ABSTRACT
Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis. New rapid, sensitive and specific methods for detection of GBS in pregnant women are needed in order to provide timely treatment of neonates. The sensitivity, specificity and cost of a LightCycler PCR method was compared with selective culture for the detection of GBS from 400 vaginal swabs. In addition, two DNA extraction methods (simple boiling and automated DNA extraction by Roche MagNA Pure LC) were compared for a subgroup of 100 clinical samples. The sensitivity of the LightCycler PCR assay for the detection of GBS from vaginal swabs was significantly higher than that of culture. There were no culture-positive, LightCycler PCR-negative cases. The efficiencies of the two DNA extraction procedures were not significantly different. The detection of GBS from vaginal swabs by the molecular method (including simple boiling extraction) required the same hands-on time, but the procedure was completed in 1.5 h, compared with c. 48 h for the culture-based approach. Disadvantages of the molecular method are the increased costs (45%) and the absence of antibiogram data. The LightCycler PCR is a promising tool for sensitive, specific and rapid detection of GBS directly from clinical specimens of pregnant women.
Subject(s)
DNA, Bacterial/analysis , Polymerase Chain Reaction/methods , Streptococcus agalactiae/isolation & purification , Bacterial Typing Techniques , Cell Culture Techniques , Culture Media , Female , Humans , Polymerase Chain Reaction/economics , Pregnancy , Streptococcus agalactiae/classification , Streptococcus agalactiae/genetics , Vagina/microbiology , Vaginal SmearsABSTRACT
Nod factor [NodRm-IV(Ac,S)], isolated from the bacterium Rhizobium meliloti, induces a well-known depolarization in Medicago sativa (cv Sitel) root hairs. Analysis of this membrane response using the discontinuous single-electrode voltage-clamp technique (dSEVC) shows that anion channel, K+ channel and H+-ATPase pump currents are involved in young growing root hairs. The early Nod-factor-induced depolarization is due to increase of the inward ion current and inhibition of the H+ pump. It involved an instantaneous inward anion current (IIAC) and/or a time-dependent inward K+ current (IRKC). These two ion currents are then down-regulated while the H+ pump is stimulated, allowing long-term rectification of the membrane potential (Em). Our results support the idea that the regulation of inward current plays a primary role in the Nod-factor-induced electrical response, the nature of the ions carried by these currents depending on the activated anion and/or K+ channels at the plasma membrane.
Subject(s)
Ion Channels/drug effects , Ion Channels/metabolism , Lipopolysaccharides/pharmacology , Medicago sativa/drug effects , Medicago sativa/metabolism , Calcium Signaling/drug effects , Kinetics , Medicago sativa/microbiology , Membrane Potentials/drug effects , Plant Roots/drug effects , Plant Roots/metabolism , Plant Roots/microbiology , Potassium Channels/drug effects , Potassium Channels/metabolism , Proton Pumps/drug effects , Proton Pumps/metabolism , Sinorhizobium meliloti/metabolismABSTRACT
The effects of vitamin D on the intramuscular distribution of total and bound calcium, phosphate and on available cytosolic calcium, were investigated in skeletal muscle. Total calcium and phosphorus were measured on ashed subcellular fractions of muscles from vitamin D-repleted and vitamin D-deprived rats. The variations in available calcium were followed by determining the activities of calcium-sensitive enzymes in isolated cytosol. Bound-calcium was revealed ultra-microscopically by pyroantimonate. In vitamin D-repleted muscles, the pyroantimonate method revealed specific areas of intense bound-calcium deposition: the myofibrils, where they formed pronounced lines parallel to the Z-bands. In vitamin D-deficient muscles, the calcium-pyroantimonate deposits appeared clearly reduced. This loss was accompanied by a marked reduction in total calcium and phosphorus in all the subcellular fractions, as compared to vitamin D-repleted muscles. Unexpectedly, the activity of the Ca(+)-activated isocitrate-dehydrogenase was increased in the cytosol, while that of the Ca2(+)-inhibited pyruvate-kinase decreased. Prolonged vitamin D-administration to vitamin D-repleted rats led to an intensification of calcium-pyroantimonate deposits and a general increase in total calcium and phosphorus, but no change in the cytosolic Ca2(+)-sensitive enzyme activities. Cessation of vitamin D-administration to vitamin D-repleted rats produced a regression of calcium-pyroantimonate deposits, a general decrease of total calcium and phosphate levels, and stimulation of the Ca2(+)-activated isocitrate-dehydrogenase accompanied by lowering of the Ca2(+)-inhibited pyruvate-kinase. The results clearly indicate a correlation between vitamin D-repletion and the total and bound calcium content of skeletal muscle. In addition, they demonstrate an apparent contradiction between the decrease of total and bound calcium, and the activities of cytosolic Ca2+ sensitive enzymes during vitamin D-deprivation, which can only be explained by an increase in available calcium. It is suggested that vitamin D stimulates intramuscular mechanisms tending to lower available calcium by inactivating the cation via the formation of calcium chelates.
