Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Tetanus exotoxin inhibited Ca 2+ -dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca 2+ . The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca 2+ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca 2+ -dependent exocytotic pathway to inhibit secretion.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66452/1/j.1471-4159.1988.tb01059.x.pd

Phorbol Esters Enhance Exocytosis from Chromaffin Cells by Two Mechanisms

Treatment with phorbol esters such as 12- O -tet-radecanoylphorbol acetate (TPA) rapidly enhances [ 3 H]norepinephrine secretion from digitonin-permeabilized adrenal chromaffin cells. When TPA treatment was prolonged for several hours, a second distinct enhancing effect was observed. This later enhancement was most prominent at intracellular Ca 2+ concentrations of 3–30 ΜM , and did not require the continued presence of membrane-bound protein kinase C for its expression. The effect could be elicited by as little as 30-min exposure to TPA, followed by several hours in TPA- free medium. This effect of TPA was blocked by actinomycin D and cycloheximide, indicating a requirement for RNA and protein synthesis. Similar effects were seen when intact cells that had been pretreated with TPA were stimulated to secrete by depolarizing concentrations of K + . Thus, protein kinase C enhances secretion by two mechanisms. One is rapid and probably reflects the effects of immediate protein phosphorylation. The other occurs over several hours and requires gene transcription and protein synthesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66341/1/j.1471-4159.1990.tb13302.x.pd

Mechanisms Involved in Calcium-Dependent Exocytosis a

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74760/1/j.1749-6632.1991.tb36506.x.pd

Isolated Light Chain of Tetanus Toxin Inhibits Exocytosis: Studies in Digitonin-Permeabilized Cells

Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca 2+ -dependent secretion of [ 3 H]norepinephrine from digitonin-permeabilized adrenal chromaffin cells. Preincubation of tetanus toxin with monoclonal antibodies to the light chain prevented the inhibition by tetanus toxin. Preincubation of tetanus toxin with nonimmune ascites fluid or with monoclonal antibodies directed against the C fragment (the C-terminal of the heavy chains or the heavy-chain portion of the B fragment did not prevent inhibition by tetanus toxin. The data indicate that the light chain is responsible for the intracellular blockade of exostosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65694/1/j.1471-4159.1989.tb11800.x.pd

Influence of Role Models and Hospital Design on the Hand Hygiene of Health-Care Workers

We assessed the effect of medical staff role models and the number of health-care worker sinks on hand-hygiene compliance before and after construction of a new hospital designed for increased access to handwashing sinks. We observed health-care worker hand hygiene in four nursing units that provided similar patient care in both the old and new hospitals: medical and surgical intensive care, hematology/oncology, and solid organ transplant units. Of 721 hand-hygiene opportunities, 304 (42%) were observed in the old hospital and 417 (58%) in the new hospital. Hand-hygiene compliance was significantly better in the old hospital (161/304; 53%) compared to the new hospital (97/417; 23.3%) (p<0.001). Health-care workers in a room with a senior (e.g., higher ranking) medical staff person or peer who did not wash hands were significantly less likely to wash their own hands (odds ratio 0.2; confidence interval 0.1 to 0.5); p<0.001). Our results suggest that health-care worker hand-hygiene compliance is influenced significantly by the behavior of other health-care workers. An increased number of hand-washing sinks, as a sole measure, did not increase hand-hygiene compliance

Information Technology to Support Improved Care For Chronic Illness

BackgroundIn populations with chronic illness, outcomes improve with the use of care models that integrate clinical information, evidence-based treatments, and proactive management of care. Health information technology is believed to be critical for efficient implementation of these chronic care models. Health care organizations have implemented information technologies, such as electronic medical records, to varying degrees. However, considerable uncertainty remains regarding the relative impact of specific informatics technologies on chronic illness care.ObjectiveTo summarize knowledge and increase expert consensus regarding informatics components that support improvement in chronic illness care.DesignA systematic review of the literature was performed. "Use case" models were then developed, based on the literature review, and guidance from clinicians and national quality improvement projects. A national expert panel process was conducted to increase consensus regarding information system components that can be used to improve chronic illness care.ResultsThe expert panel agreed that informatics should be patient-centered, focused on improving outcomes, and provide support for illness self-management. They concurred that outcomes should be routinely assessed, provided to clinicians during the clinical encounter, and used for population-based care management. It was recommended that interactive, sequential, disorder-specific treatment pathways be implemented to quickly provide clinicians with patient clinical status, treatment history, and decision support.ConclusionsSpecific informatics strategies have the potential to improve care for chronic illness. Software to implement these strategies should be developed, and rigorously evaluated within the context of organizational efforts to improve care

Emerging concepts in biomarker discovery; The US-Japan workshop on immunological molecular markers in oncology

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations

Phosphatidylinositol-4,5-bisphosphate: actin dynamics and the regulation of ATP-dependent and -independent secretion

ABSTRACT It has long been believed that the cortical actin cytoskeleton plays an important role in regulating the secretion of hormones and neurotransmitters. In this study, we investigated the control of actin dynamics in primary neuroendocrine cells and determined the relationship of actin dynamics to various components of the secretory response. The amount of cortical f-actin in chromaffin cells was quantified in confocal images of cells stained with Alexa Fluor 568 phalloidin. Manipulations that decreased levels of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) (e.g., removal of ATP, the expression of a protein that can sequester PIP 2 ) rapidly reduced the amount of cortical actin. In contrast, cytoskeletal disruptors such as latrunculin were much less able to reduce cortical actin levels, indicating that the amount of cortical f-actin depends more strongly on PIP 2 than on the availability of g-actin. Not only does PIP 2 regulate actin, but actin regulates the level of PIP 2 , as revealed by PIP 2 -labeling studies. Manipulation of cortical actin had differing effects on the ATP-dependent and -independent components of secretion. ATP-dependent secretion was particularly sensitive to changes in cortical actin stability and was inhibited by expression of a protein (Yersinia pestis protein kinase A) that disassembles cortical f-actin and by pharmacological agents that promote either disassembly or stabilization of actin. The data suggest that an ATP-dependent component of secretion requires rapid changes in actin dynamics. These results point to a complex web of interactions involving PIP 2 , actin, and the secretory response. The inositol phospholipid PIP 2 has long been known to be important in exocytosis in neuroendocrine cells There is also evidence for the regulation of PIP 2 at sites of actin assembly. Rac and Rho (low-molecular-weight GTPases that stimulate membrane ruffling and stress-fiber formation, respectively) both recruit the enzyme responsible for PIP 2 synthesis, PIP 5-kinase, to the plasma membrane Although PIP 2 is an important regulator of actin, other factors, such as the availability of actin monomers, may also modify actin dynamics. Cytoskeletal disrupters (latrunculin, mycalolide, and cytochalasin) ABBREVIATIONS: PIP 2 , phosphatidylinositol-4,5-bisphosphate; Alexa-phalloidin, Alexa Fluor 568 phalloidin; DMPP, dimethylphenylpiperazinium; GFP, green fluorescent protein; CAPS, Ca 2ϩ -dependent activator protein for secretion; KGEP, potassium glutamate-, EGTA-, and PIPEScontaining solution; PIPES, piperazine-N,NЈ-bis(2-ethanesulfonic acid); PH-GFP, pleckstrin homology domain of phospholipase C␦ 1 fused to green fluorescent protein; YpkA, Yersinia pestis protein kinase A; PIP, phosphatidylinositol phosphate