Cyclic antibody formation to polyglycerophosphate in normal and injected rats.

Normal adult Sprague-Dawley rats were bled serially over a 30-week period and their sera were examined for antibodies to polyglycerophosphate (PGP) by a standardized passive hemolysis test. Levels of "natural" antibodies to PGP fluctuated during this period with a majority of animals exhibiting pronounced cycling of serum antibody levels, however, the individual cycles were not synchronized with each other. Feeding of radiolabeled Gram-positive bacilli to these animals and examination of lymphoid tissue by liquid scintillation counting revealed that the antigen persisted mainly in the mesenteric lymph nodes. A second group of rats was injected i.v. with radiolabeled Gram-positive bacilli and tissues were examined for plaque-forming cells (PFC) of PGP specificity, and the sera were examined by passive hemolysis. Cycling of both anti-PGP antibodies and PFC became synchronized in the injected animals with peaks of serum antibody evident at 16 and 28 days post-injection and splenic PFC peaks at 4 and 16 days post-injection. Cycling was also observed in the mesenteric lymph nodes and bone marrow Examination of lymphoid tissue from the rats injected i.v. revealed that antigen introduced by this route also perisisted mainly in the mesenteric lymph nodes, This report demonstrates individual cycling of natural responses to environmental antigen and to the same determinant in secondary responses, indicating its importance as a regulatory mechanism.     

Mechanisms of calcium signaling in smooth muscle cells explored with fluorescence confocal imaging

A rise in the intracellular concentration of ionized calcium ([Ca²⁺]_i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca²⁺ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca²⁺ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca²⁺]_i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP₃Rs) is an important determinant of the [Ca²⁺]_i dynamics and recruits neighboring Ca²⁺-release sites to generate [Ca²⁺]_i waves; (v) [Ca²⁺]_i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca²⁺-dependent K⁺ and Cl^- channels sense the local changes in [Ca²⁺]_i during a Ca²⁺ spark and thereby may couple changes in [Ca²⁺]_i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI _cat) does not mirror changes in [Ca²⁺]_i, thus reflecting the complexity of [Ca²⁺]_i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca²⁺ release, either spontaneous or caffeine-induced, does not augment mI _cat; (ix) intracellular flash release of Ca²⁺ is less effective in augmentation of mI _cat than flash release of IP₃, suggesting that IP₃ may sensitize muscarinic cationic channels to Ca²⁺; (x) intracellular flash release of IP₃ fails to augment mI _cat in SMCs, in which [Ca²⁺]_i was strongly buffered, suggesting that IP₃ exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca²⁺]_i rather than depletion of the IP₃-dependent Ca²⁺ store; and (xi) predominant expression of IP₃R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP₃R-mediated Ca²⁺ release and muscarinic cationic channel opening.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 455–465, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

Excision vs sponge swabbing - a comparison of methods for the microbiological sampling of beef, pork and lamb carcasses

AIMS: The aim of this research was to compare excision sampling with polyurethane and cellulose acetate sponge swabbing for the recovery of total viable counts and Enterobacteriaceae on meat carcasses. METHODS AND RESULTS: Two sample types were used to compare the methods: (i) individual samples, taken from four sites on each carcass and (ii) composite samples, created by pooling the samples from four sites from an additional set of carcasses. When the polyurethane sponge and excision method were compared for individual sites, there were no significant differences in bacterial recovery on beef and pork carcasses and on two of four sites on lamb carcasses. However, when samples from each site were pooled, the excision method was more efficient than either swabbing method across the three animal species. CONCLUSIONS: Sampling using the polyurethane sponge represents an equivalent alternative method to excision for the bacteriological sampling of carcass surfaces which is nondestructive and less labour intensive. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the scientific basis for using sponge swabbing instead of excision in compliance with 2001/471/EC.     

Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus

It is well documented that long term potentiation (LTP) is impaired in the hippocampus of the aged animal. Among the changes that contribute to this impairment is an increase in hippocampal concentration of the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and increased IL-1beta-induced signaling. In this study we investigated the possibility that these changes were a consequence of decreased concentration of the anti-inflammatory cytokine, IL-4, and decreased IL-4-stimulated signaling. We report that functional IL-4 receptors are expressed on granule cells of the dentate gyrus and that receptor activation results in phosphorylation of JAK1 and STAT6. Hippocampal IL-4 concentration was decreased with age, and this was accompanied by a decrease in phosphorylation of JAK1 and STAT6. The evidence indicates that IL-4 modulates expression of IL-1beta mRNA and protein and that it attenuates IL-1beta-induced impairment of LTP and phosphorylation of JNK and c-Jun. We argued that, if a decrease in hippocampal IL-4 concentration significantly contributed to the age-related impairment in LTP, then restoration of IL-4 should restore LTP. To test this, we treated rats with VP015 (phospholipid microparticles-incorporating phosphatidylserine), which increases IL-4 concentration in hippocampus. The data indicate that the VP015-induced increase in IL-4 concentration in hippocampus of aged rats and lipopolysaccharide (LPS)-treated rats was accompanied by a reversal of the age-related and LPS-induced impairment in LTP in perforant path granule cell synapses. We propose that interplay between pro-inflammatory and anti-inflammatory responses impact significantly on synaptic function in the hippocampus of the aged rat.