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Biomedical subjects

C M Wilson

Publications and source records attributed to C M Wilson.

At least 19 recordsLinked to original sources
PubMed1995

Regulation of cell surface GLUT1, GLUT3, and GLUT4 by insulin and IGF-I in L6 myotubes.

The effects of insulin and IGF-I on the cell surface quantities of GLUT1, GLUT3 and GLUT4 glucose transporters in L6 myotubes were determined with the exofacial bis-mannose phololabel (ATB-BMPA). In basal cells, an equal molar quantity of each transporter isoform was found at the cell surface. Insulin stimulated the translocation of all three glucose transporter isoforms to the plasma membrane fraction from the light microsome fraction, resulting in equal molar quantities on the cell surface. IGF-I stimulated a similar translocation of all isoforms, augmented by an increase in surface GLUT3 as assessed by ATB-BMPA.

Affinity Labels↗
PubMed1995

Thyroid hormone increases the partitioning of glucose transporters to the plasma membrane in ARL 15 cells.

The stimulation of glucose transport by 3,5,3'-triiodo-L-thyronine (T3) in the liver-derived ARL 15 cell line is only partly attributable to increased GLUT-1 glucose transporter gene expression. To test the hypothesis that T3 increases the partitioning of GLUT-1 to the cell surface, we quantitated surface GLUT-1 using the photolabel ATB-[3H]BMPA. In control cells only approximately 20% of total cellular GLUT-1 was present at the cell surface. T3 treatment (100 nM) for 6 h increased the rate of 2-deoxy-[3H]glucose (2-DG) uptake by 30, 92, and 95% in three experiments and increased surface GLUT-1 photolabeling by 17, 81, and 72%, respectively, with no increase in total cellular GLUT-1. T3 treatment for 48 h increased 2-DG uptake by 143, 172, and 216% in three experiments and increased cell surface GLUT-1 photolabeling by 88, 161, and 184%, respectively, with smaller increases in total cellular GLUT-1. T3 treatment for 48 h thus increased the fraction of cellular GLUT-1 at the plasma membrane from 21 +/- 2 to 35 +/- 3% (SE). We conclude that most of the early (6-h) stimulation of glucose transport by T3 in ARL 15 cells is mediated by an increase in the partitioning of GLUT-1 to the plasma membrane. With more chronic T3 treatment (48 h), the enhanced surface partitioning of GLUT-1 is persistent and is superimposed on an increase in total cellular GLUT-1, accounting for a further increase in glucose transport.

Affinity Labels↗
PubMed1994

Regulation of the GLUT1 glucose transporter in cultured myocytes: total number and subcellular distribution as determined by photoaffinity labelling.

We have used the impermeant photoaffinity label 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-[2-3H] 1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) to identify and quantify the glucose transporters on the surface of BC3H-1 cells, a continuously cultured skeletal-muscle cell line lacking the MyoD transcription factor required for cell fusion. ATB-[2-3H]BMPA was used in combination with immunoprecipitation of the GLUT1 glucose transporter, the only isoform expressed in these cells. The total cellular GLUT1 content was also determined by photolabelling and immunoprecipitation after cell permeabilization with digitonin (0.025%). In glucose-starved cells, 85% of the glucose transporters were present at the cell surface in the basal state, with little change in response to insulin (200 nM), correlating with lack of additional 2-deoxyglucose uptake in response to insulin. Feeding the cells with glucose (25 mM) for 24 h resulted in an 80% decrease in the total GLUT1 content relative to starved cells, of which only 25% were present on the cell surface. This was associated with an 85% decrease in 2-deoxyglucose uptake. In addition, acute stimulation of the fed cells with insulin or phorbol 12-myristate 13-acetate (PMA) led to an increase in GLUT1 at the cell surface, and, in correspondence, an increase in 2-deoxyglucose uptake by approx. 2- and 4-fold respectively. We conclude that exofacial photoaffinity labelling of glucose transporters with ATB-[2-3H]BMPA in the presence and absence of digitonin, followed by specific immunoprecipitation, provides an accurate measure of total and cell-surface glucose transporters in differentiated BC3H-1 muscle cells. This technique demonstrates that glucose pre-feeding (1) decreases the total number of GLUT1 and (2) redistributes the majority of the remaining transporters to an intracellular site, where they can now be translocated to the cell surface in response to insulin and PMA.

Affinity Labels↗
PubMed1994

Glucose transporter localization in rat skeletal muscle. Autoradiographic study using ATB-[2-3H]BMPA photolabel.

Surface glucose transporters of intact muscles were photolabeled with the membrane impermeant ATB-[2-3H]BMPA reagent and localized by autoradiography. We found sparse labeling of the glucose transporters by ATB-[2-3H]BMPA on the sarcolemmal membrane around the muscle fiber. The majority of label was on the interior of the muscle fiber, at a discrete site which matched the distribution of AI junctions and which was presumed to be on the exterior surface of T-tubules. The amount of photolabel on the T-tubule was increased in response to insulin and was blocked by cytochalasin B. These results support the concept that glucose transport may occur predominantly across the T-tubule membrane under basal and insulin-stimulated conditions.

Affinity Labels↗
PubMed1994

A and B forms of the androgen receptor are present in human genital skin fibroblasts.

Two forms of the androgen receptor (AR) protein (apparent molecular masses, approximately 110 kDa and approximately 87 kDa) are present in human genital skin fibroblasts. The 87-kDa isoform (AR-A) contains an intact C terminus but lacks the normal N terminus found in the 110-kDa isoform (AR-B). AR-A is the same size as the mutant form of AR produced in fibroblasts from an androgen-resistant individual (R776) by initiation of AR synthesis at the internal Met-188 residue of AR-B. The ratio of AR-B to AR-A in fibroblasts derived from normal individuals is approximately 10:1. The AR isoforms detected in these experiments resemble the A and B forms of the human progesterone receptor, which also are encoded by a single gene and differ by the absence or presence of an N-terminal segment. The A and B forms of the human progesterone receptor differ in their ability to activate target genes and are regulated differently in various types of cells. The identification of similar forms of human AR raises the possibility that AR-A and AR-B also subserve different functions.

Blotting, Western↗
PubMed1994

Asystolic arrest as a presentation of sarcoidosis.

A 41-year-old man who presented with a history of sudden loss of consciousness suffered two further episodes during which asystole was documented. Subsequent investigations included exercise stress testing, thallium scintigraphy, electrophysiological studies, CT-scan of chest, Kveim test and a gallium-67 scan, which led to a presumptive diagnosis of averted sudden death as a first presentation of sarcoidosis with primary cardiac involvement.

Adult↗
PubMed1994

Identification of Hox genes in newborn lung and effects of gestational age and retinoic acid on their expression.

Hox genes are sequence-specific DNA transcription factors, which are important in embryonic development and are expressed in a number of fetal tissues, including the lung. Additionally, retinoic acid (RA) has been shown to modulate Hox gene expression in a number of cell types. The specific aims of this study were to 1) identify those Hox genes expressed in newborn mouse lung using reverse transcription-polymerase chain reaction (RT-PCR), 2) study the ontogeny of Hox gene expression in fetal mouse and rat lung by Northern analysis using cDNAs for mouse Hox genes, and 3) study the effects of RA on whole lung Hox mRNA levels in cultured fetal rat lung explants. Our data show that 16 different homeobox genes are expressed in newborn mouse lung. This includes seven Hox genes not previously identified in lung, as well as the divergent homeobox gene Hex. Steady-state mRNA levels of Hox A5 (Hox 1.3), B5 (Hox 2.1), B6 (Hox 2.2), and B8 (Hox 2.4) decrease with advancing gestational age in mouse lungs (E14 to adult). Similarly, Hox A5, B5, and B6 follow the same decreasing pattern of expression with advancing gestational age in rat lungs (E15 to adult). RA treatment of E17 rat lung explants in culture resulted in a significant dose- and time-dependent increase in Hox A5, B5, and B6 mRNA levels. The highest mRNA levels were seen in explants treated with 1 x 10(-5) M RA for 4-16 h. We conclude that there are many homeobox genes expressed in developing rodent lung and that their mRNA levels are affected by both gestational age and RA.

Amino Acid Sequence↗
PubMed1994

Butyrate modulates surfactant protein mRNA in fetal rat lung by altering mRNA transcription and stability.

We examined the effects of Na butyrate, a known regulator of gene expression, on surfactant protein mRNA concentration, transcription, and degradation. Exposure of explants of 18-day fetal rat lung to Na butyrate resulted in a decrease in surfactant protein A (SP-A) mRNA concentration to 7% of control after 6 h and to 18% of control after 24 h. The reduction in SP-A mRNA concentration was associated with decreased mRNA transcription and stability at both these times. The effects on SP-B mRNA were similar to those on SP-A, but quantitatively less. In contrast, butyrate had a biphasic effect on SP-C mRNA concentration. There was an initial decrease to 30% of control at 6 h, followed by an increase to control levels by 24 h. Transcription of SP-C was increased at both these times, whereas degradation was enhanced at 6 h, but not at 24 h. The level of surfactant protein mRNA after butyrate treatment therefore depends on the balance between induced changes in transcription and degradation. Butyrate had no effect on gamma-actin mRNA concentration in this system. Circulating levels of butyric acid analogues are elevated in the mothers and fetuses in diabetic pregnancies. Some of these fetuses have delayed lung maturation and decreased amniotic fluid SP-A levels. We speculate that butyric acid analogues partially mediate the changes in pulmonary maturation induced by maternal diabetes.

Animals↗
PubMed1994

Amino acid substitutions in the hormone-binding domain of the human androgen receptor alter the stability of the hormone receptor complex.

We have investigated the basis of androgen resistance in seven unrelated individuals with complete testicular feminization or Reifenstein syndrome caused by single amino acid substitutions in the hormone-binding domain of the androgen receptor. Monolayer-binding assays of cultured genital skin fibroblasts demonstrated absent ligand binding, qualitative abnormalities of androgen binding, or a decreased amount of qualitatively normal receptor. The consequences of these mutations were examined by introducing the mutations by site-directed mutagenesis into the androgen receptor cDNA sequence and expressing the mutant cDNAs in mammalian cells. The effects of the amino acid substitutions on the binding of different androgens and on the capacity of the ligand-bound receptors to activate a reporter gene were investigated. Substantial differences were found in the responses of the mutant androgen receptors to incubation with testosterone, 5 alpha-dihydrotestosterone, and mibolerone. In several instances, increased doses of hormone or increased frequency of hormone addition to the incubation medium resulted in normal or near normal activation of a reporter gene by cells expressing the mutant androgen receptors. These studies suggest that the stability of the hormone receptor complex is a major determinant of receptor function in vivo.

Androgen-Insensitivity Syndrome↗
PubMed1994

Respiratory distress caused by parasites.

Parasitic etiologies of pulmonary symptoms generally represent some degree of immunocompromise or occur as uncommon presentations of common parasitic infections. In regions where HIV infection is more prominent, PCP probably will be the most common parasitic respiratory disease seen. In geographic regions where helminthic infections are more common, patients with respiratory symptoms and eosinophilia probably will be seen more often and require a search for a specific etiology. In more rare circumstances, travel to an endemic area may suggest a more exotic parasitic etiology for respiratory symptoms.

Animals↗
PubMed1993

Characterization of the pfmdr2 gene for Plasmodium falciparum.

We report the characterization of the pfmdr2 gene which is a gene related to the P-glycoprotein family but with a somewhat different structure than the mdr genes. Based on DNA sequence analysis of genomic clones, we have discovered that the pfmdr2 gene has 10 predicted transmembrane domains and a single ATP-binding site. In a homology search using GenBank sequences, we discovered that the pfmdr2 gene has a significant homology with the hmt1 gene in yeast. The yeast hmt1 gene is involved in cadmium resistance and is hypothesized to transport cadmium containing complexes from the cell. We have further characterized the pfmdr2 gene expression by northern analysis and discovered that it is expressed in a stage-specific manner, only at the trophozoite stage and not in ring stages. We have prepared a rabbit antibody to a recombinant fusion protein expressing a portion of the pfmdr2 coding region. In IFA analysis, this antibody stains trophozoites and not ring stages. Western analysis reveals a protein of approximately 110 kDa which is consistent with the size of the predicted open reading frame based on DNA sequence analysis. Based on this analysis and previous work, there is no evidence for a change in pfmdr2 expression in drug-resistant versus drug-sensitive parasites.

ATP-Binding Cassette Transporters↗
PubMed1993

Stage-specific transcripts of the Plasmodium falciparum pfmdr 1 gene.

Genes have been identified in Plasmodium falciparum which belong to the ATP-binding cassette superfamily of transport systems based upon sequence and structural homology. One of these genes, the pfmdr 1 gene, is homologous to the mammalian mdr 1 gene, which encodes the P-glycoprotein product. Strong phenotypic data suggests the involvement of a P-glycoprotein-like molecule in the mediation of drug resistance in P. falciparum. The goal of this work was to characterize the expression of the pfmdr 1 messenger RNA: both the stage specific expression and the level of expression in mefloquine sensitive or resistant parasites. We identified two messenger RNA homologous to this gene, one of 8.5 kb expressed in ring and trophozoite stages, and a second messenger RNA of 7.5 kb expressed only in trophozoites. Previously we had reported an increased expression of messenger RNA for pfmdr 1 in a mefloquine-resistant clone. Here we extend this and demonstrate that overexpression of the pfmdr 1 gene is consistent with the mefloquine resistance phenotype in a panel of recent isolates from Thailand.

Animals↗
PubMed1992

Risk of rupture of postangiographic femoral false aneurysm.

The surgical management of 50 false aneurysms caused by transfemoral arterial catheterization was reviewed to document the incidence and effects of rupture before repair. Twelve false aneurysms ruptured, leading to shock in six patients, distal ischaemia in three and stroke in one. The mean(s.d.) time from catheterization to rupture was 2.8(1.7) (range 1-6) days. Postoperative complications occurred in seven patients with ruptured and eight with non-ruptured aneurysms (P < 0.04). The mean(s.d.) age of patients with ruptured aneurysms was 67.2(6.3) (95 per cent confidence interval 63.5-70.8) years and those without 58.5(9.1) (95 per cent confidence interval 55.3-61.7) years (P < 0.008). On multiple regression analysis, age, peripheral vascular disease and raised plasma liver enzyme levels on admission were found to be significant independent predictive variables for rupture (all P < 0.05). It is recommended that patients with these risk factors undergo urgent operative correction of femoral false aneurysm.

Aneurysm, False↗
PubMed1992

Expression and characterization of full-length and partial human androgen receptor fusion proteins. Implications for the production and applications of soluble steroid receptors in Escherichia coli.

We have expressed fusion proteins encoding defined segments of the coding segment of the human androgen receptor (hAR) in Escherichia coli using the pGEX-2T expression vector. Large quantities of fusion proteins containing glutathione-S-transferase (GST) linked to the amino or carboxy terminal region of the receptor and a fusion protein containing the complete amino acid sequence of the androgen receptor were produced in soluble form. The GST hAR fusion proteins containing the hormone-binding domain of the androgen receptor exhibit high affinity specific binding for a variety of natural and synthetic androgens. Analysis of the binding properties of the complete and truncated androgen receptor fusion proteins revealed that the amino terminus affects the Kd of the fusion proteins for mibolerone (0.89 vs. 3.43 nM for the truncated and complete fusion proteins, respectively). Despite these differences, both the truncated and complete hAR fusion proteins exhibit a higher affinity for dihydrotestosterone than for testosterone, implying that the preferential affinity for dihydrotestosterone observed in androgen receptor prepared from native sources is a measure of the inherent structure of the hormone-binding domain. Furthermore, the ligand-receptor complex is stable, as the ligand is not easily displaced with unlabelled competitor and is stable to mild heat denaturation. Fusion proteins containing the DNA-binding domain demonstrate specific DNA binding, as evidenced by studies using segments of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) and synthetic glucocorticoid response elements. These studies establish that GST hAR fusion proteins exhibit physical properties similar to those of native androgen receptor. Affinity purification using a glutathione affinity resin and cleavage of the fusion proteins at a thrombin cleavage site permits a marked enrichment using a two-step purification. The use of such methods will facilitate the study of the normal and mutant receptor proteins.

Base Sequence↗
PubMed1992

Electron beam versus photon beam radiation therapy for the treatment of orbital lymphoid tumors.

Shielding the lens of the eye while treating orbital lymphoid tumors and achieving a relatively homogeneous dose can be a dosimetric challenge. There are many clinical cases of tumors close to one or both eyes, where it is desirable to spare the lens as much as possible. We will show isodose curves comparing direct anterior fields using various electron beam energies to a single 6 mv photon beam to a multifield combination. The presence of an eye shield in a photon beam may impair the dose distribution at the back of the eye to an undesirable degree. The strong in-depth dose drop-off in electrons and the ease with which the beam can be intercepted suggests that an electron beam with an eye shield may offer advantages. We designed a suspended movable lens shield .8 cm wide with a height of 4.5 cm made of cerrobend surrounded by a brass cylinder mounted to a thin plexiglass plate to insert into a Varian electron 6 x 6 cone. A 1.5 cm gap between block and eye surface was used. Verification of dose distribution under the lens shield was obtained by film dosimetry.

Humans↗