Glycosylation of myelin glycoproteins in peripheral nerve via lipid intermediates

Membrane preparations from chick peripheral nervous system (PNS) catalyzed the transfer of [³H]glucose from UDP-[³H]glucose into glucosylphosphoryl dolichol. The initial rate of glucosylphosphoryl dolichol formation in a non-myelin membrane fraction from actively myelinating chick PNS was 11 fold higher than that from adult. Exogenous dolichyl monophosphate stimulated glucosylphosphoryl dolichol synthesis in both fractions. The higher level of glucosylphosphoryl dolichol synthesis corresponded to the onset of myelination in chick PNS. Exogenous dolichyl monophosphate also stimulated the labeling of glucosylated oligosaccharide lipids and glycoproteins in the fraction. On SDS polyacrylamide gel electrophoresis, the relative mobility of the major and minor radioactive glycoprotein corresponded with that of the P0 and PASII glycoprotein in PNS myelin, respectively. The results suggest that myelin glycoproteins in PNS are glycosylated via lipid intermediates.     

Mode of action of furosemide on the chloride-dependent short-circuit current across the ciliary body epithelium of toad eyes

Summary The effects of furosemide on the chloride-dependent short-circuit current across the toad ciliary epithelium were examined. Under control conditions, the short-circuit current obeyed Michaelis-Menten kinetics against medium chloride concentration, the Michaelis constant (K _m ) for chloride being 90mm and the maximal short-circuit current (V _max) 128 A/cm². Furosemide added to the aqueous side of the epithelium rapidly reduced the short-circuit current; the effect was reversible. The effect of furosemide addition to the stromal side was much smaller and slower than that from the aqueous side. The dose-dependent range of furosemide action was from 0.1 m to 1mm with 50% inhibition occurring at about 3 m. Line-weaver-Burk plot of the short-circuit current against the chloride concentration showed that furosemide decreased the value ofV _max and increased theK _m ; the inhibition being of mixed type. A Hill plot of the dose-response curve yielding a slope of unity suggested one furosemide molecule combines with one chloride transport site. Probenecid, a competitive inhibitor of organic acid transport, reduced the effects of furosemide significantly when added simultaneously. The involvement of organic acid transport system in the mechanism of furosemide action on chloride transport was suggested.Department of Ophthalmology.     

Regional differences in blood flow and oxygen consumption in resting muscle and their relationship during recovery from exhaustive exercise.

This investigation evaluated regional differences in blood flow and oxygen consumption and their relationship in exercised muscle during recovery from exhaustive exercise. Five healthy men performed exhaustive one-legged cycling exercise. Positron emission tomography was used to measure blood flow, oxygen uptake, and oxygen extraction in the quadriceps femoris muscle before and after exercise. Regions of interest included five areas of the muscle (two proximal, one central, and two distal), which were evenly spaced across the muscle. Before exercise, blood flow and oxygen consumption decreased significantly (P < 0.05) in the direction from the proximal to the distal portions; blood flow declined from 2.0 +/- 0.5 to 1.4 +/- 0.3 ml x 100 g-1 x min-1, and oxygen consumption decreased from 0.21 +/- 0.04 to 0.17 +/- 0.02 ml.100 g-1x min-1. In contrast, these gradients in blood flow and oxygen consumption diminished during recovery after exercise. Consequently, there was a positive relationship between changes in blood flow and oxygen consumption in an exercised muscle during recovery after exercise (r = 0.963, P < 0.01). These changes became larger in the direction from proximal to distal portions: blood flow increased from 2.9 +/- 0.7 to 3.9 +/- 0.8 and oxygen consumption from 1.4 +/- 0.1 to 1.8 +/- 0.4 times resting values. These results suggest that hemodynamic variables are heterogeneous within a muscle both at rest and during recovery from exercise and that there is a systematic difference in these variables in the direction from proximal to distal regions within the quadriceps femoris muscle.     

Suppression of heat- and polyglutamine-induced cytotoxicity by nonsteroidal anti-inflammatory drugs.

We have shown that sodium salicylate activates the heat shock promoter and induces the expression of heat shock proteins (hsps), with a concomitant increase in the thermotolerance of cells. To determine whether these effects are generally displayed by nonsteroidal anti-inflammatory drugs (NSAIDs), we examined the effects of a cyclooxygenase inhibitor, indomethacin, and a lipoxygenase inhibitor, nordihydroguaiaretic acid. Both inhibitors up-regulated the hsp promoter at 37 degrees C through the activation of heat shock factors, and increased cellular levels of hsps in mammalian cells, although the degree of the expression of hsps and thermotolerance of cells differed depending on the drugs. Furthermore, NSAIDs such as sodium salicylate and indomethacin suppressed the protein aggregation and apoptosis caused by an expanded polyglutamine tract in a cellular model of polyglutamine disease. These findings suggest that NSAIDs generally induce the expression of hsps in mammalian cells and may be used for the protection of cells against deleterious stressors and neurodegenerative diseases.     

Specific inactivation of glutathione S-transferases in class Pi by SH-modifiers

Treatment of Class Pi glutathione S-transferases (GST) such as rat GST P (7-7), human GST pi and mouse GST MII with 0.05-0.1 mM N-ethylmaleimide (NEM) in 0.1 M Tris-HCl (pH 7.8) resulted in almost complete inactivation of these forms, whereas no or less inactivation occurred for GSTs in Class Alpha and Mu under the same conditions. Inactivated GST P lost its S-hexyl-GSH-Sepharose column affinity. About 0.8 mol of [14C]NEM was found to be covalently bound to 1 mol of GST P subunit when 80% of the activity was lost. Similar treatment with N-dimethyl-amino-3,5-dinitrophenyl maleimide, a colored analogue of NEM, followed by trypsin digestion, HPLC and amino acid sequence analysis revealed that one cysteine residue at the 47th position from the N-terminal of the GST P subunit was preferentially modified. Subunits of GST P and GST pi are known to have 4 cysteine residues at the same corresponding positions. The present results suggest that the 47th cysteine residue may be located in the vicinity of the active site of Class Pi GSTs.     

P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells.

The expression of a functional P-glycoprotein (P-gp) which pumps drugs out of brain capillary endothelial cells (BCEC) into blood was studied by evaluating the steady-state uptake and efflux of vincristine (VCR) by primary cultured bovine BCEC. The steady-state uptake of VCR was increased in the presence of metabolic inhibitors, and an anti-P-gp monoclonal antibody, MRK16, as well as verapamil and steroid hormones which are known to reverse multidrug resistance in tumor cells. Furthermore, efflux of VCR from BCEC was inhibited by verapamil. By immunohistochemistry, P-gp was localized at the luminal side of the capillary endothelial cells in both gray matter of bovine brain and primary cultured BCEC. These data suggest that P-gp functions as a drug efflux pump at the luminal side of BCEC and regulates the transfer of certain lipophilic drugs from the blood into the brain.     

Dynamics of “neuron ring”

Summary Dynamics of neuron ring has been studied by means of computer simulation. The ring is formed of some number of identical neurons which loop together with lateral inhibitions and are stimulated by a command neuron. In this model, we assumed that each neuron has individual activity intrinsically without any inputs. The function of each neuron is essentially the same as that of Reiss' model; each neuron receives one excitatory and two inhibitory signals and when the membrane potential exceeds the instantaneous values of threshold, the neuron fires and sends out signals. Fatigue effect is also introduced in this model; after firing, the threshold level rises up for a period. This report is mainly concerned with the mechanism by which the individual activities of each neuron transit into an ordered behavior as a whole when they are externally stimulated. Results can be summarized as follows: The individual random activities of neurons can be organized and transfered into an ordered behavior on a completely symmetrical neuron ring if externally stimulated and the following conditions are satisfied; 1. the individual inherent activity is within a moderate range, 2. the fatigue effect is large enough, 3. external stimulation is high enough and 4. neurons in the ring are laterally inhibited. The individual activity and the lateral inhibition are essential to generate a meaningful output pattern as a group and fatigue effect acts as a stabilizing factor of the generated pattern.     

Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by alpha-tocopherol

The oxidation of rat red blood cells (RBC) by molecular oxygen was performed in an aqueous suspension with an azo compound as a free-radical initiator. The RBC were oxidized at a constant rate by a free-radical chain mechanism, resulting in hemolysis. The extent of hemolysis was proportional to the concentration of free radical. alpha-Tocopherol in RBC membranes suppressed the oxidation and hemolysis to produce an induction period. Tocopherol was constantly consumed during the induction period, and hemolysis developed when tocopherol concentrations fell below a critically low level. Among the membrane lipids, phosphatidylethanolamine, phosphatidylserine, and arachidonic acids were predominantly oxidized in the absence of tocopherol. In the presence of tocopherol, however, such lipid changes were suppressed during a 120-min incubation even when hemolysis started. Membrane proteins as well as lipids were oxidized. The formation of proteins with high molecular weight and concomitant decrease of the low-molecular-weight proteins were observed on gel electrophoresis with the onset of hemolysis. This study clearly showed the damage of RBC membranes caused by oxygen radical attack from outside of the membranes, and suggested that membrane tocopherol even below a critically low level could suppress lipid oxidation but that it could not prevent protein oxidation and hemolysis.