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C Watts

Publications and source records attributed to C Watts.

At least 73 records · Page 4Linked to original sources
PubMed1997

Characterization of transport of newly assembled, T cell-stimulatory MHC class II-peptide complexes from MHC class II compartments to the cell surface.

In human B cells MHC class II molecules acquire antigenic peptides in lysosome-related compartments called the MHC class II compartments (MIIC). How assembled complexes, capable of activating T cells, then reach the cell surface has not been fully resolved. We have used selective ablation of early and recycling endosomes to determine whether newly peptide-loaded class II molecules require functional recycling endosomes to exit to the cell surface. Cellular compartments accessed by transferrin-horseradish peroxidase conjugates were functionally inactivated by cross-linking with diaminobenzidine and hydrogen peroxide. Cells with ablated endosomal compartments were unable to recycle transferrin to the cell surface and could not deliver exogenous Ag for processing and presentation to T cells. In contrast, cells that had taken up Ag and assembled intracellular class II-peptide complexes before endosome ablation were still able to deliver class II-peptide complexes to the cell surface and stimulate T cell proliferation. This delivery was abolished in the presence of brefeldin A. These data show that the parts of the endocytic apparatus necessary for Ag delivery to the MIIC are not required for functional class II-peptide complexes to reach the cell surface. Moreover, the brefeldin A sensitivity of this final step in the class II molecule biosynthetic pathway suggests a vesicular intermediate for transport between the MIIC and the plasma membrane.

B-Lymphocytes↗
PubMed1997

Antigen endocytosis and presentation mediated by human membrane IgG1 in the absence of the Ig(alpha)/Ig(beta) dimer.

Membrane immunoglobulin (mIg) M and D heavy chains possess minimal (KVK) cytoplasmic tails and associate with the Ig alpha/Ig beta (CD79) dimer to achieve surface expression and antigen presentation function. In contrast, the cytoplasmic tail of mIgG is extended by 25 residues (gamma ct). We have tested the possibility that mIgG can perform antigen capture and presentation functions independently of the Ig(alpha)/beta dimer. We show that CD4/(gamma)ct chimeras are efficiently endocytosed partially dependent on a tyrosine residue in (gamma)ct. In addition, human mIgG was expressed on the surface of Ig(alpha)/Ig(beta)-negative non-lymphoid cells and mediated antigen capture and endocytosis. Antigen-specific human mIgG targeted antigen to MIIC-type vesicles in the Ig(alpha)/beta negative melanoma Mel JuSo and augmented antigen presentation 1000-fold, identical to the augmentation seen in Ig(alpha)/beta-positive B-cells expressing the same transfected mIgG. Thus, unlike mIgM, mIgG has autonomous antigen capture and presentation capacity, which may have evolved to reduce or eliminate the BCR's dependence on additional accessory molecules.

Amino Acid Sequence↗
PubMed1997

Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase.

Legumain is a cysteine endopeptidase that shows strict specificity for hydrolysis of asparaginyl bonds. The enzyme belongs to peptidase family C13, and is thus unrelated to the better known cysteine peptidases of the papain family, C1 (Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 461-486). To date, legumain has been described only from plants and a blood fluke, Schistosoma mansoni. We now show that legumain is present in mammals. We have cloned and sequenced human legumain and part of pig legumain. We have also purified legumain to homogeneity (2200-fold, 8% yield) from pig kidney. The mammalian sequences are clearly homologous with legumains from non-mammalian species. Pig legumain is a glycoprotein of about 34 kDa, decreasing to 31 kDa on deglycosylation. It is an asparaginyl endopeptidase, hydrolyzing Z-Ala-Ala-Asn-7-(4-methyl)coumarylamide and benzoyl-Asn-p-nitroanilide. Maximal activity is seen at pH 5.8 under normal assay conditions, and the enzyme is irreversibly denatured at pH 7 and above. Mammalian legumain is a cysteine endopeptidase, inhibited by iodoacetamide and maleimides, but unaffected by compound E64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane). It is inhibited by ovocystatin (cystatin from chicken egg white) and human cystatin C with Ki values < 5 nM. We discuss the significance of the discovery of a cysteine endopeptidase of a new family and distinctive specificity in man and other mammals.

Amino Acid Sequence↗
PubMed1997

Proteolytic cleavage of cellubrevin and vesicle-associated membrane protein (VAMP) by tetanus toxin does not impair insulin-stimulated glucose transport or GLUT4 translocation in rat adipocytes.

Acute insulin stimulation of glucose transport in fat and skeletal muscle occurs principally as a result of the hormonal induced translocation of the GLUT4 glucose transporter from intracellular vesicular stores to the plasma membrane. The precise mechanisms governing the fusion of GLUT4 vesicles with the plasma membrane are very poorly understood at present but may share some similarities with synaptic vesicle fusion, as vesicle-associated membrane protein (VAMP) and cellubrevin, two proteins implicated in the process of membrane fusion, are resident in GLUT4-containing vesicles isolated from rat and murine 3T3-L1 adipocytes respectively. In this study we show that proteolysis of both cellubrevin and VAMP, induced by electroporation of isolated rat adipocytes with tetanus toxin, does not impair insulin-stimulated glucose transport or GLUT4 translocation. The hormone was found to stimulate glucose uptake by approx. 16-fold in freshly isolated rat adipocytes. After a single electroporating pulse, the ability of insulin to activate glucose uptake was lowered, but the observed stimulation was nevertheless nearly 5-fold higher than the basal rate of glucose uptake. Electroporation of adipocytes with 600 nM tetanus toxin resulted in a complete loss of both cellubrevin and VAMP expression within 60 min. However, toxin-mediated proteolysis of both these proteins had no effect on the ability of insulin to stimulate glucose transport which was elevated approx. 5-fold, an activation of comparable magnitude to that observed in cells electroporated without tetanus toxin. The lack of any significant change in insulin-stimulated glucose transport was consistent with the finding that toxin-mediated proteolysis of both cellubrevin and VAMP had no detectable effect on insulin-induced translocation of GLUT4 in adipocytes. Our findings indicate that, although cellubrevin and VAMP are resident proteins in adipocyte GLUT4-containing vesicles, they are not required for the acute insulin-induced delivery of GLUT4 to the plasma membrane.

Adipose Tissue↗
PubMed1997

Constitutive macropinocytosis allows TAP-dependent major histocompatibility complex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells.

Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage-colony-stimulating factor have potent T cell-stimulatory properties both in vitro and in vivo. This has been well documented for major histocompatibility complex (MHC) class II-restricted responses, and more recently using peptide-loaded and protein-pulsed DC for CD8 responses following adoptive transfer in mice. An unresolved question concerns the capacity of BMDC to present exogenous antigen on MHC class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis driven by constitutive membrane ruffling activity. Up to one-third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15-min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell-for-cell basis. Presentation was TAP dependent, brefeldin A sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA-stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen-specific cytotoxic T lymphocyte response following adoptive transfer in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Effect of embryonic donor age and dissection on the DARPP-32 content of cell suspensions used for intrastriatal transplantation.

The aim of this study was to determine in vitro the DARPP-32 content of donor cells used for striatal transplantation in vivo. The effect of selective embryonic dissection of the lateral ganglionic eminence (LGE) was compared with the standard dissection of the whole ganglionic eminence (WGE) at each of three embryonic ages (14, 15, and 16 days of gestation) in the rat. The resultant cell suspensions were cultured for up to 7 days and incubated with antibodies against DARPP-32, a marker of striatal medium spiny neurons; beta-tubulin III, a neuronal marker; GFAP, a marker of reactive astrocytes; and Gal-C, a marker of oligodendrocytes. LGE dissection gave rise to more DARPP-32 neurons compared to WGE; but this relationship was only observed in the younger embryos. When older (16 days gestation) embryos are used there is no difference in the yield of DARPP-32 cells obtained from LGE and WGE. LGE dissections were also observed to contain fewer glial cells. There was no beneficial effect of LGE over WGE on survival of striatal neurons in vitro. These results have important implications for the selection and dissection of fetal donor material used in clinical trials of intrastriatal transplantation as a potential treatment for Huntington's disease.

Animals↗
PubMed1997

Disfluency in spasmodic dysphonia: a multivariate analysis.

This study examined visual analog scaling (VAS) judgments of disfluency by normal listeners in response to oral reading by speakers with spasmodic dysphonia (SD) and by nondysphonic controls, as well as the variables of frequency of occurrence of disfluencies, speaking rate, number of reading errors, and temporal acoustic measures of interword interval duration and articulation time. MANOVA yielded statistically significant differences between SD and control speakers for all variables except reading errors. Although no significant fluency-related differences were observed in terms of type of vocal spasm or voice tremor, significant differences in disfluency measures were obtained for clinical ratings of severity of dysphonia. Greater dysphonia severity ratings were associated with decreased fluency, but milder ratings were not necessarily associated with disfluency. Stepwise multiple regression analysis demonstrated that frequency of disfluency occurrence, speaking rate, and reading errors accounted for more than three fourths of the variability in VAS judgments of disfluency. Findings suggest that although disfluency is not a defining feature of SD, it does contribute significantly to the overall clinical impression of severity of the disorder.

Adult↗
PubMed1997

Neuronal cell transplantation for Parkinson's and Huntington's diseases.

The brain constitutes a privileged transplantation site. Under appropriate conditions neuronal tissues can survive transplantation into the damaged brain, integrate with the host, and alleviate functional impairments associated with neurological disease. The experimental techniques have been developed to the point of clinical application with demonstrable benefit in Parkinson's disease, and similar applications in Huntington's disease appear to be imminent. Nevertheless, present techniques require use of embryonic/fetal tissues which will limit the availability of donors for the foreseeable future. There is an active search for alternative sources of tissue that are equally effective but more readily available, including engineered cells, expanded stem/precursor cells, and xenografts.

Adrenal Glands↗
PubMed1997

A new keratin 2e mutation in ichthyosis bullosa of Siemens.

Ichthyosis bullosa of Siemens (IBS) is a rare autosomal dominant skin condition with features similar to epidermolytic hyperkeratosis (EH). Clinical symptoms are characterized by mild hyperkeratosis with an acral distribution. Histology shows epidermolysis of upper spinous and granular cells, whereas ultrastructurally, tonofilaments form perinuclear aggregates. IBS has been linked to the type II keratin cluster on chromosome 12q, and K2e mutations have recently been identified in IBS patients. We have studied genomic DNA from two IBS families and in both cases heterozygous point mutations were found in the 2B helical domain of K2e. One family had an established mutation in codon 493 (E493K), whereas the other had an unreported mutation in the adjacent codon (E494K). Both mutations were confirmed by allele-specific PCR. These data reinforce the hypothesis that mutations in the TYRKLLEGEE motif of the 2B helix are deleterious to keratin filament network integrity and provide further evidence for the involvement of K2e mutations in IBS.

Adolescent↗
PubMed1997

Capture and processing of exogenous antigens for presentation on MHC molecules.

Class I and class II MHC molecules bind peptides during their biosynthetic maturation and provide a continuously updated display of intracellular and environmental protein composition, respectively, for scrutiny by T cells. Receptor-mediated endocytosis, phagocytosis, and macropinocytosis all contribute to antigen uptake by class II MHC-positive antigen-presenting cells. Capture of antigenic peptides by class II MHC molecules is facilitated because antigen catabolism and class II MHC maturation take place in the same compartments or in communicating compartments of the endosome/lysosome system. These class II MHC-rich, multivesicular endosomes receive incoming antigen and can support not only antigen processing and class II MHC peptide loading but also the export of peptide/class II MHC complexes to the cell surface. A balance between production and destruction of antigenic peptides is achieved by the activity of local proteases and may be influenced by binding of antigen to other proteins both prior to the onset of processing (e.g. antibodies) and during antigen unfolding (e.g. MHC molecules). T cell determinants that can be released for MHC binding without a substantial processing requirement may be able to utilize a distinct minor population of cell surface class II MHC molecules that become available during peripheral recycling. Although peptides derived from exogenous protein sources are usually excluded from presentation on class I MHC molecules, recent evidence shows that this embargo may be lifted in certain professional antigen-presenting cells to increase the spectrum of antigens that may be displayed on class I MHC.

Animals↗
PubMed1996

Incorporating psychometric measures in selecting and developing surgeons.

Provides a review of the recent literature on the selection and development of surgical trainees and surgeons, and discusses findings on the validity of psychometric measures of ability and personality in such processes. Drawing on this body of research, outlines the pilot project recently completed by the authors at St George's Hospital in London, which sought to test the appropriateness of incorporating psychometrics in supporting the career development of junior doctors applying for surgical training. The results of this pilot study tend to confirm those provided by similar research on surgeons, as well as other clinical groups--that psychometric measures have a significant role to play in aiding the assessment and career development of doctors.

Attitude of Health Personnel↗
PubMed1996

Persistent back pain and sciatica in the United States: patient characteristics.

Low back pain is an extremely common, seriously disabling, nonfatal public health problem worldwide. The National Low Back Pain Study was a multicenter study of a large, heterogeneous group of patients who have been referred to either a neurosurgeon or an orthopedic surgeon for the evaluation and treatment of a persistent complaint of low back pain. In this paper, we characterize persistent low back pain patients and their complaints, describe the impact of persistent low back pain on the patients' functional and psychological status, report on the patients' medical characteristics, and identify treatments that are currently prescribed for these patients. Persistent low back pain is most common among people in their mid-to-late thirties and early-to-mid forties. The patients are mostly white, well educated, and generally affluent. The majority are gainfully employed, but some quit working because of pain and those who do tend to be less educated, and more likely to be involved in litigation. The average patient has had low back pain intermittently for 10 years. The pain is usually well localized but its severity varies considerably. Besides pain, most persistent low back pain patients report a variety of motor and sensory deficits. Patients also report significant functional impairment at work, at play, and at home. The typical patient does not, however, display significant psychological distress. Most patients have consulted multiple health care providers, have received a variety of treatments, and have used a variety of medications to alleviate pain; a few have been subjected to more aggressive treatment measures including surgery, intradiscal therapy, and narcotic and psychoactive drugs. None of these treatments has been effective. Physical examinations of these patients do not provide significant clues for making a definitive diagnosis. Nonspecific abnormalities such as muscle spasm, tenderness, and trigger points are quite common, but motor weakness and sensory deficits in the lower extremities, and reflex changes in the knees and ankles, are much less common. The classic combination of reflex changes, motor weakness, and sensory deficits associated with specific protruded discs are extremely rare even though one of three patients had a diagnosis of disc herniation. Diagnostic imaging studies revealed that the majority of persistent low back pain patients have spondylotic abnormalities involving root compression or lumbar instability or both, with root compression as the primary cause of the complaint. Myofascial syndrome and lumbar instability were the next most common diagnoses. After a thorough evaluation by specialists in spinal disorders, three of five persistent low back pain patients were prescribed an additional course of conservative therapy, one of five was prescribed surgery, and the rest were prescribed no treatment. Persistent low back pain patients appear to be a distinct group of low back pain patients who are different from patients who have similar nonpersistent acute symptoms and those who have the chronic pain syndrome characterized by significant behavioral and psychological co-morbidities.

Adult↗