Renal brush border membrane lipid composition in Basenji dogs with spontaneous idiopathic Fanconi syndrome.

To comprehend the renal defect underlying idiopathic Fanconi syndrome in the Basenji dog, we have focused on delineating the lipid profiles of renal brush border membranes isolated from affected and normal Basenji dogs to establish any physical or compositional changes underlying previously observed transport and membrane-fluidity changes. The lipid composition was studied with respect to total lipid, cholesterol, and phospholipid content, cholesterol to phospholipid ratio, distribution of the major phospholipid classes, and fatty acid composition. Total phospholipid of the isolated renal brush border membranes from Fanconi syndrome dogs analyzed by 31P nuclear magnetic resonance showed no difference compared with that of normal dogs.

The effect of glucose and galactose toxicity on myo-inositol transport and metabolism in human skin fibroblasts in culture.

Myo-inositol transport and metabolism were studied in cultured human skin fibroblasts exposed to potentially toxic levels of glucose or galactose. Although variable among 11 different cell lines, the myo-inositol level in confluent cells, ranging from 10-50 nmol/mg protein, was constant with passage. A high-affinity transport system for myo-inositol had an apparent Kt of 55 microM and Vmax of 16 pmol/min/mg protein.

myo-inositol transport and metabolism in fetal-bovine aortic endothelial cells.

The myo-inositol transport system in confluent fetal-bovine aortic endothelial cells was characterized after 7-10 days in subculture, at which time the myo-inositol levels and rates of myo-[2-3H]-inositol uptake and incorporation into phospholipid had reached steady state. Kinetic analysis indicated that the uptake occurred by both a high-affinity transport system with an apparent Kt of 31 microM and Vmax. of 45 pmol/min per mg of protein, and a non-saturable low-affinity system.

Kinetic evidence for compartmentalization of myo-inositol in hepatocytes.

myo-Inositol uptake and conversion to phosphatidylinositol (PI) was studied in isolated rat hepatocytes. Uptake of myo-[2-3H]-inositol into the trichloroacetic acid (TCA)-soluble fraction showed no evidence of saturation, while incorporation into lipid had an apparent Km of 0.28 mmol/L for external myo-inositol.

Maturational regulation of inositol 1,4,5-trisphosphate metabolism in rabbit airway smooth muscle.

Airway reactivity has been shown to vary with age; however, the mechanism(s) underlying this process remain unidentified. To elucidate the role of ontogenetic changes in phosphoinositide-linked signal transduction, we examined whether age-related differences in tracheal smooth muscle (TSM) contractility to carbachol (CCh) are associated with developmental changes in the production and metabolism of the second messenger, inositol 1,4,5-trisphosphate (Ins (1,4,5)P3). In TSM segments isolated from 2-wk-old and adult rabbits, both the maximal isometric contractile force and sensitivity (i.

Phosphatidylinositol:myo-inositol exchange activity in intact nerve endings: substrate and cofactor dependence, nucleotide specificity, and effect on synaptosomal handling of myo-inositol.

Micromolar concentrations of CMP produced a large increase in Mn2+-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve endings or synaptosomes. The apparent Km for CMP was 2 microM, and that for myo-inositol was 38 microM. Only cytidine nucleotides were capable of enhancing activity, and this effect is probably specific for CMP, because the synaptosomal preparation rapidly converted CTP or CDP to CMP.

Effect of elevated potassium on phospholipid and inositol metabolism of isolated nerve endings.

Synaptosomes were isolated from rat cerebra, and incubated in the presence of labelled phosphate and inositol. When the potassium concentration of the medium was increased by replacing NaCl with KCl, there was a marked increase in phosphate labeling of phosphatidic acid (PA) and phosphatidylinositol (PI). This was evident with [K(+)] above 12 mM and peaked at about 40 mM KCl.

High-performance liquid chromatography of phospholipids: quantitation by phosphate analysis.

Quantitation of individual phospholipids separated by HPLC from tissue extracts by colorimetric analysis of phosphate was investigated. Elution of inorganic phosphate and breakthrough of lecithin were determined using radioisotopes. A substance which interfered with sample phosphate determinations was found in the column eluant, and a method to minimize its effect was developed.

CMP-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve-endings.

Greatly enhanced manganese-dependent phosphatidylinositol:myo-inositol exchange activity was observed when isolated, intact nerve-endings were incubated with the nucleotide, CMP, suggesting that the enzyme, CDP-diglyceride:inositol phosphatidyl transferase, catalyzes this exchange. CMP, at 10 microM, produced as much myo-[2-3H] inositol incorporation into phosphatidylinositol as did 1 mM. This CMP-stimulated exchange activity may reside on the plasma membrane.

The effect of phentolamine on synaptosomal phosphatidylinositol in experimental galactose toxicity.

Abnormal myo-[2-3H]inositol incorporation into phosphatidylinositol has been found in phentolamine-treated synaptosomes that were isolated from the cerebral hemispheres of galactose toxic rats and incubated with [33P]Pi and myo-[2-3H] inositol. In galactose toxic rats phentolamine-stimulated myo-[2-3H]inositol labeling of phosphatidylinositol was 70% greater than in normal animals. This enhanced labeling of synaptosomal phosphatidylinositol in galactose toxic rats during stimulation with phentolamine is in marked contrast to the depressed myo-inositol labeling of phosphatidylinositol reported with acetylcholine stimulation.

High-performance liquid chromatography of phospholipids with UV detection: optimization of separations on silica.

Chromatography of phospholipids was performed on silica columns with detection by absorbance at 205 nm using mixtures of hexane--isopropanol--water in which the role of water and isopropanol in elution was investigated. One system was developed which provided adequate separation of most major phospholipid species. However, lipids with several ionizable groups were not well separated and gave multiple broad peaks.

Experimental galactose toxicity: effects on synaptosomal phosphatidylinositol metabolism.

Experimental galactose toxicity was induced by weaning rats onto an isocaloric 40% galactose diet. Synaptosomes were prepared from cerebra of rats at 2-9 weeks post-weaning and incubated with [33P][i and myo-[2-3H]inositol in the presence or absence of 0.2 mM-acetylcholine.

Evaluation of the binding of serotonin by isolated CNS acidic lipids.

Binding of serotonin by rat lipids was examined in an organic solvent-aqueous partition system. Only phospholipids and sulfatide were found to have appreciable activity; this technique was unsuitable for gangliosides due to their poor extractibility. Binding by phospholipid was abolished and that by sulfatide was greatly inhibited by increasing ionic strength in the aqueous phase.

Evaluation of continuous solvent extraction of organic acids from biological fluids.

We evaluated the efficiency of continuous solvent extraction with ether for the analysis of organic acids by gas chromatography using a representative group of organic acids and urine from several normal children. Variables examined were the time of extraction, volume of sample, and the quantity and chemical class of acid present. In terms of the decreased time required and invariant extraction of acids of pathologic significnace continuous solvent extraction compares favorably with the more time consuming albeit less discriminatory ion-exchange procedure.

Beta-methylcrotonic aciduria associated with lactic acidosis.

A patient is described in whom lactic acidosis of very severe degree was found to coincide with the presence of beta-methylcrotonic acid and rho-hydroxyphenyllactic acid in urine in large amounts, while beta-hydroxyisovaleric acid was found to be a relatively minor excretion product. Beta-methylcrotonic acid is demonstrated, for the first time, to be present in blood and CSF. These findings are discussed in relation to the patients previously reported to have beta-methylcrotonylglycinuria and raise the possibility that our patient's organic aciduria may be secondary to acquired disease rather than to an inborn error of metabolism.

Subcellular fractionation of pig platelets.

Subcellular components were obtained from pig platelets, disrupted by means of a French press and separated into 4 primary fractions. The granule fraction (10 000 g) was subjected to a sucrose gradient fractionation. Primary fractions and the granule subfractions were studied electron microscopically and biochemically by following the distribution of markers of membranes, lysosomes or alpha-granules, mitochondria and dense granules.