Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in bovine psoas muscle before and after electrical stimulation. In association with an accelerated glycolytic rate on stimulation, there was a significant increase in the binding of certain glycolytic enzymes, the most notable of which were phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase. From the known association of glycolytic enzymes with the I-band of muscle it is proposed that electrical stimulation of anaerobic muscle increases enzyme binding to actin filaments. Calculations of the extent of enzyme binding suggest that significant amounts of enzyme protein, particularly aldolase and glyceraldehyde 3-phosphate dehydrogenase, are associated with the actin filaments. The results also imply that kinetic parameters derived from considerations of the enzyme activity in the soluble state may not have direct application to the situation in the muscle fibre, particularly during accelerated glycolysis.     

Properties of electrical insulating materials at cryogenictemperatures: A literature review

Many insulating materials and systems for superconducting electrical equipment have been developed in response to the severe demands of safe usage, such as in cryogenic temperature, high mechanical stresses, high radiation, etc. They have been designed to fulfill very extreme service conditions, either at liquid helium or liquid nitrogen temperatures. Whatever the future industrial development, there is already abundant knowledge about insulating materials at cryogenic temperatures, and the feasibility has been proven. Many laboratories in the world continue research to increase this knowledge and to fill in the remaining gaps     

Reagentless enzyme electrode for the determination of manganese through biocatalytic enhancement.

An amperometric enzyme electrode is described for the detection and determination of manganese(II). The biosensor is based on the stimulation by manganese of the aerobic oxidation of substrates by horseradish peroxidase. A mediator, 1,2-naphthoquinone, is used as the substrate and is incorporated with the enzyme into a carbon-paste electrode. The resulting electrode acts as an enzyme-based oxygen sensor, which is sensitive to manganese. Electrochemical control of enzyme activity is achieved through substrate promotion of catalysis. Enzyme modulation by manganese can be switched on and off or adjusted through the appropriate selection of the applied potential. Currents are generated due to the bioelectrocatalytic reduction of oxygen in response to the introduction of manganese sulfate. A sustained current is achieved which is dependent on manganese concentration. Concentrations of 0.5 microM manganese or greater can be measured, and the sensor is reversible, as demonstrated by manganese removal. Biological selectivity for manganese provides a sensor which does not respond to other divalent cations tested, with the possible exception of cobalt. Reagentless, continuous sensing is achieved through substrate cycling.     

Severe B cell deficiency and disrupted splenic architecture in transgenic mice expressing the E41K mutated form of Bruton's tyrosine kinase

To identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse effects on B cell function, and essentially corrected the X-linked immunodeficiency (xid) phenotype in Btk- mice. In contrast, expression of a constitutively activated form of Btk carrying the E41K gain-of-function mutation resulted in a B cell defect that was more severe than xid. The mice showed a marked reduction of the B cell compartment in spleen, lymph nodes, peripheral blood and peritoneal cavity. The levels in the serum of most immunoglobulin subclasses decreased with age, and B cell responses to both T cell-independent type II and T cell-dependent antigens were essentially absent. Expression of the E41K Btk mutant enhanced blast formation of splenic B cells in vitro in response to anti-IgM stimulation. Furthermore, the mice manifested a disorganization of B cell areas and marginal zones in the spleen. Our findings demonstrate that expression of constitutively activated Btk blocks the development of follicular recirculating B cells.     

HeLa cells as a model to study the invasiveness and biology of Legionella pneumophila

HeLa cells were established as a model system to study the invasiveness and biology of Legionella pneumophila. In this model, invasion could be distinguished from adherence; virulent strains of L. pneumophila were adherent and invasive, whereas nonvirulent strains were adherent but poorly invasive. Invasion was rapid and did not require de novo bacterial protein synthesis, suggesting that the invasion factor is constitutively expressed by virulent strains. Entry into HeLa cells required actin polymerization and an intact microtubule cytoskeleton and was only moderately inhibited by the presence of 100 mM glucose or galactose. Intracellular replication of virulent L. pneumophila took place in ribosome-studded complex endosomes and led to the formation of free bacteria-laden vesicles presumably released from lysed HeLa cells. These free vesicles (referred to as mature vesicles) were isolated in continuous density gradients of Percoll. The bacteria contained in the isolated mature vesicles had a unique envelope structure and were highly adherent to HeLa cells, characteristics that correlated with a bright red appearance after the Giménez stain (Giménez positive). Plate-grown legionellae and replicating legionellae, harboured in complex endosomes, displayed a typical Gram-negative envelope and stained green after the Giménez stain (Giménez negative). Chronically infected cultures of HeLa cells were also established that may be a useful tool for studying long-term interactions between virulent L. pneumophila and mammalian cells. HeLa cells constitute a valuable model system that offers unique opportunities to study parasite-directed endocytosis, as well as stage specific-parasite interactions.     

Constitutive activity and structural instability of the wild-type human H2 receptor

Stable expression of the human H2 receptor in Chinese hamster ovary cells resulted in an increase in basal cyclic AMP (cAMP) production, which was inhibited by the inverse agonists cimetidine, famotidine, and ranitidine with potencies similar to those found for the rat H2 receptor. Burimamide, a neutral antagonist at the rat H2 receptor, behaved as a weak partial agonist at the human H2 receptor. Burimamide competitively antagonized both the histamine-induced increase in cAMP and the cimetidine-induced reduction of the basal cAMP level with apparent K(B) values that were similar to its H2 receptor affinity. Investigation of the modulation of receptor expression after long-term drug treatment revealed that at low concentrations histamine induced a significant reduction in H2 receptor expression, whereas at high concentrations receptor expression was slightly increased. The partial agonist burimamide induced, like inverse agonists, an up-regulation of the human H2 receptor after prolonged treatment. These findings suggest a structural instability of the constitutively active human H2 receptor in transfected Chinese hamster ovary cells. Occupation of the H2 receptor by any ligand reduces the instability, thus resulting in higher cellular expression levels.     

Pharmacokinetics and anti-HIV-1 efficacy of negatively charged human serum albumins in mice

Changes of blood metabolites and hormones were studied in female breeding calves before, during and after weaning from 4 to 18 weeks of age. Calves were initially fed increasing amounts of whole milk (up to 7 kg/day in week 8 of life). Milk intake was then gradually decreased up to the age of 16 weeks, when calves were completely weaned and only fed hay and concentrates. Average daily gain was 0.85 kg. Postprandial concentrations of glucose, insulin, insulin-like growth factor-I and 3.5.3'-triiodothyronine concentrations gradually decreased (P < 0.05) with age, while those of beta-hydroxybutyrate, protein, albumin, haemoglobin and iron increased (P < 0.05). Concentrations of cholesterol transiently increased, whereas those of urea reversibly decreased. Non-esterified fatty acids, triglycerides and growth hormone did not consistently change during the duration of the study. In conclusion, changes of glucose, beta-hydroxybutyrate, haemoglobin, iron, insulin, insulin-like growth factor-I and 3.5.3'-triiodothyronine were markedly different from those usually seen in veal calves of the same age.