Search PubMed⌕ Search

Biomedical subjects

S M Wilson

Publications and source records attributed to S M Wilson.

At least 91 records · Page 5Linked to original sources
PubMed1996

Osteopontin expression in lung cancer.

Osteopontin (OPN), an integrin-binding, transformation-associated protein, is secreted by tumor cell lines in culture and is associated with increased malignancy in some experimental tumor systems. Little is known, however, about the significance of OPN expression in human cancers. The aims of this study were to determine if OPN was expressed in a series of surgically resected lung cancers, and if there was a relationship between OPN expression and clinico-pathologic findings or outcome. Twenty-five patients who underwent curative pulmonary resection were studied prospectively. RNA was extracted from primary tumor and distant normal lung tissue for each patient. OPN RNA levels were evaluated by northern blotting. Immunohistochemistry on paraffin-embedded tissue, using an anti-OPN monoclonal antibody, was performed to assess tissue distribution of OPN protein. OPN RNA and protein were over-expressed in the majority of tumors, relative to paired normal tissue. There was variation in the cells of the tumor that were OPN-immunopositive. In some cases OPN was present in tumor cells, while in the majority of cases OPN was detected primarily in tumor-infiltrating macrophages and necrotic areas. Over-expression of OPN RNA or protein generally was not related to clinico-pathological findings. However, there was a statistically significant association between OPN-immunopositivity in the tumor and patient survival. These findings suggest that OPN levels in lung tumors have the potential to provide clinically important predictive information on patient outcome, and that OPN may play a role in the biology of lung cancer.

Adult↗
PubMed1996

Activation of apical P2U purine receptors permits inhibition of adrenaline-evoked cyclic AMP accumulation in cultured equine sweat gland epithelial cells.

Experiments were undertaken using cultured equine sweat gland epithelial cells that express purine receptors belonging to the P2U subclass which allow the selective agonist uridine triphosphate (UTP) to increase the concentration of intracellular free Ca2+ ([Ca2+]i). Experiments using pertussis toxin (Ptx), which inactivates certain guanine-nucleotide-binding proteins (G-proteins), showed that this response consisted of Ptx-sensitive and Ptx-resistant components, and immunochemical analyses of the G-protein alpha subunits present in the cells showed that both Ptx-sensitive (alpha i1-3) and Ptx-resistant (alpha q/11) G-proteins were expressed. P2U receptors may, therefore, normally activate both of these G-protein families. Ptx-sensitive, alpha i2/3 subunits permit inhibitory control of adenylate cyclase, and UTP was shown to cause Ptx-sensitive inhibition of adrenaline-evoked cyclic AMP accumulation, suggesting that the receptors activate Gi2/3. Experiments using cells grown on permeable supports suggested that P2U receptors became essentially confined to the apical membrane in post-confluent cultures. Polarised epithelia may, therefore, express apical P2U receptors which influence two centrally important signal transduction pathways. It is highly improbable that these receptors could be activated by nucleotides released from purinergic nerves, but they may be involved in the autocrine regulation of epithelial function.

Adenylate Cyclase Toxin↗
PubMed1995

Sr2+ can become incorporated into an agonist-sensitive, cytoplasmic Ca2+ store in a cell line derived from the equine sweat gland epithelium.

We have explored the properties of a Ca(2+)-dependent cell-signalling pathway that becomes active when cultured equine sweat gland cells are stimulated with ATP. The ATP-regulated, Ca(2+)-influx pathway allowed Sr2+ to enter the cytoplasm but permitted only a minimal influx of Ba2+. Experiments in which cells were repeatedly stimulated with ATP suggested that Sr2+, but not Ba2+, could become incorporated into the agonist-sensitive, cytoplasmic Ca2+ store. Further evidence for this was provided by experiments using ionomycin, a Ca2+ ionophore which has no affinity for Sr2+.

Adenosine Triphosphate↗
PubMed1995

The effect of a phorbol ester upon the cholinergic regulation of potassium permeability in the rat submandibular gland.

Acetylcholine releases calcium from cytoplasmic stores and permits an influx of calcium in salivary acinar cells. The resultant rise in [Ca2+]i causes an increase in potassium permeability which is an important part of the secretory response. We have investigated the effects of 12-O-tetradecanoyl phorbol-13-acetate, a potent activator of protein kinase C, upon this regulation of potassium permeability in superfused pieces of rat submandibular salivary gland. This compound inhibited the initial [Ca2+]O-independent component of the response of acetylcholine but had no effect upon the subsequent [Ca2+]O-dependent phase. This compound does not, therefore, appear to inhibit receptor-regulated calcium influx.

Acetylcholine↗
PubMed1995

Twin analysis of odor identification and perception.

Findings from the first twin analysis using the University of Pennsylvania Smell Identification Test (UPSIT) and a phenyl ethyl alcohol (PEA) threshold detection test are presented. A genetic influence on odor identification was suggested for males, but not for females, consistent with previous twin research on physical measures. In addition, females scored significantly higher on the UPSIT than males. A curvilinear age trend for odor identification was detected for males, but not for females. In contrast, neither genetic, age, nor gender effects were suggested for PEA sensitivity. Performance on the olfactory tests was unrelated to scores on standard measures of intelligence.

Adolescent↗
PubMed1995

Expression of muscle actins in diffuse mesotheliomas.

Ultrastructural examinations have shown myofibroblastoid differentiation in sarcomatoid/desmoplastic mesotheliomas, but immunohistochemical expression of muscle actins seldom has been documented. We examined 10 sarcomatoid, 12 epithelial, and five biphasic mesotheliomas immunohistochemically for the expression of muscle-specific actin (MSA) and smooth muscle actin (SMA) and compared it with that in 12 specimens of lung cancer. All of the sarcomatoid mesotheliomas were found to be positive for both MSA and SMA. The epithelial cells in nine epithelial and two biphasic mesotheliomas were positive for MSA, but SMA was only positive in one epithelial mesothelioma. Conversely, the lung cancers were negative for both MSA and SMA in the epithelial cells, except for one specimen that was weakly positive for MSA. The stromal cells in both the epithelial mesotheliomas and lung cancers were negative for cytokeratin but were positive for MSA and SMA, whereas the sarcomatoid and biphasic mesothelioma spindle cells were positive for all three antibodies. We concluded that sarcomatoid mesothelioma was positive for MSA and SMA, which is in support of its myofibroblastic differentiation, and that positivity for MSA in some epithelial mesotheliomas might be of diagnostic value in differentiation from lung cancers.

Actins↗
PubMed1995

Calcium-dependent regulation of membrane ion permeability in a cell line derived from the equine sweat gland epithelium.

We measured the rates of 125I- and 86Rb+ efflux from preloaded, cultured equine sweat gland cells. The calcium ionophore ionomycin increased the efflux of both isotopes. Anion efflux was unaffected by Ba2+, but this cation inhibited 86Rb(+)-efflux, suggesting that [Ca2+]i-activated potassium channels were present. Activation of these channels was not, however, important for the efflux of anions. We measured 125I- efflux from valinomycin-depolarised cells in which anion cotransport was inhibited. Changes in 125I- efflux reflect changes in anion permeability under these conditions, and ionomycin caused a clear permeability increase that was abolished by the anion channel blocker diphenylamine-2-carboxylate. ATP and UTP increased the efflux of both isotopes, suggesting that type P2U purine receptors allow these nucleotides to regulate membrane permeability.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1995

Detection of parasites of the Leishmania donovani-complex by a polymerase chain reaction-solution hybridization enzyme-linked immunoassay (PCR-SHELA).

A polymerase chain reaction (PCR) based on the detection of the Lmet2 repeat sequence specific to members of the Leishmania donovani-complex is described. To improve PCR specificity, a post-PCR hybridization step is often performed but this usually involves an entirely new procedure with additional manipulations, expense and time. We have simplified this post-PCR hybridization by developing a strategy which includes the probe in the PCR and enables the hybridization to be performed automatically as part of the PCR programme. The hybrids are afterwards detected by capture in microtitre wells and colorimetric visualization. This method, which we have termed PCR-solution hybridization enzyme-linked immunoassay (PCR-SHELA), is rapid, able to detect less than 5 cultured parasites and is specific for parasites of the Leishmania donovani-complex. We also describe the application of PCR-SHELA to the detection of amastigotes in various tissues of infected laboratory animals.

Animals↗
PubMed1995

Simplified PCR for detection of Haemophilus ducreyi and diagnosis of chancroid.

A simplified PCR was developed for detection of Haemophilus ducreyi in samples from chancroid patients. The strategy included a straightforward chloroform extraction sample preparation method, a one-tube nested PCR to minimize contamination risks, and a colorimetric method for detection of products. Primers were designed from published nucleotide sequences of the 16S rRNA gene of H. ducreyi, with longer outer primers for annealing at a higher temperature and shorter inner primers labelled with biotin and digoxigenin for binding with avidin and colorimetric detection. The PCR technique detected all 35 strains of H. ducreyi tested, from four different geographical regions, and was negative for other, related strains of bacteria and for the common contaminating bacteria tested. Of 25 samples from H. ducreyi culture-positive chancroid patients, 24 were PCR positive and 1 produced a weak reaction. Of 83 samples from clinical cases of chancroid in the Republic of South Africa, 69 were PCR positive. The sensitivity of PCR compared with that of clinical diagnosis was 83%. All 50 negative control samples were negative. Encouraging results were also obtained with a consecutive series of 25 genital ulcer patients in Tanzania, of whom 9 were PCR positive. The adaptations of this simplified PCR strategy, at the sensitivity and specificity levels obtained, mean it will be useful for detection of H. ducreyi in areas where the organism is endemic, particularly where testing by culture is difficult or impossible.

Base Sequence↗
PubMed1995

DNA-based methods in the detection of Leishmania parasites: field applications and practicalities.

As conventional methods for the detection and/or diagnosis of infections with Leishmania parasites have limitations, the DNA-based alternatives have received much attention. By targeting multi-copy sequences such as kinetoplast DNA, ribosomal RNA genes, mini-exon-derived RNA genes or genomic repeats, the sensitivity of these systems can be increased. Similarly, by targeting conserved or variable regions of these targets, the specificity can be tailored to the genus, complex, species or even the individual isolate level. There are two main approaches to DNA-based detection: DNA probes involving hybridization; and amplification approaches such as PCR. DNA probes are less sensitive than amplification but are useful for large-scale screening of sandfly vectors or reservoir hosts, for example. PCR is much more sensitive and has been used for patient diagnosis with a sensitivity greater than microscopy or culture. The application of DNA probes and PCR can be simplified using chemiluminescent or colorimetric end-points, respectively, but both techniques require some specialized equipment and a certain degree of technical expertise. For this reason, their use is limited to research laboratories or central diagnostic facilities.

Animals↗
PubMed1994

Reduced malignancy of ras-transformed NIH 3T3 cells expressing antisense osteopontin RNA.

Osteopontin (OPN) is a secreted, calcium-binding phosphoprotein that frequently has been associated with the transformed phenotype. To clarify the function of OPN in tumor cells, we designed experiments to: (a) express antisense OPN RNA in murine PAP2 cells (metastatic, ras-transformed NIH 3T3 cells) and (b) examine the effects of antisense OPN expression on the tumorigenic and metastatic properties of the cells. PAP2 cells were transfected with pNMH-asOPN, an inducible, mammalian expression vector that can generate antisense OPN RNA complementary to the OPN mRNA. Two clones have been identified that expressed antisense OPN RNA in vitro. While reduced OPN protein secretion was not detected when the cells were grown in vitro, the in vivo expression of antisense OPN RNA was associated with reduced tumorigenicity. Tumors that did arise, with greatly extended lag time, had lost expression of antisense OPN RNA in vivo, suggesting that antisense OPN RNA expression was associated with reduced tumorigenicity of these cells.

3T3 Cells↗
PubMed1994

The effect of removing external sodium upon the cholinergic regulation of potassium permeability in the rat submandibular gland in vitro.

Fragments of rat submandibular gland were loaded with 86Rb+ and superfused so that the rate of 86Rb(+)-efflux could be quantified as an indicator of potassium permeability. Acetylcholine evoked an increase in permeability consisting of a transient, calcium-independent response and a sustained, calcium-dependent. Total removal of external sodium significantly inhibited both phases of this response. The results thus confirm that the cholinergic regulation of potassium permeability is compromised by removal of external sodium but do not support the view that this is due, exclusively, to an effect on calcium influx.

Acetylcholine↗
PubMed1994

Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae.

To study the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs), we have characterized nuclear polyadenylated RNA-binding (Nab) proteins from Saccharomyces cerevisiae. Nab1p, Nab2p, and Nab3p were isolated by a method which uses UV light to cross-link proteins directly bound to poly(A)+ RNA in vivo. We have previously characterized Nab2p, and demonstrated that it is structurally related to human hnRNPs. Here we report that Nab1p is identical to the Np13p/Nop3p protein recently implicated in both nucleocytoplasmic protein shuttling and pre-rRNA processing, and characterize a new nuclear polyadenylated RNA-binding protein, Nab3p. The intranuclear distributions of the Nab proteins were analyzed by three-dimensional immunofluorescence optical microscopy. All three Nab proteins are predominantly localized within the nucleoplasm in a pattern similar to the distribution of hnRNPs in human cells. The NAB3 gene is essential for cell viability and encodes an acidic ribonucleoprotein. Loss of Nab3p by growth of a GAL::nab3 mutant strain in glucose results in a decrease in the amount of mature ACT1, CYH2, and TPI1 mRNAs, a concomitant accumulation of unspliced ACT1 pre-mRNA, and an increase in the ratio of unspliced CYH2 pre-mRNA to mRNA. These results suggest that the Nab proteins may be required for packaging pre-mRNAs into ribonucleoprotein structures amenable to efficient nuclear RNA processing.

Amino Acid Sequence↗
PubMed1994

The regulation of membrane 125I- and 86Rb+ permeability in a virally transformed cell line (NCL-SG3) derived from the human sweat gland epithelium.

We have explored the factors that may regulate membrane permeability in a cell line (NCL-SG3) derived from the human sweat gland epithelium. Ionomycin increased the rate of 125I-efflux from preloaded cells and this action appeared to be due to an increase in intracellular free calcium ([Ca2+]i). The ionomycin-evoked increase in 125I- efflux was reduced in cells that were exposed either to barium or to valinomycin in the presence of a high concentration of external potassium. It thus appears that a fraction of the ionomycin-evoked increase in 125I- efflux is due to the activation of potassium channels and experiments using 86Rb+ also suggested that ionomycin increased the rate of potassium efflux, an effect which was totally abolished by barium. Blockade of Na(+)-K(+)-2Cl- cotransport and of Cl- -HCO3- exchange reduced the basal rate of 125I- efflux and the ionomycin-evoked increase in 125I-efflux from control cells and from cells depolarized by valinomycin. These transport systems thus contribute to anion efflux, although [Ca2+]i-dependent chloride channels also appear to be present. Acetylcholine increases [Ca2+]i in the secretory cells of human sweat glands, but this neurotransmitter did not increase [Ca2+]i in NCL-SG3 cells and so membrane permeability was not under cholinergic control. Adrenaline did not increase [Ca2+]i, but this hormone did evoke cyclic-3',5'-adenosine monophosphate (cyclic AMP) production. However, membrane permeability was not under adrenergic control, as the cells did not appear to express functional, cyclic AMP-dependent anion channels. This may be because they were not fully differentiated under the culture conditions. ATP consistently evoked a dose-dependent increase in anion efflux that appeared to be mediated by [Ca2+]i. The increase in [Ca2+]i was initiated by the release of calcium from a limited internal store and was subsequently sustained by calcium influx. UTP and ADP also increased [Ca2+]i, whereas adenosine, AMP and alpha,beta-methylene ATP were without effect. These data thus suggest that a subclass of type 2 purine receptor, which is functionally coupled to phosphoinositidase C, is present in these cells.

Calcium↗
PubMed1994

Extracellular ATP can activate autonomic signal transduction pathways in cultured equine sweat gland epithelial cells.

Changes in intracellular free calcium concentration ([Ca2+]i) were monitored in a cell line that was derived from the equine sweat gland epithelium. ATP and closely related compounds could increase [Ca2+]i with a rank order of potency of UTP > or = ATP > ADP >> AMP = adenosine = alpha,beta-methylene-ATP. The responses to ATP and to UTP were initiated by the release of calcium from an internal store and subsequently sustained by calcium influx. The rise in [Ca2+]i thus seems to be mediated by P2U receptors that are coupled to phosphoinositidase C. Some desensitisation of this response developed during repeated stimulation with ATP and this was blocked by staurosporine, an inhibitor of protein kinase C, and augmented by a phorbol ester which acts as an exogenous activator of this enzyme. A protein-kinase-C-dependent inhibitory pathway thus seems to become active during repeated stimulation with ATP. ATP and related compounds could also raise cellular cyclic AMP content. The order of potency was ATP > ADP = AMP = adenosine >> UTP, suggesting that this response is mediated via a separate subclass of P2 receptor. The present results demonstrate that ATP can activate autonomic signal-transduction pathways in cultured equine sweat gland cells and suggest that there may be a purinergic component to the control of secretory activity in the equine sweat gland.

Adenosine Triphosphate↗
PubMed1994

A colorimetric PCR method for the detection of M. leprae in skin biopsies from leprosy patients.

A one-tube nested polymerase chain reaction (PCR) method for the diagnosis of paucibacillary leprosy was developed using the repetitive RLEP sequence as a target. Detection of the PCR products was simplified by the adaptation of a colorimetric method. The test was specific for Mycobacterium leprae, and the sensitivity of the assay was 1 fg of purified genomic M. leprae DNA (less than one genome). Complete concordance was seen between the development of color and resolution on agarose gels. The results of frozen skin sections from untreated patients showed that the assay could detect 100% of multibacillary samples [bacterial index (BI) of 2 or more] and 69% and 70% of the samples with BIs of 1 and 0, respectively. The use of one-tube nested PCR in assessing the effectiveness of multidrug therapy (MDT) in leprosy also was determined. The simplified colorimetric assay was found to be sensitive, rapid and specific, and is suitable for use in routing diagnostic laboratories.

Base Sequence↗
PubMed1993

Comparative analysis of two alternative first exons reported for the mouse osteopontin gene.

Two conflicting conclusions regarding the structure of the mouse osteopontin (OPN) gene were tested for their validity. Miyazaki et al. (Miyazaki, Y., Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S., and Yamamoto, S. (1990) J. Biol. Chem. 265, 14432-14438) state that the OPN gene is composed of six exons and spans approximately 4.8 kilobases. Craig and Denhardt (Craig, A. M., and Denhardt, D. T. (1991) Gene (Amst.) 100, 163-171) independently reported an additional exon 5' to the region designated as "exon 1" by Miyazaki and colleagues. To investigate this discrepancy, we generated oligodeoxynucleotide probes to three regions of these reported sequences and used them to hybridize to Northern and Southern blots of RNA and DNA from mouse fibroblasts and macrophages. Two of these regions (probes "A" and "B") represent sequences that are disputed, while one of these regions (probe "C") is predicted to be in the mRNA of both sequences. Our results are consistent only with the OPN gene structure reported by Craig and Denhardt and show that a significant portion of the exon 1 reported by Miyazaki et al. is present in murine genomic DNA but is not found in cytoplasmic message. Our results also show that OPN RNA molecules from mouse fibroblasts and macrophages do not differ significantly in the regions tested.

3T3 Cells↗