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Biomedical subjects

A Boucher

Publications and source records attributed to A Boucher.

At least 37 records · Page 2Linked to original sources
PubMed1995

Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.

Na+/H+ exchanger (NHE) activity is regulated by several types of receptors directly coupled to distinct classes (i.e. Gs, Gi, Gq, and G12) of heterotrimeric (alpha beta gamma) GTP-binding proteins (G proteins), which, upon activation, modulate production of various second messengers (e.g. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and Ca2+). Recently, four isoforms of the rat Na+/H+ exchanger were identified by molecular cloning. To examine their intrinsic responsiveness to G protein and second messenger stimulation, three of these isoforms, NHE-1, -2, and -3, were stably expressed in mutant Chinese hamster ovary cells devoid of endogenous NHE activity (AP-1 cells). Incubation of cells with either AIF4-, a general agonist of G proteins, or cholera toxin, a selective activator of G alpha s that stimulates adenylate cyclase, accelerated the rates of amiloride-inhibitable 22Na+ influx mediated by NHE-1 and -2, whereas they inhibited that by NHE-3. Similarly, short term treatment with phorbol 12-myristate 13-acetate, which mimics diacylglycerol activation of protein kinase C (PKC), or with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylmethylxanthine) that lead to activation of cAMP-dependent protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocked by depleting cells of PKC or by inhibiting PKC using chelerythrine chloride, confirming a role for PKC in modulating NHE isoform activities. Likewise, the PKA antagonist, H-89, attenuated the effects of elevated cAMPi on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMPi, elevation of cGMPi by treatment with dibutyryl-cGMP or 8-bromo-cGMP had no influence on NHE isoform activities, thereby excluding the possibility of a role for cGMP-dependent protein kinase in these cells. These data support the concept that the NHE isoforms are differentially responsive to agonists of the PKA and PKC pathways.

Animals↗
PubMed1995

[Prognostic factors in well-differentiated thyroid cancers].

Although well differentiated thyroid carcinoma is usually associated with a favorable outcome, a small percentage of patients die from their cancer. In order to determine the risks for a specific patient, various prognostic scores have been established. The more famous are AMES, AGES, the score designed by the EORTC classification and an other by WHO based on TNM classification. From these studies, two clinical criteria have the best prognostic value: age at the time of diagnosis and stage of the disease. Other factors, less effective in predicting the prognosis are the degree of histological differentiation of the tumor, sex of the patient and nodal status.

Adult↗
PubMed1995

Determinants of late allograft nephrectomy.

When loss of graft function occurs more than six months after transplantation, allograft nephrectomy is not routinely performed at the time of graft failure. It is usually performed only on those patients who subsequently develop specific complications. However, little is known about the characteristics that make patients more likely to require allograft nephrectomy. The purpose of our study was to identify risk factors for the subsequent need for allograft nephrectomy in patients with graft failure occurring more than 6 months after transplantation. Forty-one patients were studied. Inclusion criteria were: loss of graft function > or = 6 months after transplantation, resumption of dialysis and initiation of weaning from immunosuppression. Thirty patients were treated with cyclosporine + prednisone +/- azathioprine and 11 with azathioprine + prednisone. Mean follow-up time was 17.8 months, ranging from 6 months to 6.1 years. Recipient age, sex and race, original renal disease, donor, donor source (cadaveric vs living related), HLA compatibility, levels of panel reactive antibodies, occurrence of initial delayed graft function, causes of graft failure and tapering of immunosuppression were similar in patients with and without allograft nephrectomy. Using univariate analysis, allograft nephrectomy was found to be significantly more frequent in patients with a history of 2 or more episodes of acute rejection than in patients with no rejection episode: 83% vs 30% (p = 0.03). In addition, allograft nephrectomy was found to be significantly more frequent if the immunosuppressive regimen included cyclosporine (62% vs 27.3%; p = 0.04). Using multivariate analysis however, the number of previous episodes of rejection was found to be the only significant predictor for allograft nephrectomy. None of the other variables considered in the multivariate analysis, including the type of immunosuppressive therapy, was identified as a significant predictor for the need to perform allograft nephrectomy. In summary, the need for late allograft nephrectomy was correlated with the number of previous episodes of acute rejection. Patients with a history of numerous rejection episodes should thus be considered more likely to require allograft nephrectomy once immunosuppression is withdrawn. Possible interventions to reduce or prevent the need for nephrectomy include more gradual tapering of immunosuppression at the time of graft failure or indefinite low-dose immunosuppressive therapy.

Adult↗
PubMed1994

Basolateral K channel activated by carbachol in the epithelial cell line T84.

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na >> Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single KBIC channels, the carbachol-stimulated Isc was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues.

4-Aminopyridine↗
PubMed1994

Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium, nucleotides and kinases.

Agonists that elevate calcium in T84 cells stimulate chloride secretion by activating KBIC, an inwardly rectifying K channel in the basolateral membrane. We have studied the regulation of this channel by calcium, nucleotides and phosphorylation using patch clamp and short-circuit current (ISC) techniques. Open probability (Po) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 microM-5 mM), ATP (0.1 mM)+protein kinase A (PKA; 180 nM), or isobutylmethylxanthine (IBMX; 1 mM). Analysis of event durations suggested that the channel has at least two open and two closed states, and that rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or ADP. Activity was further enhanced when PKA was added in the presence of MgATP, but only if free calcium concentration was elevated (400 nM). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical membrane using alpha-toxin. Finally, protein kinase C (PKC) inhibited single KBIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phosphorylation by PKA and PKC modulate the responsiveness of the inwardly rectifying K channel to Ca-mediated secretagogues.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Two dimensional gel electrophoresis identifies minor differences in immunologically cross-reactive 64 KDa autoantigens in the thyroid and eye muscle.

Among the candidate eye muscle autoantigens proposed as being relevant to the pathogenesis of thyroid-associated ophthalmopathy (TAO), a 64 kDa membrane autoantigen appears to be most closely associated with the eye disorder. We have examined the tissue localization and some of the physicochemical properties of this molecule in 3 human tissues, namely thyroid (THY), eye muscle (EM) and skeletal muscle (SKE), and in pig eye muscle (PEM), by two-dimensional (2-D) [isoelectric focusing (IEF)/sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE)] gel electrophoresis followed by Western blotting. Antibody probes used were whole sera from patients with TAO and antibodies affinity purified from TAO sera by binding to, and elution from, a sepharose-4B column conjugated with D1, a 98 amino acid peptide fragment of a recombinant 64 kDa thyroid autoantigen. Soluble membrane proteins eluted from a slice of SDS-PAGE gel containing 60-70 kDa material was prepared from the four tissues and used as antigen for 2-D gel separation. The presence of a 64 kDa antigen in THY and EM recognized by sera from patients with TAO, but only rarely by those from normal individuals, was confirmed. Pretreatment of the eluted 60-70 kDa material with N-Glycosidase F to eliminate charge heterogeneity resulting from glycosylation differences, changed the pI and MW of molecules recognized by TAO sera, in THY and EM. This suggests that the 64 kDa molecule(s) in EM and THY targeted by sera from patients with TAO are glycoproteins and that they are different in the two tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Cross-reactive antibodies in the serum of balb/c mice immunized with thyroid or eye muscle membranes.

During the course of immunizing balb/c mice with eye muscle (EM) or thyroid (THY) membranes for monoclonal antibody (MCAB) production their sera frequently contain antibodies which react against both EM and THY membranes in enzyme-linked immunosorbent assay (ELISA) and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In order to further study this phenomenon we have analyzed sera from 27 balb/c mice, including 10 that were studied serially, and their tissues examined histologically at sacrifice. Following immunization serum and, in some cases, the corresponding MCAB produced by fusion of the mouse spleen cells with a mouse myeloma cell line, were tested for EM and THY cross-reactivity in an ELISA and by immunoblotting. The number of antibodies demonstrated in Western blotting identified as bands of reactivity, and ELISA levels, expressed as optical density--increased with time, each peaking at around 10-12 weeks. THY and EM antibody cross-reactivity was demonstrated in the majority of mice, serum from mice immunized with THY membranes reacting with these membranes as well as with pig EM membranes in both ELISA and immunoblotting and, conversely, sera from mice immunized with pig EM membranes also reacting with THY membranes in the two tests. In Western blotting a variety of THY and EM-reactive antibodies were demonstrated including those directed against a 64 kDa protein, shown to be an important autoantigen in thyroid-associated ophthalmopathy. There was also some cross-reactivity with brain membranes, used as control antigen in both tests and in immunization, although to a lesser degree, but very little to liver and orbital connective tissue membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Multimeric non-radioactive cRNA probes improve detection of potato spindle tuber viroid (PSTVd).

Experimental data showed that multimeric, complementary RNA (cRNA) probes, labelled with non-radioactive digoxigenin (DIG), improved sensitivity of detection of the potato spindle tuber viroid (PSTVd) RNA by 2- to 30-fold as compared with corresponding multimeric cDNA probes. The degree of PSTVd detectability improvement depended upon the type of alkaline phosphatase substrate (colorimetric vs. chemiluminescent) used. Use of hexameric DIG-labelled cRNA probes in combination with chemiluminescent (Lumi-Phos 530) substrate resulted in detection of 0.48 pg of PSTVd RNA. The size of the synthesized cRNA probes corresponded to the size of the respective PSTVd cDNA templates. Interestingly, there was no relationship between the size of the synthesized, DIG-labelled DNA probes and that of the PSTVd cDNA template. This type of anomaly was not observed with other plant viral cDNA templates. Monomeric or multimeric cDNA probes detected both a mild and a severe PSTVd strain in viroid-infected potato leaf extracts diluted 1024 to 2048 times. In comparison, cRNA probes exhibited a much greater dilution end point; PSTVd RNA was detectable in viroid-infected potato leaf tissue diluted up to 16,384 times. Comparable levels of PSTVd sensitivity of detection were obtained with viroid-infected potato tuber tissue.

Alkaline Phosphatase↗
PubMed1994

Molecular cloning and sequencing of the capsid and the nuclear inclusion protein genes of a North American PVYN isolate.

The sequence of the 3'-terminal 3258 nucleotides of a tobacco veinal necrosis strain of a potato virus Y (PVYN) isolate from North America was determined. The sequence revealed an open reading frame of 2931 nucleotides, of which the start codon was not identified. The nontranslated region contains 327 nucleotides upstream of a poly(A) tract. The open reading frame encodes a large polyprotein containing 976 amino acids. The data indicate that the coat protein (CP) and the nuclear inclusion protein (NIb) are derived from the large polypeptide by proteolytic cleavage similar to many other potyviruses. The putative cleavage sites of NIa/NIb and NIb/CP correspond to Q/A and Q/G sequences, respectively, as in other PVYN isolates. The CP and NIb contain 267 and 519 amino acid residues, respectively. Higher sequence homology of CP was observed with other PVYN isolates than with the PVYO isolates.

Amino Acid Sequence↗
PubMed1994

Safety and efficacy of recombinant human erythropoietin in correcting the anemia of patients with chronic renal allograft dysfunction.

Recombinant human erythropoietin (rHuEPO) is effective in correcting anemia in hemodialysis, peritoneal dialysis, and predialysis patients. Limited studies in patients with failing renal allografts suggest a similar efficacy but provide little information concerning benefits, dose requirements, or adverse events. This study examined these considerations in a group of 40 patients (18 men; 22 women) aged 40.3 +/- 13.8 yr with stable, chronic renal allograft failure. All patients had a hemoglobin < 95 g/L and a serum creatinine > 250 mumol/L at baseline. Patients received rHuEPO (50 U/kg sc) three times weekly for 24 wk along with iron po if serum ferritin was < 100 micrograms/L. Mean hemoglobin rose from 78.9 +/- 10.4 to 102.6 +/- 18.4 g/L after 24 wk. Mean rHuEPO dose at 24 wk was 129.8 +/- 81.9 U/kg per week. With oral iron supplementation only, serum ferritin fell throughout the 24 wk, whereas serum iron, transferrin saturation, and total iron-binding capacity remained stable. Quality of life was assessed by use of the general Sickness Impact Profile and the disease-specific Transplant Disease Questionnaire measures at baseline and every 8 wk during rHuEPO therapy. Significant improvement was noted in global Sickness Impact Profile scores and in four of five dimensions of the Transplant Disease Questionnaire. Serious adverse events were infrequent. No change in mean systolic or diastolic blood pressure was noted, although there was a significantly increased need for antihypertensive drugs in 18 patients (P = 0.0002). A significant inverse correlation was noted between baseline renal function and maintenance rHuEPO dose (r = -0.45; P < 0.05). Twelve patients returned to dialysis during the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1993

Pathogenesis of thyroid-associated ophthalmopathy: an autoimmune disorder of the eye muscle associated with Graves' hyperthyroidism and Hashimoto's thyroiditis.

Thyroid-associated ophthalmopathy, the progressive eye disorder which occurs frequently in patients with Graves' hyperthyroidism and, occasionally, in those with Hashimoto's thyroiditis, may be a two-stage disorder of the eye muscle. In the first stage, which may occur in the great majority of patients with Graves' hyperthyroidism and in an unknown, but probably small, proportion of those with Hashimoto's thyroiditis, antibodies and CD4+ (helper) T lymphocytes reactive with eye muscle and thyroid shared antigens, of which 64-kDa membrane proteins are good candidates, may initiate a mild eye muscle inflammation, manifested as eye muscle swelling on orbital imaging. The second stage, which occurs in about 25% of patients with Graves' hyperthyroidism and in 2% of those with Hashimoto's thyroiditis, may be due to reactivity of cytotoxic antibodies against eye muscle-specific membrane antigens, one of which at approximately 35 kDa appears a likely candidate, and, possibly, cytotoxic T cells in the context of the appropriate class I MHC molecule. Orbital connective tissue inflammation, which plays an important role in the development of progressive orbital inflammation, is likely to be secondary to the eye muscle reaction. The recent cloning of a 64-kDa thyroid and eye muscle antigen which shares significant homology with the muscle protein tropomodulin and mapping of its antibody-reactive epitopes provide structural information about one candidate eye muscle autoantigen and promise for a more rational approach to the diagnosis and management of this common, progressive eye disorder.

Graves Disease↗
PubMed1993

Antibodies reactive with an intracellular epitope of a recombinant 64 kDa thyroid and eye muscle protein in patients with thyroid autoimmunity and ophthalmopathy.

We have developed an enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies reactive with a 98 amino acid fragment, called D1, of a recombinant thyroid and eye muscle membrane protein corresponding to a MW of 64 kDa (called 1D) in the serum of patients with thyroid autoimmunity with and without ophthalmopathy. Antibodies against the D1 fragment expressed as a fusion protein with beta galactosidase, were detected in 29% of patients with thyroid-associated ophthalmopathy (TAO) of < 1 yr duration, in 33% of those with disease of > 3 yr duration, in 40% of patients with Graves' hyperthyroidism (GH) without evident eye disease, in 31% of patients with lid lag and retraction but no other signs of progressive ophthalmopathy, in 25% of patients with euthyroid Graves' disease and in 43% of patients with untreated Hashimoto's thyroiditis (HT), but in none of 14 patients with other (non-immunological) thyroid disorders. Although tests were positive in 6 out of the 15 patients with ophthalmopathy and no overt thyroid autoimmunity overall there was no close association of the antibodies with clinical features of the eye disease or its course. In those sera in which Western blotting for antibodies reactive with a 64 kDa eye muscle membrane protein and ELISA were both carried out there was no close correlation between the two tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Nature of 64 kDa eye muscle and thyroid membrane proteins and their significance in thyroid-associated ophthalmopathy--an hypothesis.

Although a variety of eye muscle antigens are recognized by autoantibodies in the serum from patients with thyroid associated ophthalmopathy (TAO) 64 kDa membrane proteins, which are also expressed in the thyroid, are most closely linked to the development of ophthalmopathy in patients with autoimmune thyroid disease. A cloned 64 kDa protein called 1D, which shares homology with tropomodulin, appears to be closely related to a 64 kDa eye muscle protein identified in immunoblotting and both may be members of a family of cytostructural proteins. The relationship between 64 kDa proteins in eye muscle and thyroid, and its equivalent in somatic skeletal muscle, will only be understood when the various proteins are cloned from expression libraries, sequenced and their consensual and unique domains identified. Since these 64 kDa antigens are expressed in both thyroid and eye muscle, a possible mechanism for the association of ophthalmopathy with autoimmune thyroid disease is immunological cross-reactivity by autoantibodies and sensitized T lymphocytes. Autoantibodies reactive with 64 kDa eye muscle proteins are associated with ophthalmopathy in patients with autoimmune thyroid disorders and predictive of the development of ophthalmopathy in patients with Graves' hyperthyroidism.

Animals↗
PubMed1992

Sequence analysis of five new field isolates demonstrates that the chain length of potato spindle tuber viroid (PSTVd) is not strictly conserved but as variable as in other viroids.

The sequence analysis of five new field isolates of potato spindle tuber viroid (PSTVd) of different virulence revealed that the length of their RNA chain is not strictly conserved to 359 nucleotides (nts), as one could have inferred from the previously sequenced PSTVd strains. It was now found that the chain length is strain-specific like in the case of practically all other viroids, and that it may vary, so far, between 356 and 360 nts. Taking our previously sequenced and least virulent mild strain PSTVd KF6-M as standard, the new mild strains PSTVd WA-M and PSTVd F-M differ from it by one or two nts. The new intermediate-severe strains PSTVd F-IS and PSTV-F 88-IS differ from the standard mild strain by eight and nine nts, respectively, whereas the new severe-lethal strain PSTVd F-SL differs in seven nts. Most of these mutations are located within the virulence-modulating (VM) region and within the variable region (VR), and only in two strains a single mutation is found in the right terminal domain.

Base Composition↗
PubMed1992

A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs.

We surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs. A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants. First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT). Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E. coli DNA polymerase I, E. coli DNA ligase, and beta-NAD+. The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme. The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E. coli strain DH5 alpha. This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence.

Autoradiography↗
PubMed1992

Significance of antibodies reactive with a 64 kDa eye muscle membrane antigen in patients with thyroid autoimmunity.

SDS-polyacrylamide gel electrophoresis and Western blotting for antibodies reactive with a 64 kDa protein in pig eye muscle membrane was carried out in patients with lid lag and retraction, but no other signs of ophthalmopathy, associated with thyroid disease or nonimmunologic goiter and in patients with Graves' hyperthyroidism without ophthalmopathy who were studied prospectively to determine the relationship of eye muscle antibodies to clinical features of the ophthalmopathy as they appeared in this group of predisposed patients. Seventy-one percent of euthyroid patients with lid lag and retraction but no established ophthalmopathy had detectable serum antibodies to a 64 kDa eye muscle membrane protein. Much smaller proportions had antibodies to proteins of other MW. In normal subjects with previously detectable antibodies to a 64 kDa protein, serum titers, determined by carrying out immunoblotting at serum dilutions of 1:25-1:6400, were low (< or = 1:100) in all cases tested. On the other hand, titers were higher (1:200-1:6400) in 16 of 22 patients with established ophthalmopathy and in 5 of 7 patients with lid lag and retraction tested. Titers tended to be lower in patients with ophthalmopathy of > or = 3 years duration than in those of < or = 1 year duration. Antibody titers were low (1:25) in 6 of 7 patients with Graves' hyperthyroidism without evident eye disease tested. Antibodies to a 64 kDa eye muscle membrane protein were predictive of the development of ophthalmopathy in patients with Graves' hyperthyroidism studied prospectively for periods of 8-42 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗