Calcium dependence of phenylephrine-, endothelin-, and potassium chloride-stimulated atrial natriuretic factor secretion from long term primary neonatal rat atrial cardiocytes

Since calcium is involved in both excitation-secretion and excitation-contraction coupling, it was of interest to evaluate its involvement in atrial natriuretic factor (ANF) release from atrial cardiocytes. In medium containing physiological levels of calcium (1.4 mM), the secretion of ANF from primary atrial cells was stimulated from 3- to 6-fold by a variety of agents including KCl, phenylephrine, and endothelium (ET). However, in medium containing 2 nM calcium, KCl was incapable of increasing ANF secretion above basal levels, while the stimulatory effects of phenylephrine and ET were only partially diminished. Nifedipine or verapamil could mimic the effects of the 2 nM calcium medium on KCl-, phenylephrine-, and ET-stimulated ANF secretion. Kinetic studies indicated that during the initial 5 min of ET-stimulated secretion the cells exhibited little requirement for extracellular calcium; however, the requirement was more apparent during the sustained secretion observed between 10 min and 2 h of secretagogue exposure. Additionally, the stimulation of ANF secretion by ET increased to a maximum of about 15-fold over basal by 10-min after ET application; subsequent to this time there was an apparent functional desensitization wherein the rate of secretion decreased by approximately 3-4-fold and remained at this level for the duration of secretagogue exposure up to 2 h. All forms of stimulated secretion could be inhibited through ionomycin-mediated depletion of intracellular calcium pools. Taken together, these results indicate that atrial cardiocytes require both extracellular and intracellular calcium to support maximal rates of stimulated ANF secretion, and that intracellular calcium pools may be used during the early phase of secretion, while the extracellular source of calcium may be important for the sustained phase of secretion.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Stress modulates calcium mobilization in immune cells

Both acute and chronic restraint stress modulated mitogen-induced increases in cytoplasmic free-calcium concentrations ([Ca2+]i) in mouse spleen cells. Dual-color analysis of lymphocyte subpopulations demonstrated that acute (2 hour) restraint stress suppressed mitogen-stimulated increases in [Ca2+]i in CD4+ T cells, but enhanced [Ca2+]i in CD8+ T cells. Chronic restraint stress (2 hours daily for up to 21 days) resulted in a significant suppression of mitogen-stimulated increases in [Ca2+]i in CD4+ T cells at 3 and 7 days, but not at 21 days. CD8+ T cells were unaffected by chronic stress. Chronic stress (for 7 days) had a modest suppressive effect on mitogen-induced Ca2+ responses in B cells. Within T lymphocyte subpopulations, both acute and chronic stress predominantly affected CD4+ T cells, which may induce a functional reversal of the CD4/CD8 ratios in vivo. Such a reversal could result in suppression of a variety of immune responses such as lymphocyte proliferation and antigen-specific antibody production. These findings indicate that the inhibitory effects of stress on calcium mobilization in lymphocytes may be an early event mediating stress-induced immunosuppression.     

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations

Summary A 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.     

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12

Summary Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.     

In vitro establishment of human fibroblasts of lysosomal diseases,GM1-gangliosidosis and Sandhoff disease,by transformation with origin-minus SV40 DNA

The permanent human cell lines preserving defects of lysosomal enzymes, G_M1-1019-SV and SA-1077-SV, were established from the respective fibroblasts from patients with G_Ml-gangliosidosis and Sandhoff disease by transfection with replication origin-minus simian virus 40 DNA. These ceils grow rapidly without entering senescence during more than 120 population doublings. The activity of -galactosidase in G_M1-1019-SV and of B-N-acetylhexosaminidase in SA-1077-SV was respectively 40- and 180-fold lower than that of normal fibroblasts.     

Miniature endplate currents of muscle fibers from rat diaphragm during galantamine-induced acetylcholinesterase inhibition

Miniature endplate currents (MEPC) were recorded in muscle fibers of rat diaphragm using voltage clamp technique during acetylcholinesterase (AChE) inhibition induced by various concentrations of galantamine. Their amplitude and time course began to increase at a galantamine concentration of 3.16·10^–8 g/ml. Increased concentrations of galantamine produced a greater effect. Maximum amplitude and time course were reached at a concentration of 10^–6 g/ml. The input resistance of muscle fibers increased under the effects of galantamine. In all cases MEPC fell exponentially. At a concentration of 10^–5 g/ml galantamine produced a curarelike effect; amplitude and time course of decay increased to a lesser extent than at a concentration of 10^–6 and the decay in MEPC became biphasic. Following washout of galantamine (10^–5 g/ml) the time course of MEPC first rose, then fell, returning to the initial level in 3 h, and decay again became exponential. Changes in MEPC parameters under the effects of different concentrations of galantamine and washout were closely correlated. A positive correlation was found between the time course of decay and MEPC amplitude both in the presence and absence of AChE inhibition. It is postulated that the functional importance of synaptic AChE in repressing the postsynaptic action of acetylcholine is limited and that parameters of postsynaptic response may therefore be used to evaluate its action.A. A. Ukhtomskii Institute of Physiology, A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 607–614, September–October, 1985.     

Assignment of the gene locus for the sixth component (C6) of complement in mice to chromosome 15

A structural locus (C-6) for the sixth component of complement in mice is assigned to chromosome 15. Three-point linkage analysis indicated that the order of loci is C-6, Gpt-1, Gdc-1, and that the map distances are 25.9±4.9 between C-6 and Gpt-1, and 36.4±5.5 between C-6 and Gdc-1. Since Gdc-1 is more distal than Gpt-1, and C-6 is 26 cM away from Gpt-1, it is estimated that the C-6 is proximal to the centromere. In addition, a new C6 form found in AKR mice is described. We propose the designation C6B for it and C-6 ^b for the allele encoding C6B.Abbreviations used in this paper IEF isoelectric focusing - GPT glutamic-pyruvic transaminase - GDC L-glycerol 3-phosphate dehydrogenase - cM centimorgan