Ageing is associated with a decrease in the number of sarcolemmal ATP-sensitive K+ channels in a gender-dependent manner

The opening of sarcolemmal K(ATP) channels is considered to be an important endogenous cardioprotective mechanism. On the other hand, age-dependent changes in the myocardial susceptibility to ischemia and hypoxia have been observed in different species, including humans. Here, we have hypothesized that aging might be associated with the changes in sarcolemmal K(ATP) channels. Therefore, the main objective of the present study was to establish whether aging changes expression of cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels. RT-PCR using primers specific for K(ATP) channel subunits, Kir6.2, Kir6.1 and SUR2A subunits was performed using total RNA from guinea-pig ventricular tissue. Whole cell electrophysiology was done on isolated guinea-pig ventricular cardiomyocytes. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Tissue and cells were harvested from young and old, male and female guinea-pigs. RT-PCR analysis did not reveal significant age-related changes in levels of Kir6.1 or Kir6.2 mRNAs. However, levels of SUR2A were significantly lower in old than in young females. Such age-differences were not observed with cardiac tissue from male animals. In both old and young males, pinacidil (100 microM) induced outward currents. The difference between current density of pinacidil-sensitive component in females, but not males, was statistically significant. Western blotting analysis revealed higher levels of Kir6.2 and SUR2A proteins in cardiac membrane fraction from young than old females. The present study demonstrates that in females, but not males, aging is associated with decrease in number of cardiac K(ATP) channels which is due to decrease in levels of the SUR2A subunit.     

Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

Limiting dilution analysis has been used in the context of allogeneic bone marrow transplantation to determine anti-recipient interleukin-2 (IL-2) producing helper T lymphocyte precursor (HTLp) frequencies, which in several studies have been predictive of graft-versus-host disease (GVHD). Recently high anti-recipient IL-4 producing HTLp frequencies have been reported and associated with a decreased risk of GVHD. The aim of the present study was to define the optimal conditions for combined determination of IL-2 and IL-4 producing anti-recipient HTLp frequencies. We have optimised the CT.h4S bioassay with regards to specificity, sensitivity, detection limit, and reproducibility. We have found the optimal assay conditions to be 1 x 10 (4) CT.h4S cells/well deprived of IL-4 for 24 h and preincubated for 7 h followed by 18 h of incubation with tritiated methyl-thymidine. In this setting the CT.h4S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack of IL-4 detection was not due to high amounts of soluble IL-4 receptor. With the use of 1x10(6) responder cells/well in HLA-mismatched MLC, we found limited IL-4 accumulation still increasing at day 12. We conclude that the CT.h4S bioassay is a reliable and specific method for quantification of IL-4 accumulation in cultures of human MNC. The difference in optimal timing for IL-2 (day 3) and IL-4 (>/=day 12) detection and evidence of very low IL-4 producing HTLp frequencies makes the relevance of a combined IL-2/IL-4 HTLp assay questionable.     

Sensitivity of T cells to anti-CD3-stimulated suicide is independent of functional phenotype.

We have examined mixed populations and cloned murine CD4+ T cell lines for their sensitivity to anti-CD3-stimulated suicide. The sensitivity of CD4+ clones to anti-CD3-stimulated suicide is independent of their interleukin-2/interleukin-4 or interferon-gamma secretion profile or their lytic efficiency in anti-CD3 redirected lytic assays.     

An investigation into the presence of secondary electrons in megavoltage photon beams

The presence of secondary electrons in photon beams of 60Co and 4, 8, 10, 15 and 25 MV x-rays has been studied by measuring surface charge using thin window ionisation chambers. Measurements have been made for square fields from 4 X 4 cm2 to 35 X 35 cm2 for locations from the collimator head to a distance of 4 m from the target. In addition, measurements have been made for rectangular fields at 10 MV and 25 MV for fields of equivalent area from 5 X 5 cm2 to 25 X 25 cm2. By eliminating the inverse square effect, the presence of contaminants from the head and the effect of build-up in air are clearly seen and well separated. Comparison between the curves at different energies indicates an increasing effect of contamination from the head with energy and a decreasing effect of electron production in air with increasing energy.     

The influence of isatin β-thiosemicarbazone on the development of neurovaccinia virus in cells as shown by electron microscopy

Summary Isatin -thiosemicarbazone (ITSC) is well known for its chemoprophylactic activity against certain poxvirus infections. The influence of 20 M ITSC on the development of neurovaccinia virus particles in infected embryo rabbit kidney cells has been studied by electron microscopy. It is shown that ITSC exerts an intracellular effect, permitting the formation of immature and abnormal forms, but preventing the normal development of these to give mature infective virus particles. The effects are compared with the normal development of mature virus particles as observed by us and as described by other authors from their electron microscopy studies.