Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (ICCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. ICCh was induced by carbachol (CCh, 50 microM) at a holding potential of -30 mV or -60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited ICCh concentration dependently in a reversible manner (53 +/- 8.6% at 1 microM, mean +/- SE, n = 11). In addition, amplitudes of ICCh were only 37 +/- 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca2+ channel current. The peak value of Ba2+ current at 0 mV was reduced to 35 +/- 7.4% (n = 9) by 3 microM of ML-7. As ICCh is known to have an intracellular Ca2+ dependence, we tried to exclude the possibility that ML-7 inhibited ICCh indirectly via suppression of Ca2+ current and the similar inhibitory effects of ML-7 on ICCh were confirmed under the following conditions: (1) clamp of membrane potential at -60 mV; (2) clamp of intracellular [Ca2+] to 1 microM by 10 mM BAPTA; (3) pre-inhibition of Ca2+ channel by verapamil. Different from the effects on ICCh, ML-7 barely inhibited the same cationic current induced by guanosine 5'-O-(3-thiotriphosphate) (GTP[gammaS], 0.2 mM) in the pipette solution. These results suggest that a Ca2+/calmodulin-MLCK-dependent pathway can modulate the activation of ICCh in guinea-pig gastric antral myocytes.     

Signal probability for reliability evaluation of logic circuits

As integrated circuits scale down into nanometer dimensions, a great reduction on the reliability of combinational blocks is expected. This way, the susceptibility of circuits to intermittent and transient faults is becoming a key parameter in the evaluation of logic circuits, and fast and accurate ways of reliability analysis must be developed. This paper presents a reliability analysis methodology based on signal probability, which is of straightforward application and can be easily integrated in the design flow. The proposed methodology computes circuit’s signal reliability as a function of its logical masking capabilities, concerning multiple simultaneous faults occurrence.     

Comparison of calcium lactate with chlorine as a washing treatment for fresh‐cut lettuce and carrots: quality and nutritional parameters

The efficacies of calcium lactate and chlorine washing treatments of fresh‐cut lettuce and carrots were compared during storage at 4 °C over 10 days. The gas composition of packages, colour, enzyme activity, texture, sensory attributes, microflora and levels of ascorbic acid and carotenoids were evaluated at 1, 3, 7 and 10 days. Calcium lactate treatment was not significantly different to chlorine treatment (p < 0.05) in terms of maintaining colour, texture and acceptability of fresh‐cut lettuce and carrots during the entire storage period. The washing treatments did not affect levels of ascorbic acid of fresh‐cut lettuce or carrots. Carotenoid levels were higher in calcium lactate‐treated carrots than chlorine‐treated samples at the end of storage. Mesophilic, psychrotrophic and lactic acid bacteria counts were not significantly different between treatments for both vegetables. Copyright © 2005 Society of Chemical Industry     

Glycosylation is essential for functional expression of a human brain ecto-apyrase

The importance of N-linked glycosylation for the function and oligomerization of an E-type ATPase was examined by using tunicamycin and peptide N-glycosidase F, two agents used to prevent and remove glycosylations, respectively. The cDNA encoding a human ecto-apyrase (HB6), predicted to have seven N-linked glycosylation sites, was transiently expressed in mammalian COS cells and the resulting membrane preparations were treated with peptide N-glycosidase F (PNGase-F). PNGase-F caused a decrease in the apparent molecular weight of the protein (consistent with glycan removal) and a decrease in enzymatic activity over time. The ecto-apyrase was also expressed in the presence of tunicamycin, which completely prevented N-linked glycosylation, resulting in a nonglycosylated core protein devoid of ATP and ADP hydrolyzing activity. However, control and tunicamycin-treated cells expressed the enzyme to similar levels and localization. Interestingly, the quaternary structure of this E-type ATPase appears to be dependent upon the presence of glycan chains. The glycosylated ecto-apyrase exists as a homodimer in situ as assessed by both size-exclusion chromatography of detergent-solubilized ecto-apyrase and cross-linking of membrane-bound ecto-apyrase, in contrast to the enzymatically deglycosylated ecto-apyrase and the tunicamycin-treated ecto-apyrase. These results suggest that glycosylation is necessary for homooligomerization and nucleotide hydrolyzing activity, but not for expression and plasma membrane localization of the E-type ATPase. Similar results were obtained with another human ecto-apyrase, CD39, suggesting that the importance of glycosylation may be generalized to all membrane-bound E-type ATPases.