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Biomedical subjects

S Watkins

Publications and source records attributed to S Watkins.

At least 37 records · Page 2Linked to original sources
PubMed2000

Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein.

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin-glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin-glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a gamma- and delta-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.

Amino Acid Sequence↗
PubMed2000

The pediatric intern retreat: 20-year evolution of a continuing investment.

For the past 22 years the interns in pediatrics at the University of Washington and Children's Hospital and Regional Medical Center have been relieved of all clinical duties in order to participate in a five-day retreat. The retreat provides an opportunity for the interns to learn more about their classmates, build stronger bonds, and provide mutual support. This retreat has been supported by the hospital, the department of pediatrics, faculty, fellows, and community physicians. The authors describe the history of the Intern Retreat, present its goals, daily activities, and faculty, and discuss how the retreat is funded and supported by the hospital and the medical community.

Humans↗
PubMed2000

Stent-based gene therapy.

Delivery of gene therapy to inhibit intimal hyperplasia has been proposed to prevent postangioplasty restenosis. We sought to apply gene therapy by using a stent-based technique. There are several hurdles that must be overcome before gene-stent therapy can be applied successfully in clinical trials. These include increasing the efficiency of gene delivery through atherosclerotic plaque; increasing intramural retention times; preventing the inflammatory reaction that stents coated with biodegradable polymers can elicit; overcoming the risk of systemic gene delivery; and accessing the adventitia via percutaneous approach. We evaluated a gene-stent delivery mechanism based on microporous metal microneedles developed with nanotechnology in an attempt to overcome some of these problems. A novel approach to the transfection of genes by microfabricated technology was evaluated in smooth muscle cells in culture. We demonstrated that microneedles can deliver gene therapy to smooth muscle cells in culture and can produce controlled penetration of the IEL and intima. We conclude that taller microneedles need to be developed to reach the media in diseased human arteries and that this technology has the potential to be incorporated in a stent to deliver gene therapy in atherosclerotic plaque.

Angioplasty, Balloon, Coronary↗
PubMed1999

Subcellular trafficking of the cytoplasmic expression system.

Cationic liposomes have provided many advantages over viral vector formulations; however, the problem of inefficient gene expression remains. This is due in part to the nuclear membrane, which limits DNA entry into the nucleus. Cytoplasmic expression systems using T7 RNA polymerase have been developed to express genes in the cytoplasm and avoid the need for nuclear import of DNA. Although these systems show improved transgene expression, little is known about how they function in transfected cells. Direct comparisons between a cytoplasmic and nuclear expression system were carried out with a 293 cell line stably expressing T7 RNA polymerase. A formulation for optimal reporter gene expression was developed and used in conjunction with a variety of subcellular trafficking inhibitors to study the process of DNA endocytosis. Transfected cells were also studied at different stages of the cell cycle to determine the dependence of each system on mitosis. These results showed that cytoplasmic and nuclear expression systems utilize similar endocytosis pathways to the point of endosomal release. Once DNA is released into the cytoplasm, the cytoplasmic expression system shows immediate expression that is proportional to the amount of DNA released. In contrast, DNA targeted for nuclear expression requires additional time for nuclear entry. The level of nuclear expression is also restricted by the limited amount of DNA that is imported into the nucleus. Finally, mitosis is required for effective nuclear expression but not for cytoplasmic expression. Therefore, the cytoplasmic expression system has considerable advantages over traditional nuclear expression systems and may be an effective method for transfecting nondividing cells. Efficient expression of genes delivered by nonviral vectors is hindered owing to poor nuclear transport of plasmid DNA. A potential solution to this problem would be to use a cytoplasmic expression system. Previous studies have shown that this method produces enhanced gene expression when compared with traditional nuclear expression systems; however, the actual mechanisms by which the cytoplasmic expression system works remains unknown. This article focuses on a direct comparison between cytoplasmic and nuclear expression in terms of optimal DNA delivery formulations, intracellular trafficking of DNA, and cell cycle dependence. These results indicate that the cytoplasmic expression system has two primary advantages over nuclear expression in that it does not rely on nuclear DNA transport or mitosis for efficient expression.

Anti-Bacterial Agents↗
PubMed1999

General practice. Variations enigma.

A study of general practice involving 23 health authorities and 2,270 practices completed last year, revealed considerable variations across England and Wales. Single-handed practices account for an average of 29 per cent of all those in the study. The majority of HAs had no practices linked to NHSnet. More than half the practices offered early morning surgeries, and many also offered appointments after six o'clock.

Benchmarking↗
PubMed1999

Human blood dendritic cell-like B cells isolated by the 5G9 monoclonal antibody reactive with a novel 220-kDa antigen.

We developed a murine IgG1 mAb, 5G9, following immunization of a BALB/c mouse with Daudi cells. By immunoprecipitation, 5G9 reacted with a 220-kDa Ag on Daudi cells, which reduced to four subunits (55, 65, 80, and 85 kDa). mAb 5G9 bound to 40-60% of peripheral blood B cells, weakly reacted with monocytes and granulocytes, and did not bind to erythrocytes, platelets, T cells, or NK cells. mAb 5G9 brightly stained scattered cells in human tonsil sections, which appeared to be dendritic cells (DC) by morphology. mAb 5G9 also stained scattered cells in cytospin slides of monocyte-derived DC with long, thin, beaded membrane processes, morphologically distinct from other monocyte-derived DC. Positive selection of blood mononuclear cells with mAb 5G9 and sheep anti-mouse IgG Dynabeads demonstrated an enriched population of DC. By flow cytometry analysis, these cells were CD19, CD20, CD22, CD40, CD44, CD83, CD86, IgD, and HLA-Dr positive and either kappa- or lambda-L chain positive. They did not express CD3, CD4, CD5, CD10, CD11b, CD13, CD25, CD56, CD14, CD33, or CD64. Isolated 5G9+ cells were potent APCs in allogeneic MLR, compared with 5G9- PBMC, 5G9- B cells, monocytes, and monocytes cultured in IL-4 and GM-CSF for 24 h. mAb 5G9 defines a novel peripheral blood cell with B cell phenotype and DC morphology and function: DC-like B cells. The significance of this cell in immune responses requires further study.

Adult↗
PubMed1999

Subcellular and cell-cycle expression profiles of CDK-inhibitors in normal differentiating myeloid cells.

A central question in hematopoiesis is how cell-cycling behavior changes during the emergence of the differentiated state. To further understand what genetic regulators might couple proliferation status to differentiation, we studied the expression of the cell-cycle inhibitors p21 and p27 during the in vitro differentiation of normal CD34(+) blast cells along the myeloid lineage. We find p27 but not p21 to be expressed in freshly harvested resting CD34(+) cells. Thereafter, p21 levels peak concurrent with cellular proliferation and then decline in expression as cells undergo terminal differentiation. In contrast, p27 levels are fairly constant but the subcellular localization of p27 changes from nuclear expression to predominantly cytoplasmic expression and finally to perinuclear localization at progressive stages of differentiation. This report discusses the implications of these findings.

Antigens, CD↗
PubMed1999

Overexpression of the human manganese superoxide dismutase (MnSOD) transgene in subclones of murine hematopoietic progenitor cell line 32D cl 3 decreases irradiation-induced apoptosis but does not alter G2/M or G1/S phase cell cycle arrest.

To determine whether overexpression of the human MnSOD transgene protected 32D cl 3 hematopoietic progenitor cells from ionizing irradiation, 32D cl 3 cells were co-electroporated with the pRK5 plasmid containing the human MnSOD transgene and SV2-neo plasmid with G418-resistant colonies selected. Two clones (1F2 and 2C6) were identified to overexpress the human MnSOD transgene by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and increased biochemical activity. Measurement of irradiation-induced damage was determined in cells removed from G418 for 1 week before irradiation. Irradiation survival curves, apoptosis tunnel assay, and Comet assay was performed. Cell cycle distribution was determined for each line at 0, 1, 3, 6, 24, and 48 hr after 500 cGy by fixing the cells in 70% ethanol, staining with propidium iodide, and analysis by flow cytometer. Biochemical MnSOD activity in U/mg protein was 2.6 for 32D cl 3 and significantly elevated to 8.4 and 6.6 (P < 0.001) U/mg protein for subclones 1F2 and 2C6, respectively. Irradiation survival curves demonstrated an increased shoulder on the irradiation survival curve for 1F2 and 2C6 cells with an n of 4.95 +/- 0.48 (P = 0.042) and 4.95 +/- 0.13 (P = 0.011), compared with 2.77 +/- 0.20 for 32D cl 3. A higher percent of 32D cl 3 cells demonstrated apoptosis at 24 and 48 hr after 1,000 cGy irradiation, compared with 1F2 and 2C6 cells (at 24 hr, 29.37% +/- 2.01% of 32D cl 3 cells were apoptotic compared with 5.21 +/- 2.61 (P = 0.018) and 5.27 +/- 2.58 (P = 0.004) for 1F2 and 2C6, respectively). Significantly more DNA strand breaks were detected by Comet assay in 32D cl 3 cells (Comet length at 600 cGy of 103.4 +/- 50.3 units, compared with 69.7 +/- 36.3 (P < 0.001) and 48.9 +/- 27.5 (P < 0.001) for 1F2 and 2C6, respectively). In contrast, irradiation-induced cell cycle arrest was similar between the cell lines with a G2/M phase arrest at 6 hr and a G1/S phase arrest at 24 and 48 hr after irradiation. While overexpression of MnSOD increases the shoulder on the irradiation survival curve of 32D cl 3 cells, decreases irradiation-induced apoptosis, and DNA strand breaks by Comet assay, irradiation-induced alterations in cell cycle distribution were not significantly altered. These 32D cl 3 subclonal lines overexpressing MnSOD provide a potentially valuable system with which to study the mechanism of irradiation-induced cell cycle arrest separate from irradiation-induced apoptosis.

Animals↗
PubMed1999

Impaired auditory frequency discrimination in dyslexia detected with mismatch evoked potentials.

Deficits in phonological skills appear to be at the heart of reading disability; however, the nature of this impairment is not yet known. The hypothesis that dyslexic subjects are impaired in auditory frequency discrimination was tested by using an attention-independent auditory brain potential, termed mismatch negativity (MMN) while subjects performed a visual distractor task. In separate blocks, MMN responses to graded changes in tone frequency or tone duration were recorded in 10 dyslexic and matched control subjects. MMN potentials to changes in tone frequency but not to changes in tone duration were abnormal in dyslexic subjects. This physiological deficit was corroborated by a similarly specific impairment in discriminating tone frequency, but not tone duration, which was assessed separately. Furthermore, the pitch discrimination and MMN deficit was correlated with the degree of impairment in phonological skills, as reflected in reading errors of regular words and nonwords. It is possible that in dyslexia a persistent sensory deficit in monitoring the frequency of incoming sound may impair the feedback control necessary for the normal development of phonological skills.

Acoustic Stimulation↗
PubMed1999

Morphogenetic events in mixed cultures of rat hepatocytes and nonparenchymal cells maintained in biological matrices in the presence of hepatocyte growth factor and epidermal growth factor.

Hepatocytes were grown in chemically defined hepatocyte growth medium (HGM) containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on collagen-coated polystyrene beads in roller bottle cultures, forming clusters of beads, and proliferating hepatocytes and nonparenchymal cells, including fenestrated endothelium-forming vascular structures. Desmin-positive cells surrounded hepatocytes. Collagen types I and III were deposited in a diffuse manner whereas collagen type IV surrounded the clusters of the epithelial cells, forming a basement membrane. When the mixed cell clusters were implanted in Matrigel (Collaborative Research, Bedford, MA), hepatocytes grew in three dimensions, forming plates and ducts. Many single, long plates of hepatocytes were seen, suggesting progressive linear assembly guided by hepatocyte specific structural parameters. HGF, EGF, and transforming growth factor-alpha (TGF-alpha) enhance these phenomena. HGF plus EGF elicited maximal response. TGF-beta1 suppressed formation of the ducts and plates. Within three months in Matrigel, the cultures established monolayers composed of plates, ducts, and a well-delineated canalicular network. The mixed cultures expressed albumin, A1AT, AFP, transferrin, and CYPIIB1. Following implantation of the cell clusters in Matrigel, there was decreased expression of c-met, urokinase, urokinase receptor, and TGF-beta1. Electron microscopy showed differentiated hepatocytes with nearly normal ultrastructure. The proliferating cell nuclear antigen (PCNA) labeling index was high (more than 80%) whereas the Bromo-deoxyaridine labeling index of ongoing DNA synthesis varied from 10% to 15%. These results show that the mixed cultures of proliferating hepatocytes and nonparenchymal cells can reproduce the hallmark structures of hepatic histological architecture while maintaining differentiation and the capacity to proliferate. (HEPATOLOGY 1999;29:90-100.)

Animals↗
PubMed1999

Longitudinal evaluation of transport kinetics in children receiving peritoneal dialysis.

Functional stability of the peritoneal membrane is necessary for maintenance of peritoneal dialysis (PD) as a therapeutic option. Few studies have investigated this issue in children. We evaluated the peritoneal membrane solute transport capacity longitudinally in 26 children (mean age 11.0+/-5.5 years) receiving long-term PD. Each patient underwent a standardized peritoneal equilibration test on two occasions (mean interval between studies 19.8+/-5.9 months) to determine solute dialysate to plasma (D/P) ratios, dialysate glucose to initial dialysate glucose (D/D(0)) ratios, and mass transfer area coefficients (MTAC). The correlation of transport capacity with peritonitis history was also assessed. No significant change in MTAC, D/P, or D/D(0) values were found when comparing original and follow-up data of the group overall. However, transport of creatinine and glucose was significantly (P<0.05) greater in the peritonitis group compared with the group without peritonitis, and differences in the change over time between the peritonitis groups was significant for MTAC creatinine (P=0.035) and D/D(0) glucose (P=0.020). In summary, this experience demonstrates functional stability of the peritoneal membrane in pediatric patients receiving PD. However, follow-up assessments of peritoneal solute kinetics may be necessary in patients with a history of peritonitis in order to permit early identification of those who may be at risk for ultrafiltration failure and sclerosing peritonitis.

Adolescent↗
PubMed1999

Optimal care of the pediatric end-stage renal disease patient on dialysis.

This manuscript is an effort on behalf of the American Society of Pediatric Nephrology to provide recommendations designed to optimize the clinical care of pediatric patients with end-stage renal disease (ESRD). Although many of the recommendations are evidenced-based with the supporting data being derived from a variety of sources, including patient registries, others are opinion-based and derived from the combined clinical experience of the authors. In all cases, it is recommended that the decision to initiate dialysis should be made only after an assessment of a combination of biochemical and clinical characteristics. Irrespective of the choice of dialysis modality (hemodialysis v peritoneal dialysis), dialysis efficacy should be measured regularly, and the dialysis prescription should be designed to achieve target clearances. Attention to dialysis adequacy, control of osteodystrophy, nutrition, and correction of anemia is mandatory, because all may influence patient outcome in terms of growth, cognitive development, and school performance. Finally, the availability of a multidisciplinary team of pediatric specialists is desirable to provide all facets of pediatric ESRD care, including renal transplantation, in an optimal manner. Future clinical research efforts intended to address topics such as dialysis adequacy, anemia management, and growth should be encouraged.

Adaptation, Psychological↗
PubMed1999

High polymorphism at the human melanocortin 1 receptor locus.

Variation in human skin/hair pigmentation is due to varied amounts of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eu- and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many mammals. We have sequenced the MC1R gene in 121 individuals sampled from world populations with an emphasis on Asian populations. We found variation at five nonsynonymous sites (resulting in the variants Arg67Gln, Asp84Glu, Val92Met, Arg151Cys, and Arg163Gln), but at only one synonymous site (A942G). Interestingly, the human consensus protein sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency (7%) in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. The MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon was sequenced to study the evolution of MC1R. The ancestral human MC1R sequence is identical to the human consensus protein sequence, while MC1R varies considerably among higher primates. A comparison of the rates of substitution in genes in the melanocortin receptor family indicates that MC1R has evolved the fastest. In addition, the nucleotide diversity at the MC1R locus is shown to be several times higher than the average nucleotide diversity in human populations, possibly due to diversifying selection.

Alleles↗
PubMed1999

NOX, a novel nitric oxide scavenger, reduces bacterial translocation in rats after endotoxin challenge.

Endotoxemia promotes gut barrier failure and bacterial translocation (BT) by upregulating inducible nitric oxide synthase (iNOS) in the gut. We hypothesized that administration of a dithiocarbamate derivative, NOX, which scavenges nitric oxide (NO), may reduce intestinal injury and BT after lipopolysaccharide (LPS) challenge. Sprague-Dawley rats were randomized to receive NOX or normal saline via subcutaneously placed osmotic pumps before or after LPS challenge. Mesenteric lymph nodes, liver, spleen, and blood were cultured 24 h later. Transmucosal passage of Escherichia coli C-25 or fluorescent beads were measured in an Ussing chamber. Intestinal membranes were examined morphologically for apoptosis, iNOS expression, and nitrotyrosine immunoreactivity. NOX significantly reduced the incidence of bacteremia, BT, and transmucosal passage of bacteria and beads when administered before or up to 12 h after LPS challenge. LPS induced enterocyte apoptosis at the villus tips where bacterial entry was demonstrated by confocal microscopy. NOX significantly decreased the number of apoptotic nuclei and nitrotyrosine residues. NOX prevents LPS-induced gut barrier failure by scavenging NO and its toxic derivative, peroxynitrite.

Animals↗
PubMed1998

Structural characterization of three genetic variants of human serum albumin modified in subdomains IIB and IIIA.

Three new genetic variants of human serum albumin have been detected in Italy by routine clinical electrophoresis. Albumin Milano Slow is common in Northern Italy, while albumins Liprizzi and Trieste, which are fast migrating, are rare and local variants. Isoelectric focusing analysis of the CNBr fragments obtained from the carboxymethylated alloalbumins in all cases localized the mutation to fragment CB5 (residues 330-446). The modified CNBr fragments were isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abnormal tryptic peptides revealed that all the variants are caused by single point mutations: Trieste, Lys359-->Asn, Milano Slow, Asp375-->His, and Liprizzi, Arg410-->Cys. These results were confirmed by sequence determination of a variant V8 peptide in the case of Trieste, and by DNA sequence analysis for the other two variants. The DNA analysis showed a G-->C transversion at nucleotide position 11969 for albumin Milano Slow, and a C-->T transition at position 13251 for Liprizzi. The latter represents a mutation at a hypermutable CpG dinucleotide site. Albumins Trieste and Milano Slow, as most of the variants thus far described, have mutations involving residues on the surface of the molecule. In contrast, albumin Liprizzi represents the first example of a mutation in the most active binding pocket of the molecule, placed in subdomain IIIA.

Amino Acid Sequence↗
PubMed1998

Pharmacokinetics and pharmacodynamics of zolmitriptan in patients with mild to moderate hypertension: a double-blind, placebo-controlled study.

Zolmitriptan is a potent selective 5HT1B/1D receptor agonist for acute migraine therapy. Zolmitriptan has vasoconstrictor activity in cerebral vessels and may cause slight elevations of blood pressure in subjects without hypertension. Therefore, the pharmacokinetics and pharmacodynamics of zolmitriptan (5, 10, and 20 mg) were evaluated in 16 patients with mild to moderate hypertension (controlled by hydrochlorothiazide 50 mg once daily) and 17 healthy age- and sex-matched control subjects in a randomized, placebo-controlled, double-blind, four-period crossover study. The pharmacokinetics of zolmitriptan and its metabolites were dose proportional. Although area under the concentration-time curve (AUC0-infinity) and maximum concentration (Cmax) were slightly higher in patients with hypertension at all doses, this was only statistically significant for AUC at the 20-mg dose. Differences between subjects with and without hypertension were not clinically significant. Zolmitriptan produced a small increase in blood pressure, but this was similar in subjects with and without hypertension and was of no clinical significance. Zolmitriptan was well tolerated in both groups. Zolmitriptan plasma concentrations were higher in women than in men, with higher values of AUC and Cmax and lower total clearance in women. These results indicate that zolmitriptan can be administered for treatment of migraine in patients with controlled hypertension without dose adjustment.

Area Under Curve↗
PubMed1998

Apoptosis in esophageal cancer following induction chemoradiotherapy.

BACKGROUND: The poor survival of patients with esophageal cancer following esophagectomy has led to intense investigation into combined modality therapy. Based on results from clinical trials examining chemoradiotherapy alone without surgery, resection has come under increased scrutiny and its necessity as a component of a multimodal approach has been questioned. In this study, we examined whether residual tumor cells in esophagectomy specimens following induction chemoradiotherapy are viable and, therefore, provide putative evidence for the appropriateness of esophagectomy. MATERIALS AND METHODS: Between August 1991 and January 1995, 46 patients were entered into an induction chemoradiotherapy trial consisting of 5-fluorouracil, cisplatin, alpha-interferon, and concurrent external beam radiotherapy followed by esophagectomy. Response was determined histologically and apoptosis assessed with a terminal deoxytransferase assay system. p53 status was determined by immunohistochemistry and mutational analysis. RESULTS: Thirty-eight patients underwent esophagectomy, 33 of whom had either a complete (n = 10) or partial (n = 23) response. None of the 28 patients with residual tumor in the resected specimen had 100% apoptotic cells and the vast majority of specimens had less than a 10% apoptotic rate. The percentage of apoptotic cells did correlate with tumor differentiation but not with histologic type nor presence of p53 mutations. CONCLUSIONS: These data suggest that resection following upfront chemoradiotherapy is a necessary component of a multimodality approach to esophageal cancer and will ultimately provide superior local-regional control to a nonsurgical approach.

Adult↗