The site-specific deoxyribonuclease from Bacillus pumilus (endonuclease R.Bpu1387).
(49/12477)
A new site-specific endonuclease (DNase) was isolated from the cells of Bacillus pumilus AHU 1387 strain. This enzyme (endonuclease R.Bpu 1387) introduced double-stranded scissions at unique sites on DNA's of coli phage lambda, lambdadvl, coli phage T7, Bacillus phage phi105C, Bacillus phage SP10, and Simian Virus 40, in the presence of magnesium ion. The activity was stimulated by the presence of NaCl. (+info)
Assay of intercellular adhesiveness using cell-coated Sephadex beads as collecting particles.
(50/12477)
A simple, rapid and precise method, based on a previous method, for measuring relative rates of intercellular adhesion is described. DEAE-Sephadex beads were treated with nitrocellulose in order to allow cells to grow on their surfaces. Balb/c 3T3 and Balb/c 3T12 cells were used to characterize the assay. They formed confluent cell layers on nitrocellulose-treated DEAE-Sephadex. These cell-coated beads were employed to collect 32P-labelled cells from single cell suspensions. Since they formed statistically uniform, large collecting surfaces, the collection of labelled cells was markedly improved as compared to the original assay. The cell-coated beads collected a large percentage of the labelled cells in a short time. The percentage of cells collected was independent of the concentration of labelled cells in the assay mixture, and the collection was linear for approximately 60 min. The variability between replicate assays was usually +/- 5%. The assay allows the rapid and precise determination of intercellular adhesion in large numbers of individual samples. These features make it useful to screen for effects of different treatments on intercellular adhesions. (+info)
Primitive nervous systems: electrophysiology of the pharynx of the polyclad flatworm, Enchiridium punctatum.
(51/12477)
1. Electrical activity accompanying motor activity can be recorded from the excised pharynx of Enchiridium punctatum. Multiple stimuli elicit behaviour which consists of an initial aperture closure followed by extension and then peristalsis. If the stimulus parameters are increased the preparation bends from side to side instead of proceeding through the behavioural sequence. Bending appears to inhibit other movements differentially. 2. The conduction involved with peristalsis is polarized and proceeds in a proximal direction. 3. With stimulus intensities greater than those needed to produce the behavioural response an initial muscle potential (IMP) is evoked. The IMP is frequency sensitive. Maximum facilitation occurs within 100 ms and drops to 50% of maximum within 250 ms. 4. Conduction velocities of the IMP range from 0-05 m s-1 to 1-9 m s-1. Conduction velocities appear to increase with facilitation. (+info)
Ceramide generation in nitric oxide-induced apoptosis. Activation of magnesium-dependent neutral sphingomyelinase via caspase-3.
(52/12477)
Sodium nitroprusside (SNP), a NO donor, has been recognized as an inducer of apoptosis in various cell lines. Here, we demonstrated the intracellular formation of ceramide, a lipid signal mediator, in SNP-induced apoptosis in human leukemia HL-60 cells and investigated the mechanisms of ceramide generation. The levels of intracellular ceramide increased to, at most, 160% of the control level in a time- and dose-dependent manner when the cells were treated with 1 mM SNP. SNP also decreased the sphingomyelin level to approximately 70% of the control level and increased magnesium-dependent neutral sphingomyelinase (N-SMase) activity to 160% of the control activity 2 h after treatment. Neither acid SMase nor magnesium-independent N-SMase was affected by SNP. Caspases are thought to be key enzymes in apoptotic cell death. Acetyl-Asp-Glu-Val-Asp-aldehyde, a synthetic tetrapeptide inhibitor of caspases, inhibited magnesiumdependent N-SMase, ceramide generation, and apoptosis. Moreover, recombinant purified caspase-3 increased magnesium-dependent N-SMase in a cell-free system. These results suggest that the findings that SNP increased ceramide generation and magnesium-dependent N-SMase activity via caspase-3 are interesting to future study to determine the relation between caspases and sphingolipid metabolites in NO-mediated signaling. (+info)
Kinetics of the interaction of rabbit skeletal muscle phosphorylase kinase with glycogen.
(53/12477)
The kinetics of the interaction of rabbit skeletal muscle phosphorylase kinase with glycogen was studied by the turbidimetric method at pH 6.8 and 8.2. Binding of phosphorylase kinase by glycogen occurs only in the presence of Ca2+ and Mg2+. The initial rate of complex formation is proportional to the enzyme and polysaccharide concentration; this suggests the formation of a complex with 1:1 stoichiometry in the initial step of phosphorylase kinase binding by glycogen. The kinetic data suggest that phosphorylase kinase substrate--glycogen phosphorylase b--favors the binding of phosphorylase kinase with glycogen. This conclusion is supported by direct experiments on the influence of phosphorylase b on the interaction of phosphorylase kinase with glycogen using analytical sedimentation analysis. The kinetic curves of the formation of the complex of phosphorylase kinase with glycogen obtained in the presence of ATP are characterized by a lag period. Preincubation of phosphorylase kinase with ATP in the presence of Ca2+ and Mg2+ causes the complete disappearance of the lag period. On changing the pH from 6.8 to 8.2, the rate of phosphorylase kinase binding by glycogen is appreciably increased, and complex formation becomes possible even in the absence of Mg2+. A model of phosphorylase kinase and phosphorylase b adsorption on the surface of the glycogen particle explaining the increase in the strength of phosphorylase kinase binding with glycogen in the presence of phosphorylase b is proposed. (+info)
Effect of moderate improvement in metabolic control on magnesium and lipid concentrations in patients with type 1 diabetes.
(54/12477)
OBJECTIVE: To evaluate the effect of clinically obtainable improvements in metabolic control in patients with type 1 diabetes on biochemical cardiovascular risk factors. RESEARCH DESIGN AND METHODS: Blood and 24-h urinary samples were obtained from 49 patients with type 1 diabetes before and after a run-in period and after 3 months of intervention, with frequent adjustment of insulin dosage according to measured blood glucose concentrations. RESULTS: The intervention caused a mean insulin dosage increment of 10%, a 20% decrease in fasting plasma glucose concentration, a 10% decrease in albumin corrected serum fructosamine, and a somewhat lesser decrease in HbAlc.A 14% decrease in the renal excretion of magnesium (Mg) was observed, but without a change in average serum Mg concentration. Serum HDL cholesterol increased 4%, and serum triglycerides decreased 10% as an average. Looking at individual patients, the decrease in serum triglycerides correlated with both the change in serum total Mg concentration and with the increase in insulin dosage. Using the change in serum total Mg concentration and in insulin dosage as independent variables in a multiple regression analysis, the coefficient of correlation with the decrease in serum triglycerides was 0.52. CONCLUSIONS: Moderate but clinically obtainable improvement of metabolic control in patients with type 1 diabetes seems to reduce the loss of Mg, increase serum HDL cholesterol, and decrease serum triglycerides. The decrease in serum triglycerides was associated with the change in serum total Mg concentration. These reductions in Mg loss and serum triglycerides might reduce the risk of developing cardiovascular disease in patients with type 1 diabetes. (+info)
Identification of the Mg2+-binding site in the P-type ATPase and phosphatase members of the HAD (haloacid dehalogenase) superfamily by structural similarity to the response regulator protein CheY.
(55/12477)
The large HAD (haloacid dehalogenase) superfamily of hydrolases comprises P-type ATPases, phosphatases, epoxide hydrolases and L-2-haloacid dehalogenases. A comparison of the three-dimensional structure of L-2-haloacid dehalogenase with that of the response regulator protein CheY allowed the assignment of a conserved pair of aspartate residues as the Mg2+-binding site in the P-type ATPase and phosphatase members of the superfamily. From the resulting model of the active site, a conserved serine/threonine residue is suggested to be involved in phosphate binding, and a mechanism comprising a phosphoaspartate intermediate is postulated. (+info)
Luminal dissociation of Ca2+ from the phosphorylated Ca2+-ATPase is sequential and gated by Mg2+.
(56/12477)
Transport of Ca2+ across the membrane by the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum involves the transfer of two Ca2+ ions from a pair of cytoplasmic sites to a pair of luminal sites, driven by phosphorylation of the ATPase. The ATPase is inhibited by Mg2+ at alkaline pH values. Inhibition follows from a decrease in the rate of release of Ca2+ from the phosphorylated ATPase. Phosphorylation-induced release of Ca2+ from the ATPase is biphasic at alkaline pH, which is consistent with sequential release of Ca2+ from the phosphorylated ATPase; the rates of both components decrease with increasing Mg concentration. The effect of Mg2+ on the slow phase of release follows from the binding of Mg2+ at the empty outer luminal site, vacated by the release of the first Ca2+ ion. The effect of Mg2+ on the rate of release of the first Ca2+ ion could follow from binding to a gating site also affecting the binding of Ca2+ to the cytoplasmic sites. (+info)