Release Of Intracellular Proteins Research Articles

Evidence using immunoelectron microscopy for regulated and constitutive pathways in the transport and release of endothelin.

We investigated the distribution of endothelin (ET)-like immunoreactivity in the human coronary artery and examined sites linked to the storage and release of intracellular proteins. Intense ET-like immunoreactivity was observed at the light-microscope level in luminal coronary artery endothelial cells. A low level of staining also was detected in the outer medial smooth-muscle layer and diffusely within the adventitia. Immunoelectron microscopy was used to determine the ultrastructural localisation of ET in the endothelium. Positive ET-like immunoreactive staining was detected in secretory vesicles at the ultrastructural level. Quantitative immunoelectron microscopy revealed that ET-like immunoreactivity was predominantly localised to the cytoplasmic matrix (including secretory vesicles) and Weibel-Palade bodies (endothelial cell-specific storage granules). Labelling was detected in 44% of Weibel-Palade body profiles positively identified by using antisera to von Willebrand factor and in cytoplasm surrounding these structures. A low level of immunoreactive staining was associated with mitochondria, whereas the cell nucleus and Golgi complex showed little or no positive staining. These findings indicate that ET is released from human coronary artery endothelial cells via two distinct secretory pathways. We propose that ET is continuously transported in and released from secretory vesicles by the constitutive secretory pathway. ET may also be stored in Weibel-Palade bodies and released after an appropriate stimulus by the regulated pathway. Positive immunoreactivity was also observed in plasmalemmal vesicles (50- to 60-nm diameter), indicating a role for these structures in endocytosis.

Long-term effects of fatty acids on cell viability and gene expression of neonatal cardiac myocytes

Fatty acids are the most important source of energy for the adult heart. However, cardiac substrate preference changes during development and alters in pathophysiological states. Fatty acids have also been shown to be involved in signal transduction pathways, thereby affecting gene expression in various cell systems. In the present paper the significance of changes in substrate preference and the potential role of fatty acids in signal transduction in the cardiomyocyte are briefly reviewed. Furthermore, the development of a cellular model system, useful in exploring the long-term effects of fatty acids on the normal and hypertrophic cardiomyocyte, is described. Some aspects of this model system are illustrated by showing the effects of different fatty acid species on cell viability and the effects of fatty acids on the expression of heart type fatty acid-binding protein (H-FABP), a 15 kDa protein thought to be involved in intracellular trafficking of fatty acids. To this end primary cultures of rat neonatal ventricular myocytes were kept in defined medium containing various (combinations of) substrates for up to 48 h. First, the effects of prolonged exposure to different fatty acid species, complexed to BSA, on cell viability were investigated. Exposure of the cells to saturated fatty acids (C16:0 or C18:0), but not mono-unsaturated (C16:1 or C18:1) fatty acids, resulted in cell death, as evidenced by the release of intracellular proteins like lactate dehydrogenase. The detrimental effects of saturated fatty acids were nullified by the co-addition of mono-unsaturated fatty acids. Accordingly, the combination of C16:0 C18:1 was used to examine the effects of fatty acids on the expression of H-FABP. Therefore, the cells were incubated with either (i) glucose only, (ii) fatty acids only, or (iii) glucose plus fatty acids. Incubation with fatty acids (with or without glucose) resulted in a nearly four-fold increase of the H-FABP mRNA level. Similarly, at the protein level the cellular H- FABP LDH ratio increased almost two-fold. In hypertrophic cardiomyocytes (stimulated with the α 1-adrenergic agonist phenylephrine) the stimulatory effect of fatty acids on H-FABP expression was mitigated. These findings strongly suggest that fatty acids are able to modulate gene expression in the context of the cardiac muscle cell.

Autolysis of Lactococcus lactis caused by induced overproduction of its major autolysin, AcmA.

The optical density of a culture of lactococcus lactis MG1363 was reduced more than 60% during prolonged stationary phase. Reduction in optical density (autolysis) was almost absent in a culture of an isogenic mutant containing a deletion in the major autolysin gene, acmA. An acmA mutant carrying multiple coples of a plasmid encoding AcmA lysed to a greater extent than the wild-type strain did. Intercellular action of AcmA was shown by mixing end-exponential-phase cultures of an acmA deletion mutant and a tripeptidase (pepT) deletion mutant. PepT, produced by the acmA mutant, was detected in the supernatant of the mixed culture, but no PepT was present in the culture supernatant of the acmA mutant. A plasmid was constructed in which acmA, lacking its own promoter, was placed downstream of the inducible promoter/operator region of the temperate lactococcal bacteriophage r1t. After mitomycin induction of an exponential-phase culture of L. lactis LL302 carrying this plasmid, the cells became subject to autolysis, resulting in the release of intracellular proteins.

Review article: platelets in inflammatory bowel disease--pathogenetic role and therapeutic implications.

An elevated platelet count is well recognized as a marker of inflammatory bowel disease activity. There is an increased incidence of systemic thromboembolism in this disease. Recent work indicates that platelets exhibit several proinflammatory properties including release of inflammatory mediators, and recruitment, chemotaxis and modulation of the activity of other inflammatory cells. Furthermore there is evidence that microvascular thrombosis and a procoagulant state may play a role in the pathogenesis of inflammatory bowel disease. These observations prompted recent studies of platelet activity in inflammatory bowel disease, which indicate enhanced platelet aggregation in vivo and in vitro, and increased platelet activation as measured by increased release of intracellular proteins into plasma and expression of platelet surface markers, including P-selectin and GP53. These abnormalities could contribute to the pathogenesis of inflammatory bowel disease by enhancing inflammation and promoting microinfarction. Aminosalicylates reduce platelet activity although they also have many other additional properties to explain their efficacy in inflammatory bowel disease. There are however several specific antiplatelet drugs now available which may provide new therapeutic possibilities in the management of this disease.

Use of Hydrodynamic Cavitation for Large Scale Microbial Cell Disruption

A variety of disruption techniques are available for the release of intracellular proteins. Analysis of present day equipment in terms of possible cavitating conditions indicates that, to get efficient disruption of cells, the equipment must operate very near or below the cavitation inception number. A simple set-up has been described which generates cavitating flow. The effect of various parameters such as discharge pressure, and the number of passes through the cavitating zone were studied. Also, the effect of cell type, growth stage of cell and cell concentration on the cell disruption were investigated. The effect of these cavitating conditions on the activity of the macromolecules was also studied. The energy effciency of the hydrodynamic cavitation was compared with that of established techniques such as ultrasonication and mixer-blender methods. It was observed that the hydrodynamic cavitation set-up described is almost an order of magnitude more energy effcient than the established techniques.

The effect of freeze-thawing on the release of intracellular proteins from Escherichia coli by means of a bead mill

Escherichia coli cell pastes were freeze-thawed prior to milling, to determine its effect on the efficiency of the bead mill. The quantity of the released proteins and the extracted activity of a periplasmic enzyme, penicillin acylase (EC 3.5.1.11), increased with the application of freeze-thawing. Freezing, but not the frozen storage that follows was the main reason for increased bead mill efficiency.

Improvement of cell lysis activity of immobilized lysozyme with reversibly soluble-insoluble polymer as carrier

Hen egg white lysozyme was immobilized by covalent binding to a polymer showing reversibly soluble-insoluble characteristics with pH change. The retention of the specific activity of the immobilized enzyme can be as high as 41% of that of the free enzyme. The immobilized enzyme could be used in repeated batch lysis of M. lysodeikticus cells and to enhance the release of intracellular proteins 1.4 folds when compared with batch operation.

The use of zeolite y in the purification of intra cellular accumulated proteins from genetically engineered cells

Fast and efficient release of intracellular proteins by the use of detergents followed by immediate removal of the detergent by adsorption to zeolite Y is described. The removal of the detergent makes it possible to continue the purification process without interference by the detergent and the process is exemplified by the purification of protein A fromE. coli encoded by the plasmid pRIT-2T. Compared to disruption of the cells by sonication, increased stability against proteolytic degradation was obtained.

3 The Metabolic Effects of Exercise-Induced Muscle Damage

Exercise-induced skeletal muscle damage results in a remarkable number of localized and systemic changes, including release of intracellular proteins, delayed onset muscle soreness, the acute-phase response, and an increase in skeletal muscle protein turnover. These exercise-induced adaptations appear to be integral to the repair of the damaged muscle and may be essential for hypertrophy. Chronic exercise produces adaptations in skeletal muscle, resulting in increased capacity of oxidative metabolism; the repair of damaged muscle resulting in hypertrophy may be an important mechanism for protection against further exercise-induced damage. Although the release of CK from skeletal muscle following damage is a commonly observed phenomenon, circulating CK activity is not a quantitative and, in some cases, even a qualitative indicator of skeletal muscle damage. Eccentric exercise-induced skeletal muscle damage offers an opportunity to investigate the signals and modulators of the repair of muscle damage, a process that may be central to the adaptations in muscle as a result of chronic activity.

Physiological mechanisms contributing to increased interleukin-1 secretion.

Interleukin-1 (IL-1) is a monocyte-derived polypeptide that mediates many host defense adaptations to environmental and infectious stresses. This investigation was intended to characterize further IL-1 activity found in human plasma following exercise (3) and to identify physiological initiators of IL-1 secretion. IL-1 activity was measured by the ability of plasma fractions to stimulate lymphocyte proliferation. This activity appeared in plasma several hours after exercise on a cycle ergometer (1 h at 60% of aerobic capacity, n = 8 subjects) and was neutralized with a specific antiserum to human IL-1. The hypothesis that IL-1 release from monocytes was initiated by phagocytosis of material from cells damaged by exercise was tested. The increase in IL-1 activity did not correlate significantly (r = 0.55) with creatine kinase activity, a marker for release of intracellular proteins into the circulation, and IL-1 secretion by monocytes was not stimulated by incubation with red blood cell lysates in vitro. Thus the stimulus for IL-1 secretion did not appear to be related to a scavenging function of monocytes. The possibility that IL-1 secretion may be mediated by stress hormones associated with exercise was examined. IL-1 secretion by monocytes was increased up to 48 +/- 18% (P less than 0.01) by addition of physiological concentrations of epinephrine in vitro. Low concentrations of hydrocortisone (1 ng/ml) also augmented IL-1 secretion by 58 +/- 20%. Higher concentrations in the physiological range had no effect, and combinations of epinephrine and hydrocortisone suppressed IL-1 secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

A general method for the quantitation of immune cyotysis.

THE cytolytic effect of immune serum has been the subject of study by a number of investigators. It has been shown1 that cells other than erythrocytes undergo lytic changes in the presence of antibody and complement. It has been further shown2, using cells labelled with phosphorus-32, that the lysis of such cells is accompanied by a release of intracellular proteins, similar to the release of haemoglobin from lysed erythrocytes. It appeared that the immune lysis of tissue cells is quite analogous to the immune lysis of erythrocytes. On this basis, a test was developed using radioactive labelled cells which provides a means of quantitating immune lysis for cells derived from a variety of tissues.