Biomedical subjects

B S Wilson

Publications and source records attributed to B S Wilson.

At least 55 records · Page 3Linked to original sources

PubMed1994

Cellular variations in heterotrimeric G protein localization and expression in rat pituitary.

The secretory cells of the anterior pituitary are regulated by hypothalamic and target endocrine hormones. In many cases, intracellular signaling after ligand binding to cell surface receptors is mediated by heterotrimeric G proteins. In this study, cells from the rat pituitary and two pituitary cell lines (GH3 and AtT-20 cells) were doubly labeled by immunofluorescence with specific rabbit antibodies to the alpha-subunits of G proteins (Gs, Gi1-3, or Gq) and monoclonal antibodies against pituitary hormones (GH, PRL, beta LH, beta TSH, or beta FSH) to identify specific cell types. Gs, Gi, and Gq were detected in all secretory types, but differences were found in their levels of expression and cellular distribution. The cell periphery was the predominant site of localization of all these G proteins. In addition, both Gi3 and at least one member of the Gq family were seen by immunofluorescence on intracellular sites typical of the Golgi region. By immunogold labeling, alpha i3 was localized to membranes of Golgi cisternae in sections of rat pituitary and GH3 cells, where it was concentrated on the cis-side of the Golgi stack. These findings demonstrate that G proteins are widely expressed in anterior pituitary cells and further support a role for Gi alpha 3, and possibly Gs, in intracellular membrane trafficking.

Animals↗

PubMed1994

Increased protein expression through improved ribosome-binding sites obtained by library mutagenesis.

This report describes a method whereby library mutagenesis combined with drug selection was used to generate unique and efficient ribosome-binding sites (RBS) for expressing recombinant proteins in Escherichia coli. The RBS was deleted from a vector expressing beta-lactamase and replaced with a 16-base sequence containing a library of mutations. Selection of the library with ampicillin yielded several unique RBS sequences that were more efficient than ompA RBS for expressing a bacterial (beta-lactamase) and a mammalian protein (single-chain Fv antibody). The described approach provides a practical means to improve recombinant protein expression and, also, provides new sequences to further evaluate the complex regulatory mechanism underlying translation initiation.

Animals↗

PubMed1993

Endoplasmic reticulum-through-Golgi transport assay based on O-glycosylation of native glycophorin in permeabilized erythroleukemia cells: role for Gi3.

An assay for endoplasmic reticulum (ER)-through-Golgi transport has been developed in streptolysin O-permeabilized murine erythroleukemia (MEL) cells. The reporter proteins are metabolically labeled native murine glycophorins, which display a distinctive shift in electrophoretic mobility after acquisition of O-linked oligosaccharides. The O-linked sugars are acquired at a site distal to a brefeldin A block, presumably in a cis Golgi compartment, and sialylation occurs in middle and/or trans Golgi compartments. In permeabilized cells supplemented with cytosolic proteins and an ATP-generating system, 20-50% of the radiolabeled precursor glycophorins can be converted to the mature, sialylated form. This maturation process is ATP- and cytosol-dependent and is blocked by guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]). Electron microscopy of permeabilized MEL cells shows retention of ER elements, stacked Golgi cisternae, free polysomes, and other subcellular components. In the presence of GTP[gamma S], dilated vesicles accumulate around the Golgi stacks. Antisera to the carboxyl terminus of the Golgi resident alpha subunit of Gi3 inhibit maturation of glycophorin. To our knowledge, a transport assay utilizing O-glycosylation of an endogenous protein as a monitor of ER-through-Golgi traffic in permeabilized cells has not been reported previously. Furthermore, the data provide evidence for heterotrimeric GTP-binding protein involvement in Golgi function.

Adenosine Triphosphate↗

PubMed1993

Increased antibody expression from Escherichia coli through wobble-base library mutagenesis by enzymatic inverse PCR.

We tested the value of a new library mutagenesis approach, called library enzymatic inverse PCR (LEIPCR), for expression-level enhancement of antibody Fv fragments produced in Escherichia coli. The production level of active, metal chelate-specific antibody from our constructs is limited by a low expression level of the second, heavy-chain cistron. To increase the production level, LEIPCR was applied to the wobble bases of the second cistron leader peptide. In LEIPCR mutagenesis, the entire plasmid is amplified using mutagenic primers with class-IIS restriction endonuclease (ENase) sites at their 5' ends. The PCR product is digested with the class-IIS ENase (here, BsaI; GGTCTCN[symbol: see text]NNNN[symbol: see text]), which removes its own recognition sequence, and the ends are self-ligated. Thus, LEIPCR can be used to make plasmid mutant libraries regardless of the nucleotide sequence, and independent of available ENase sites. The resulting library of 10(7) wobble mutants was screened for active Fv by a colony filter lift. A selected mutant was shown to produce between four- and elevenfold more active Fv than the wild type (wt), and fivefold more heavy chain. Mutations outside of the leader peptide were shown not to be involved. The mutated areas of the mRNAs of two different up-mutants may have less secondary structure than the wt. Thus, the sequence of the mRNA of the second leader peptide was limiting to the expression level of heavy-chain and active Fv.

Amino Acid Sequence↗

PubMed1993

New processing strategies for multichannel cochlear prostheses.

Various strategies for representing speech information with multichannel cochlear prostheses were compared in tests with implant patients. The strategies included the compressed analog (CA) approach of a standard clinical device, and alternative interleaved pulses (IP) and continuous interleaved sampling (CIS) strategies. CA and IP strategies had been compared in previous studies with a wide range of subjects. The present studies compared all three types in tests with one subject and CA and CIS strategies in tests with six additional subjects. Subjects for the present studies were selected for their excellent performance with the clinical CA processor, and the tests included closed-set identification of consonants and open-set recognition of words and sentences. For every test, every subject obtained his or her highest score, or repeated a score of 100% correct, using a CIS strategy. In the comparisons of all three approaches, IP processor scores were between those obtained with CA and CIS processors. The results are discussed in terms of their implications for processor design.

Cochlear Implants↗

PubMed1993

Importance of patient and processor variables in determining outcomes with cochlear implants.

Within-subjects comparisons of processing strategies for cochlear implants are reviewed. Compressed analog strategies were compared to interleaved pulses strategies in tests with one group of 8 subjects, and to continuous interleaved sampling strategies in tests with another group of 11 subjects. The tests included open-set recognition of words and sentences. The results show that, while different strategies may produce quite different outcomes across subjects, individual performances with one processing strategy are significantly correlated with those of alternative strategies. These findings emphasize the importance of patient variables in determining outcomes across a variety of prosthesis designs.

Acoustic Stimulation↗

PubMed1993

Driving performance in persons with mild senile dementia of the Alzheimer type.

OBJECTIVE: To assess the effect of mild senile dementia of the Alzheimer type (SDAT) on driving ability. DESIGN: Cross-sectional study with correlation analysis. SETTING: A university-based Alzheimer's Disease Research Center, which evaluates community-living older adult volunteers, and the university's Program in Occupational Therapy. PARTICIPANTS: Healthy elderly controls (n = 13) and subjects with very mild (n = 12) and mild (n = 13) SDAT. Dementia severity was staged by the Washington University Clinical Dementia Rating. MEASUREMENTS: The driving ability of participants on the in-car road test was scored independently by a driving instructor, blinded to the study design and to the dementia status of the subjects, and an unblinded occupational therapist. Interview-based perceptions of driving ability were obtained independently from the subjects and their collateral sources. Attentional and visuospatial performances of the subjects were assessed prior to the road test. RESULTS: All control and very mild SDAT subjects were judged to be "safe" drivers (ie, passed the in-car road test), but five (40%) of the mild SDAT subjects had driving impairment sufficient to "fail" the road test. Neither subject self-assessment nor caregiver perceptions of driving ability consistently predicted driving performance. Attentional task performance correlated well with road test results. CONCLUSIONS: Some SDAT subjects retain "safe" driving skills. The greater the dementia severity, the greater the likelihood of poor driving ability. Performance-based (road test) evaluations are necessary to properly determine driving skills at present, but attention and other cognitive screening measures should be developed.

Aged↗

PubMed1993

White matter lesions and cerebral atrophy on MR images in patients with and without AIDS dementia complex.

OBJECTIVE: The objective of this study was threefold: to determine if the frequency of deep white matter changes and cerebral atrophy seen on MR images is significantly different between patients with and without AIDS dementia complex, to determine if certain patterns of white matter changes are more closely associated with AIDS dementia complex, and to determine if focal lesions within the white matter of the splenium are more common in AIDS dementia complex. MATERIALS AND METHODS: Forty-five patients with AIDS were clinically examined for AIDS dementia complex. MR images from these patients were retrospectively reviewed without knowledge of the clinical results. The presence or absence of white matter abnormalities and cerebral atrophy was evaluated by using graded scales and correlated with the presence or absence of AIDS dementia complex. RESULTS: Ten patients met the criteria for AIDS dementia complex. Eight of 25 patients in whom MR images showed abnormal signal intensity in deep white matter had dementia compared with two of 20 in whom MR showed no changes in deep white matter. The presence of these deep white matter abnormalities was not significantly different between groups with and without dementia (p = .08), although higher grades of deep white matter abnormality were more likely to be associated with AIDS dementia complex. Nine of 19 patients in whom MR images showed atrophy had dementia compared with one of 26 in whom MR showed no atrophy. Atrophy was significantly associated with AIDS dementia complex (p = .001). Eight of 15 patients in whom MR images showed abnormal signal intensity within the white matter of the splenium had dementia compared with two of 30 in whom MR showed normal signal intensity in this area. The degree of abnormality in the splenium was weakly associated with AIDS dementia complex (Kendall's tau = .471, p = .001). CONCLUSION: MR findings of cerebral atrophy and abnormal signal intensity in the splenium are associated with AIDS dementia complex. The presence of generalized deep white matter abnormalities does not differ significantly between patients with and without dementia, although more severe grades of white matter abnormality are more likely to be seen in patients with AIDS dementia complex.

AIDS Dementia Complex↗

PubMed1993

Design and evaluation of a continuous interleaved sampling (CIS) processing strategy for multichannel cochlear implants.

Two approaches for representing speech information with multichannel cochlear prostheses are being compared in tests with implant patients. Included in these studies are the compressed analog (CA) approach of a standard clinical device and research processors utilizing continuous interleaved sampling (CIS). Initial studies have been completed with nine subjects, seven of whom were selected on the basis of excellent performance with the Ineraid clinical processor, and the remaining two for their relatively poor performance with the same device. The tests include open-set recognition of words and sentences. Every subject has obtained a higher score--or repeated a score of 100% correct--on every test when using a CIS processor. These results are discussed in terms of their implications for processor design.

Acoustic Stimulation↗

PubMed1993

Selection of an active single chain Fv antibody from a protein linker library prepared by enzymatic inverse PCR.

Enzymatic inverse PCR mutagenesis was developed as a simple and reliable method for the construction of large libraries of site-directed mutants. Enzymatic inverse PCR library mutagenesis uses a single PCR fragment and is restriction-site independent. The usefulness of the technique was demonstrated by the design of a single chain linker for an antibody Fv fragment without computer modeling. The Fv fragment of an antibody specific for a metal chelate was expressed in active form in the periplasm of E. coli. The light and the heavy chains of the Fv are expressed as a bicistronic mRNA. Enzymatic inverse PCR mutagenesis was used to construct a library of 3 x 10(5) Fv mutants, in which the C-terminus of the light chain was connected to the N-terminus of the heavy chain by a 15-amino acid peptide linker of variable composition. After plating, active mutant colonies were identified by screening colony filter lifts with a radiolabeled hapten, N'-(2-hydroxyethyl)-p-thioureidobenzyl EDTA. About 0.2% of the mutants were positive, and a selected sFv clone was shown to have the same affinity as the Fv (9 x 10(9)) and was similar to the whole antibody (11 x 10(9)). This example compares favorably with both of the other approaches to constructing sFv's; namely, molecularly modeled linkers as well as universal linkers, which have often yielded significantly lower affinities than whole antibodies or Fabs. The enzymatic inverse PCR library mutagenesis approach is simple and reliable and can be used to obtain linkers for the great majority of antibodies for which no structural data are available. More generally, it can be used to modify DNA coding for any structural protein or regulatory element.

Base Sequence↗

PubMed1992

High level expression of transfected G protein alpha i3 subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts.

The alpha subunits of pertussis toxin-sensitive G proteins Gi1, Gi2 and Gi3 have been shown to inhibit adenylyl cyclase in transfected cells. However, Gi3 has recently been associated with protein transport and localized to the Golgi apparatus in a number of cell lines, rather than to the plasma membrane. We studied NIH 3T3 clones stably expressing different levels of a constitutively activated mutant of the alpha subunit of Gi3 (alpha i3-Q204L). Transfected alpha i3 subunits were localized to the Golgi apparatus in all NIH 3T3 clones. In clones expressing alpha i3-Q204L at high levels, alpha i3 subunits were also localized to the plasma membrane. Those clones which demonstrated expression of alpha i3 at the plasma membrane showed a 40% to 60% inhibition of forskolin-induced cAMP accumulation. Transfected NIH 3T3 clones in which plasma membrane alpha i3 was undetectable, did not show inhibition of forskolin-induced cAMP accumulation. These data suggest that, unless high expression is achieved in transfected cells, alpha i3 is targeted predominantly to the Golgi, not to the plasma membrane, and does not control adenylyl cyclase activity in NIH 3T3 fibroblasts.

3T3 Cells↗

PubMed1991

Better speech recognition with cochlear implants.

HIGH levels of speech recognition have been achieved with a new sound processing strategy for multielectrode cochlear implants. A cochlear implant system consists of one or more implanted electrodes for direct electrical activation of the auditory nerve, an external speech processor that transforms a microphone input into stimuli for each electrode, and a transcutaneous (rf-link) or percutaneous (direct) connection between the processor and the electrodes. We report here the comparison of the new strategy and a standard clinical processor. The standard compressed analogue (CA) processor presented analogue waveforms simultaneously to all electrodes, whereas the new continuous interleaved sampling (CIS) strategy presented brief pulses to each electrode in a nonoverlapping sequence. Seven experienced implant users, selected for their excellent performance with the CA processor, participated as subjects. The new strategy produced large improvements in the scores of speech reception tests for all subjects. These results have important implications for the treatment of deafness and for minimal representations of speech at the auditory periphery.

Cochlear Implants↗

PubMed1991

Regulation of IgE receptor-mediated secretion from RBL-2H3 mast cells by GTP binding-proteins and calcium.

In RBL-2H3 rat basophilic leukemia cells, cholera toxin does not per se stimulate secretion but it enhances secretion stimulated by antigens that crosslink IgE receptors, by the Ca2+ ionophore, ionomycin, and by thapsigargin, a tumor promoter that releases cytoplasmic Ca2+ stores. Calmodulin inhibitors reduce both the basal and cholera toxin-enhanced secretory responses to antigen and Ca2(+)-mobilizing agents. These synergistic effects suggest that the activation of a Gs-like GTP-binding protein, together with a (probably calmodulin-dependent) event activated by an increase in cytoplasmic Ca2+ levels, may jointly provide a sufficient signal for secretion. Antigen-stimulated secretion is inhibited by depleting cells of GTP with mycophenolic acid but is maximal in cells treated with mycophenolic acid plus cholera toxin. The simplest explanation is that cholera toxin selectively reactivates the Gs-coupled pathway leading to secretion in GTP-depleted cells without restoring the activity of a separate GTP-binding protein(s) that constrains antigen-stimulated secretion.

Animals↗

PubMed1991

Impaired secretion and increased insolubilization of IgE-receptor complexes in mycophenolic acid-treated (guanine nucleotide-depleted) RBL-2H3 mast cells.

In RBL-2H3 rat leukemic mast cells, cross-linking anti-DNP IgE-receptor complexes with multivalent antigen (DNP-BSA) activates a signal transduction pathway leading to Ca2+ influx and secretion. Cross-linking IgE-receptor complexes also stimulates a pathway that inactivates (desensitizes) receptors; this pathway becomes important at high concentrations of cross-linking antigen. Recent evidence that antigen-induced secretion is impaired by mycophenolic acid (MPA), an inhibitor of guanine nucleotide synthesis de novo, has implicated a GTP-binding protein (G protein) in the signaling pathway. Other recent studies have indicated that the conversion of cross-linked receptors to a detergent-insoluble (cytoskeleton-associated) form at high antigen concentrations is correlated with the loss of signaling activity. Here we show that secretion elicited by an optimal concentration of antigen (0.05 micrograms/ml DNP-BSA) is only inhibited by about 25% in guanine nucleotide-depleted cells, whereas secretion elicited by 5 micrograms/ml DNP-BSA, a concentration in the range that causes the high-dose inhibition of secretion, is inhibited by more than 60%. We also show that IgE-receptor complexes are insolubilized in response to 5 but not 0.05 micrograms/ml DNP-BSA in both control and guanine nucleotide-depleted cells. Importantly, the extent of insolubilization elicited by 5 micrograms/ml DNP-BSA is increased by more than 60% in the guanine nucleotide-depleted samples. These results raise the possibility that guanine nucleotide depletion reduces the secretory response to high antigen concentrations in two ways: by inhibiting the G protein-coupled signaling pathway and by increasing the availability of receptors to the pathway leading to receptor insolubilization and inactivation.

Animals↗

PubMed1991

Anatomy of the major fissure: evaluation with standard and thin-section CT.

The major fissures of the right and left lungs were studied with standard computed tomography (CT) (10-mm-thick sections) and thin-section CT (2-mm-thick sections) in 50 patients. On standard CT scans, the major fissures were seen in 90%-100% of cases at each of three selected levels. They usually appeared as hypoattenuating bands and less often as lines or hyperattenuating bands. Although in most cases the major fissure was seen as a line on thin-section CT scans, this appearance was more common in the upper portion of the left major fissure than in the upper portion of the right major fissure. A "double-fissure sign" was most frequently seen at the base of the left lung; however, the sign was also seen at higher levels, with approximately equal frequency in the right and left lungs. An incomplete major fissure was noted in the right lung in 32 cases (64%) and in the left lung in 26 cases (52%). The upper and middle portions of the left major fissure were less frequently incomplete than were the comparable portions of the right major fissure. Thin-section CT provided better delineation of the major interlobar fissures than did standard CT.

Aged↗

PubMed1991

Coding strategies for multichannel cochlear prostheses.

Comparisons of analog and pulsatile coding strategies for multichannel cochlear prostheses are reviewed. The results are related to design considerations for pediatric implants, including efficacy, safety, ease of fitting, and access to future improvements.

Adult↗

PubMed1990

Radiolocalization of human small cell lung cancer and antigen-positive normal tissues using monoclonal antibody LS2D617.

The murine monoclonal antibody LS2D617, which reacts with an antigen associated with human small cell lung carcinoma (SCLC), was tested in preclinical models to assess its potential for specific targeting of tumors in human SCLC cancer patients. LS2D617 detects a cell antigen on the surface of cultured SCLC and neuroblastoma cell lines. Scatchard analysis of the binding of LS2D617 to NCIH69 SCLC cells indicates an affinity constant of about 1 x 10(8) M-1 and an epitope expression level of approximately 2 x 10(6) antigenic sites/cell. Molecular weight analysis of the target antigen and antibody competition experiments showed that LS2D617 should be classified as a SCLC Cluster 1 antibody (i.e., reacts with the neural cell adhesion molecule). LS2D617 was labeled with 111In and tested for biodistribution (4, 24, 48, 72, and 96 h postinjection) in nude mice bearing the human SCLC NCIH69 tumor. Tumor values peaked at about 35% injected dose/g (Day 3) compared with about 8% injected dose/g for an irrelevant IgG1 antibody while normal tissue accumulation for both antibodies was about 2-8% injected dose/g. Immunohistochemical studies demonstrated that LS2D617 reacts with the central nervous system, peripheral nerves, endocrine tissues, and heart tissue of rabbits as it does in human tissues. The ability of LS2D617 to accumulate in vivo in normal tissues that express the specific target antigen was tested in rabbits. Rabbits given i.v. injections of 111In-LS2D617 or control labeled antibody were sacrificed at 48 h and tissues were examined by gamma well counting, autoradiography, and immunohistochemical staining for murine immunoglobulin. Specific uptake was seen in all sites defined as antigen positive by immunohistology (i.e., heart, liver bile duct, peripheral nerves, pituitary, adrenal), excepting the central nervous system (brain and spinal cord) which was inaccessible to antibody because of the blood brain barrier. The use of preclinical in vivo targeting models to assess tumor as well as antigen-positive normal tissue targeting should aid in the strategy of antibody-based therapeutic intervention of human cancer by providing insight into the potential for tumor targeting and normal tissue toxicity that may be encountered in the clinic.

Animals↗

PubMed1990

Lymphoscintigraphy in melanoma: initial evaluation of a low protein dose monoclonal antibody cocktail.

A low protein dose (73 +/- 10 micrograms total) 131I-labeled monoclonal antibody cocktail made of equal microgram quantities of 225.28S (IgG2a) and 763.24T (IgG1) murine monoclonal antibodies, which bind additively to a high molecular weight antigen of melanoma, was evaluated as a lymphoscintigraphic agent in 17 patients with intermediate to thick (mean Breslow depth, 3.39 +/- 0.64 mm) melanomas or clinical Stage II disease scheduled for nodal dissection. Eleven of the patients were clinically Stage I while 6 were clinically Stage II. 131I antibody cocktail, 258 +/- 10 microCi, was administered s.c. at the site of the primary melanoma or its scar following surgical removal. In eight patients, 63 +/- 8 microCi of 125I nonspecific normal sheep IgG was coadministered s.c. Gamma camera imaging was conducted beginning immediately after and continuing for several days following injection. Surgical resection, weighing, and gamma counting of the draining lymph nodes were undertaken in all patients. On gamma scans, early nodal uptake of antibody was most pronounced and of longest duration in the tumor pathologically positive patients (5 of 7 had visible nodal uptake, 4 of 7 visually stable or rising with time), with the t 1/2 of nodal clearance by gamma scan significantly (P less than 0.05) longer than in the negative patients in whom 4 of 10 showed some, although generally transient (0 of 10 stable or rising), nodal uptake. Scans were not easily interpretable when the injection site was very near the draining nodal group, in part due to the detection of scatter activity from the injection site. In several instances the scan was correct and the clinical examination was incorrect as regards nodal disease. Quantitative analysis of the surgically excised draining nodes showed significantly (P less than 0.001) more 131I anti-melanoma antibody uptake in the 21 tumor-involved nodes [0.01217% injected dose (ID)/node median] than in the 512 tumor-negative nodes (0.00051% ID/node median). Median percentage ID/g of anti-melanoma antibody in tumor-involved nodes was significantly greater (P less than 0.01) than in tumor-negative nodes (0.01984 versus 0.003215% ID/g). 125I-labeled nonspecific antibody did not accumulate significantly more in the tumor-involved nodes on a per node or per g basis in the 283 of 533 nodes studied using the dual-label approach (0.0036 versus 0.00092% ID/g). These data demonstrate that by external imaging and by tissue counting that a radiolabeled anti-melanoma monoclonal antibody cocktail can specifically accumulate to melanoma-involved lymph nodes following s.c. administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗