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Biomedical subjects

R B Kelly

Publications and source records attributed to R B Kelly.

At least 19 recordsLinked to original sources
PubMed1992

Sorting of chromogranin B into immature secretory granules in pheochromocytoma (PC12) cells.

Two tyrosine sulfated proteins, chromogranin B and secretogranin II, are targeted into regulated secretory granules, which are stored until stimulation causes them to fuse with the plasma membrane. In PC12 cells, [35S]-sulfate labels chromogranin B, secretogranin II and proteoglycans just before they are sorted into the regulated and constitutive secretory pathways. Chromogranin B was secreted by both the regulated and constitutive secretory pathways. In contrast, very little secretogranin II was secreted constitutively. Formation of regulated and constitutive secretory vesicles from the trans Golgi network was reconstituted in vitro. Mechanically permeabilized PC12 cells released regulated and constitutive vesicles containing sulfated markers. At early chase times, chromogranin B was predominantly released from permeabilized cells in small vesicles that comigrated with constitutive secretory vesicles containing proteoglycans. At intermediate chase times the vesicles containing chromogranin B resembled immature secretory granules containing secretogranin II. At very long chase times chromogranin B and secretogranin II were found in mature secretory granules that did not escape the permeabilized cells in vitro. We conclude that chromogranin B is sorted into immature regulated secretory granules with lower efficiency than secretogranin II.

Animals↗
PubMed1992

The membrane IgM-associated proteins MB-1 and Ig-beta are sufficient to promote surface expression of a partially functional B-cell antigen receptor in a nonlymphoid cell line.

The B-cell antigen receptors consist of membrane immunoglobulins (mIgs) noncovalently associated with two accessory proteins, MB-1 and Ig-beta. We used transfection into a nonlymphoid cell line to test whether MB-1 and Ig-beta were sufficient to promote cell surface expression of mIgM capable of signal transduction. Expression of MB-1 and Ig-beta, but not MB-1 alone, allowed high-level surface expression of mIgM in the AtT20 endocrine cell line, which presumably lacks other B-cell-specific components. The reconstituted antigen receptor was capable of mediating some of the signaling reactions characteristic of mIgM in B lymphocytes. Crosslinking mIgM on transfected AtT20 cells stimulated tyrosine phosphorylation of MB-1 and Ig-beta and also increased the amount of phosphatidylinositol 3-kinase activity that could be precipitated with anti-phosphotyrosine antibodies. When total cell lysates were analyzed by anti-phosphotyrosine immunoblotting, however, no induced phosphorylation of more abundant proteins was detected. Moreover, crosslinking of the receptor in AtT20 cells did not stimulate inositol phospholipid breakdown. Thus, the transfected B-cell antigen receptor could initiate some signal transduction events but AtT20 cells may lack components required for other signaling events associated with mIgM.

Animals↗
PubMed1992

Patients' knowledge about fats and cholesterol in the Community Cholesterol Survey Project.

OBJECTIVE: The Community Cholesterol Survey Project assessed attitudes, knowledge, and behaviors relating to cholesterol. DESIGN: Survey. SETTING: Six outpatient primary care practice sites (two urban, two suburban, and two rural) in northeast Ohio. PARTICIPANTS: Four hundred seventy-seven site-, age-, and gender-stratified adult patients were enrolled from a total of 604 approached (79% recruitment). INTERVENTIONS: Self-administered questionnaire and structured dietitian interview. MAIN OUTCOME MEASURES: A knowledge score derived from responses to multiple-choice questions and a knowledge rating given by the study dietitian. Motivation and dietary health were similarly measured. RESULTS: Subjects did worse than random guessing for seven of 12 knowledge questions regarding label reading, fats, and cholesterol. In particular, the meaning of "hydrogenated" and the relative energy content of fats was poorly understood. Knowledge scores and ratings were significantly correlated (r = .52). Knowledge ratings were higher in those who were receiving a cholesterol-lowering diet or who had received other advice or treatment from their physician for high cholesterol level. By analysis of variance, knowledge measures were found to have significant independent positive associations with higher social status (P < .001) and living in a suburban area (P < .05). Motivation and dietary health demonstrated similar relationships to social status. CONCLUSIONS: To make use of patients' motivation for change, it will be essential to provide education at an effective level. Instruction in label reading or creation of more meaningful food labels may have the greatest impact. A particular challenge is the education of less advantaged patients to promote healthy nutrition practices.

Adult↗
PubMed1992

Immunocytochemical analysis of secretion mutants of Tetrahymena using a mucocyst-specific monoclonal antibody.

Dense-core granules represent an adaptation of specialized secretory cells to facilitate stimulus-regulated release of stored proteins. Such granules are a prominent feature of mammalian neuroendocrine and exocrine cells and are also well developed in the ciliates. In Tetrahymena thermophila, the ability to generate mutants in dense-core granule biosynthesis and fusion presents a versatile system for dissecting steps in regulated exocytosis. We have previously shown that defective granules in such mutants could be characterized by several biochemical criteria, including buoyant density, which increases during maturation, and the degree of proteolytic processing of the content precursors. We have now used indirect immunofluorescence, taking advantage of a monoclonal antibody directed against a granule protein, to visualize the morphology and distribution of both granules and putative granule intermediates in mutant and wild-type cells. The results are consistent with the biochemical analysis and extend our characterization of the mutants, allowing us to distinguish four classes. In addition, the assay represents a powerful technique for diagnosis of new mutants.

Animals↗
PubMed1992

Low density lipoprotein receptor and cation-independent mannose 6-phosphate receptor are transported from the cell surface to the Golgi apparatus at equal rates in PC12 cells.

Efficient transport of cell surface glycoproteins to the Golgi apparatus has been previously demonstrated for a limited number of proteins, and has been proposed to require selective sorting in the endocytic pathway after internalization. We have studied the endocytic fate of several glycoproteins that accumulate in different organelles in a variant clone of PC12, a regulated secretory cell line. The cation-independent mannose 6-phosphate receptor and the low density lipoprotein receptor, both rapidly internalized from the cell surface, and the synaptic vesicle membrane protein synaptophysin, were transported to the Golgi apparatus with equivalent, nonlinear kinetics. Transport to the Golgi apparatus (t1/2 = 2.5-3.0 h) was several times faster than turnover of these proteins (t1/2 greater than or equal to 20 h), indicating that transport of these proteins to the Golgi apparatus occurred on average several times for each protein. In contrast, Thy-1, a protein anchored in the membrane by a glycosylphosphoinositide group, was internalized and transported to the Golgi apparatus more slowly than the three transmembrane proteins. Since each of the transmembrane proteins studied showed the same t1/2 for transport to the Golgi apparatus, we conclude that transport of these proteins from the cell surface to the Golgi apparatus does not require sorting information specific to any one of these proteins. These results suggest that one of the functions of late endosomes is constitutive recycling of cell surface receptors through the Golgi apparatus if they fail to recycle to the cell surface directly from early endosomes, and that the late endosome recycling pathway is followed frequently by many rapidly internalized proteins.

Animals↗
PubMed1992

Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells.

Regulated secretory cells have two pathways that transport secreted proteins from the Golgi complex to the cell surface. To identify carrier vesicles involved in regulated and constitutive secretion, PC12 pheochromocytoma cells were labeled with [35S]sulfate to identify markers for the two secretory pathways, then mechanically permeabilized and incubated in vitro. Small constitutive secretory vesicles, containing mostly sulfated proteoglycans, accumulated during an in vitro incubation with ATP. In the presence of GTP gamma S, the constitutive vesicles became significantly more dense, suggesting that a coated intermediate was stabilized. Larger immature regulated secretory granules, enriched in sulfated secretogranin II, also escaped from the permeabilized cells in vitro. During granule maturation, their density increased and the amount of cofractionating proteoglycans diminished. The data suggest that sorting continues during secretory granule maturation.

Adrenal Gland Neoplasms↗
PubMed1992

Specific association of the proto-oncogene product pp60c-src with an intracellular organelle, the PC12 synaptic vesicle.

The protein product of the proto-oncogene c-src is a membrane-associated tyrosine kinase of unknown function. Identification of pp60c-src target membranes may elucidate the function of the c-src protein. The available evidence indicates that pp60c-src associates with distinct membranes within single cell types and has different distributions in different cell types. Our experiments demonstrate targeting of pp60c-src to an isolatable and biochemically identified membrane fraction in the neuroendocrine cell line PC12. The c-src protein was found to be specifically associated with synaptic vesicles since: (a) the pp60c-src immunofluorescent pattern overlapped with a synaptic vesicle marker, synaptophysin; (b) a significant proportion (44%) of the pp60c-src from PC12 but not fibroblast postnuclear supernatants was recovered in a small vesicle fraction; (c) an anti-synaptophysin cytoplasmic domain antibody immunodepleted all of the pp60c-src vesicles in this fraction, and (d) pp60c-src copurified during a 100-fold purification of PC12 synaptic vesicles. These results suggest a role for the c-src protein in the regulation of synaptic vesicle function.

Animals↗
PubMed1992

Selections from current literature: inhaled steroid use in asthma management.

Though our understanding of asthma has improved, its incidence, prevalence and associated death rate have been increasing in most industrialized countries. Inadequate treatment of airway inflammation may explain some of this trend. Steroids are the most effective anti-inflammatory agents and are effective and safe in inhaled form. Together with sodium cromolyn, they should be used more frequently in the management of all types of asthmatics, especially those who require chronic or acute administration of oral steroids.

Administration, Inhalation↗
PubMed1992

Adherence to screening flexible sigmoidoscopy in asymptomatic patients.

The purpose of this study was to assess patient adherence to physician-recommended screening flexible sigmoidoscopy. In the setting of a family practice residency program, adherence rates in asymptomatic patients (N = 333, age > or = 50 years) were compared among a Usual Care Group; an Intervention Group that received educational materials and a phone reminder; and a Continuity Group, which had longstanding continuity with a single physician. Data from mailed questionnaires (N = 180) were used to examine the associations of demographic factors and attitudes with adherence. Adherence was 30.3% overall, with a nonsignificant increase in the Intervention Group compared with the Usual Care Group. In a pooled analysis of the Usual Care and Continuity Groups, the half of the sample with the highest continuity had a significantly higher adherence rate than the rest of the sample (45%; P < 0.001). In a discriminant analysis (78% correct classification, P < 0.001) two history variables (family history of cancer; family history of colon problems), one measure of continuity (number of physician visits), one demographic variable (lower household income), and two attitudinal factors (perception of how painful flexible sigmoidoscopy would be; perception of how well the physician explained its importance) made statistically significant contributions to the prediction of adherence. Results of the study show that screening flexible sigmoidoscopy is acceptable to asymptomatic patients, and that continuity is likely to have a positive impact on adherence. Because attitudes offer the potential for modification, we suggest that physicians reassure patients that flexible sigmoidoscopy is not unduly painful and discuss with patients individually its importance to their health.

Aged↗
PubMed1992

Does practice location or academic connection affect recruitment of patients as research subjects?

Recruitment rates of study subjects are important planning and validity issues for many research projects. The Community Cholesterol Survey Project (CCSP) offered an opportunity to examine recruitment rates by type of study practice. The CCSP used six primary care practices (2 urban, 2 suburban, 2 rural), including a suburban academic residency program. Recruitment into the study was performed by a single dietician who followed the same procedure at each site. The overall recruitment rate of subjects was 79%. The residency site had the lowest recruitment rate (59%), which was significantly lower than any of the other sites (79% to 90%, average 84%, p less than 0.001). No demographic factors provided an explanation for this finding. The demographic factors examined included patient age, gender, race, social status, marital status, years in the practice, and presence of underlying medical conditions. These results add to the evidence that recruitment rates in nonacademic practices may be substantially better than those in residency practices and other academic sites.

Academies and Institutes↗
PubMed1991

Preventive medicine in primary care. Moving from theory to practice.

Primary care physicians are in a unique position to offer preventive services. They have access to patients when intervention may be most successful, and they see most patients over a long period, which allows for adequate follow-up. The challenges are choosing the most effective interventions and organizing records and office staff to facilitate delivery of preventive services. Preventive medicine may not be as dramatic or as immediately successful as curative medicine, but it can have a tremendous long-term impact on health and well-being.

Humans↗
PubMed1991

Tyrosine phosphorylation of components of the B-cell antigen receptors following receptor crosslinking.

Crosslinking membrane immunoglobulin (mIg), the B-cell antigen receptor, stimulates tyrosine phosphorylation of a number of proteins. Since many receptors are phosphorylated after ligand binding, we asked if components of the mIg receptor complexes were tyrosine-phosphorylated after mIg crosslinking. Both mIgM and mIgD are noncovalently associated with at least two other proteins. mIgM is associated with the MB-1 protein, which is disulfide-linked to a protein designated Ig-beta. mIgD is not associated with MB-1 but is with IgD-alpha, which is also disulfide-linked to Ig-beta. Using immunoprecipitation with a specific anti-MB-1 antiserum followed by anti-phosphotyrosine immunoblotting, we found that crosslinking mIgM stimulated tyrosine phosphorylation of MB-1, Ig-beta, and a previously unidentified 54-kDa polypeptide associated with MB-1. In mature splenic B cells that express both mIgM and mIgD, mIgM crosslinking stimulated tyrosine phosphorylation of the 32-kDa MB-1 protein, whereas mIgD crosslinking stimulated tyrosine phosphorylation of MB-1-related proteins of 33 and 34 kDa. The 32-kDa MB-1 protein was only associated with mIgM, whereas the 33- and 34-kDa MB-1-related proteins were specifically associated with mIgD and are most likely IgD-alpha. Thus, crosslinking either mIgM or mIgD stimulated tyrosine phosphorylation only of the MB-1-related proteins associated with that receptor.

Animals↗
PubMed1991

Maturation of dense core granules in wild type and mutant Tetrahymena thermophila.

Tetrahymena thermophila, a ciliated protozoan, has a well-developed pathway of regulated secretion from dense core granules called mucocysts. Since exocytosis-defective mutants are available, steps in the biogenesis of dense core granules and their fusion with the plasma membrane may be resolved genetically. To describe the steps in biochemical terms, we have generated antisera against mucocyst content proteins. One antiserum is directed against a calcium binding protein, p40, that is released on stimulation of exocytosis. p40 is shown to associate with an insoluble matrix in mature mucocysts. In addition, the antiserum recognizes a larger protein, p60, that is soluble, is not found in mature mucocysts and is not released on stimulation. Pulse-chase experiments support a precursor-product relationship between p60 and p40. Using these proteins as markers, two mutant Tetrahymena strains defective in exocytosis have been shown to accumulate the putative precursor p60 in organelles that can be distinguished from one another and from wild type mucocysts on the basis of density. The kinetics of appearance of insoluble p40 and the mutant phenotypes suggest a model of mucocyst maturation in which sorting precedes matrix condensation.

Animals↗
PubMed1991

Prediction of motivation and behavior change following health promotion: role of health beliefs, social support, and self-efficacy.

Dimensions of health beliefs (perceived risk of behavior and benefit of behavior change), social support (family and others' support for change), and self-efficacy (magnitude and strength) were examined in 215 patients undergoing a prospective trial of health promotion in a primary care medical practice. Discriminant analyses were performed to evaluate how well these dimensions predicted motivation for change and lifestyle behavior change. These relationships were examined for six lifestyle areas: cigarette smoking, dealing with stress, amount and type of food eaten, use of seat belts, and exercise habits. The analyses demonstrated a statistically and clinically significant prediction of motivation by one or more health belief and self-efficacy dimensions for most lifestyle areas. The strongest single predictors were perceived benefits and self-efficacy strength, which were each significant predictors of motivation in four lifestyle areas (P less than 0.05). Support dimensions, as measured, were not shown to have predictive value in most areas. Behavior change was poorly predicted by beliefs, support, and self-efficacy for most lifestyle areas. However, adding motivation to the discriminant function equation resulted in significant predictions in all six lifestyle areas (P less than 0.05), with an average correct classification rate of 71%. This finding strongly suggests that motivation is a very important intervening variable when evaluating health promotion and resulting behavior change.

Adolescent↗
PubMed1991

Synaptophysin is sorted from endocytotic markers in neuroendocrine PC12 cells but not transfected fibroblasts.

The targeting of synaptophysin, a major synaptic vesicle protein, in transfected nonneuronal cells has important implications for synaptic vesicle biogenesis, but has proved controversial. We have analyzed four transfected cell types by differential centrifugation and velocity gradient sedimentation to determine whether synaptophysin is targeted to endosomes or to synaptic vesicle-like structures. Synaptophysin was recovered only in vesicles that sedimented more rapidly than synaptic vesicles. The synaptophysin-containing vesicles were labeled if a surface-labeled cell was warmed to 37 degrees C, comigrated with transferrin receptor-containing vesicles on velocity and density gradients, and could be completely immunoadsorbed by anti-LDL receptor tail antibodies. These data demonstrate that synaptophysin was targeted to the early endocytotic pathway in the transfected cells and are inconsistent with the suggestion that synaptophysin expression induces a novel population of vesicles. Targeting of synaptophysin to early endosomes implicates their role in synaptic vesicle biogenesis.

Adrenal Gland Neoplasms↗
PubMed1991

Secretory granule and synaptic vesicle formation.

Sorting of secreted proteins into dense-core secretory granules may involve selective aggregation of regulated secretory proteins, rather than a conventional sortase. Synaptic vesicles, which mediate paracrine communication between adjacent cells, appear to arise by a modification of the early endosome pathway. Targeting to the cell surface involves the actin-based cytoskeleton and small GTP-binding proteins.

Animals↗
PubMed1991

Molecular architecture of the nerve terminal.

The synaptic vesicle has been the focus of recent efforts to generate a molecular description of the nerve terminal. The function and molecular composition of the synaptic vesicle are well characterized, due in part to its relative simplicity. Recent work identifies specific proteins involved in some of the functions of the synaptic vesicle and their mechanism of action.

Animals↗