamycin. In PKC assays, rapamycin was as effective as the PKC inhibitor; however, epithelial Na(+) channel (ENaC) phosphorylation was low under baseline conditions and was not altered by PKC inhibitors or activators. These results suggest that rapamycin activates Na(+) channels by binding FKBP12 and inhibiting PKC, and, in renal cells, despite binding the same immunophilin, rapamycin and FK-506 activate different intracellular signaling pathways.

Differential effects of protein kinase C on the levels of epithelial Na+ channel subunit proteins.

Regulation of epithelial Na(+) channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both beta and gamma, but not alphaENaC. Temporal regulation of beta and gammaENaC by PKC differed; gammaENaC decreased with a time constant of 3.7 +/- 1.0 h, whereas betaENaC decreased in 13.9 +/- 3. 0 h. Activation of PKC also resulted in a decrease in trans-epithelial Na(+) reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning within 4 h. Both beta and gammaENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC inhibitors attenuated the effects of PMA on ENaC subunit levels and Na(+) transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing the levels of beta and gamma but not alphaENaC protein. These results imply a PKC-dependent, long term decrease in Na(+) reabsorption.

An image analysis on pathological changes in pulmonary arteries in chronic obstructive pulmonary disease.

The changes in small pulmonary arteries of 15 patients with chronic obstructive pulmonary disease (COPD) were investigated by light and electron microscopy, image analysis etc. It was found that the structural changes in the pulmonary arteries of the patients with COPD were characterized by muscularization of non-muscular arterioles, media hypertrophy, longitudinal smooth muscle bundles in the intima and fibrosis in both the media and intima. In the course of time, these lesions resulted in thickening of the arterial wall and narrowing of the lumen. Clinically, the patients developed pulmonary hypertension causing cor pulmonale. Initial data on the structure of arterial wall at different segments were compared statistically. There was very significant difference between the COPD and control groups (P less than 0.001). By Fisher's auto-classification (automatic pattern recognition), the rate recognized was correct in more than 90% in the small arteries of less than 200 microns in diameter. It is suggested that these arteriolar changes are closely related to pulmonary hypertension. The image analysis showed that the ratios of MWA/MVA and MWT/MD were of great value in evaluating the degree of the changes in the arteries of the patients with COPD.

Reconstruction of the frequency distribution of A- and B-cell size in the pancreatic islets from several animal species--an important stereological operation in QMSOC.

The stereological algorithms for the conversion of the profile-size frequency distribution (PSFD) observed in histological section to the sphere-size frequency distribution (SSFD) in three-dimensional space was developed to a computer program and its practical application was described in this paper. The reconstructions of SSFD were based on the data of PSFD of A- and B-cells measured in sections of the pancreatic islets from ophiocephalus argus, toad, chicken, cat, rabbit and rat. The sizes of both profile and sphere were expressed by their caliper diameters. The experimental results showed that the patterns of SSFD of A- and B-cells from six animal species were somewhat different, and that all the peaks of them, however, occurred in the range from 6 to 12 microns in caliper diameter.

Information medicine and its primary functions of exploitation of medical resources.

The aim of this paper is to continue the discussion on the defects of the structures of medical resources and their applicability at the present time, and then to design the qualitative networklike subsystem of the new research Quantitatively Medicine Simulating and Operating by Computer (QMSOC) and a five-library model of the new knowledge-base of QMSOC. Finally, a set of results from the primary functions of exploitation of pancreas-glucagon-insulin information by QMSOC are presented.

The new functions of quantitatively medicine simulating and operating by computer (QMSOC).

The preliminary results from integration-evaluation on a set of 310 binary relations in current data base of the new research Quantitatively Medicine Simulating and Operating by Computer (QMSOC) are presented in this paper. Through the function derived from the reciprocity in analysis of conditions, subjects and objects, in the given biomedical events, the number of routes for observation of glucagon and insulin was increased by 38.1% and 136.4% over the conventional object-oriented searching, respectively. The intersection operation of condition sets indicates that it is possible through QMSOC to increase markedly the degree of definity of causality of biomedical events. 70 new binary quantitative relations have been created through operator 1, achieving an increment of 22.6% over the total of original binary relations in the data base. The characteristics and the significance of QMSOC are discussed.

Structure characteristics of QMSOC and the relevant operators.

This article presents a further description on the background, significance, and structure characteristics of Quantitative Medicine Simulation and Operation by Computer (QMSOC). Also some basic operators were recommended for calculations of biomedical events such as estimation of substance concentrations, exploration of etiology, evaluation of biomedical effects, etc. At last some differences of QMSOC from other artificial intelligent systems in the medical field were discussed.

Application of the method of multiple thresholding to white blood cell classification.

A new method of image segmentation based on the principle of multiple grey level thresholding has been applied to a data set consisting of 1149 white blood cells of 13 different, clinically important types, randomly chosen on 20 blood smears from leukemia patients. Classification of these cells on the basis of quantitative measurements in the segmented images yields an accuracy of 82.6%. Some of the erroneous classifications must be attributed to intrinsic problems in the assignment of a priori labels. Correcting for such cases, the performance of the method, as measured on the present data set, increases to 89.8%. This illustrates the practical applicability of the segmentation method in automated white blood cell and possibly other cytological and histological analysis systems.

Automated white blood cell classification revisited.

A novel approach to the problem of automated white blood cell classification is described. Whereas in most earlier attempts the segmentation of the cells has been recognized as the most difficult and most critical step in the sequence of operations resulting in the classification, the method described here eliminates the necessity of the detection of the contour of the nucleus and of the cytoplasm, and is therefore less sensitive to such disturbing factors as the presence of granules, of other cells touching the cell of interest, etc. The multiple sequential threshold method to be described here in two slightly different variants yields a correct classification rate of 94.7% for a 4 class problem (90 cells in the test set), and 91.8% for an 8 class problem (279 cells in the test set). Both experiments include immature cell types.