Biotinylated erythrocytes: in vivo survival and in vitro recovery.

Rabbit erythrocytes were biotinylated by reaction with N-hydroxysuccinimidobiotin; the average level of biotinylation was 25,000 molecules per erythrocyte. These biotinylated cells exhibited a normal survival rate when reinfused into rabbits. Two studies demonstrated that the biotin label was stable in vivo.

A 7-year study of the diagnostic value of rectal mucosal acetylcholinesterase measurement in Hirschsprung's disease.

Over a 7-year period, 213 children were investigated for failure to pass meconium or for chronic constipation. Of these, 45 were confirmed to have Hirschsprung's disease; in this group the acetylcholinesterase activity in rectal biopsy tissue was significantly increased (P less than .001; mean 34.

Phosphatidylinositol 4-phosphate kinase is associated with the membrane skeleton in human erythrocytes.

Phosphatidylinositol 4-phosphate kinase was eluted from human erythrocyte stroma by three separate and distinct techniques which are known to disrupt the membrane skeleton. In addition, this kinase was found to be associated with the intact skeletons prepared by Triton X-100 extraction of stroma. Phosphatidylinositol 4-phosphate kinase which has been extracted from the membrane is a freely soluble protein with poor enzymatic activity toward added phosphatidylinositol-4-phosphate; however, the enzyme was shown to reassociate with skeleton-depleted stroma and then regain full enzymatic activity toward stromal bound substrate.

Quantitation of adenosine-5'-triphosphate used for phosphoinositide metabolism in human erythrocytes.

The human erythrocyte actively phosphorylates and dephosphorylates phosphatidylinositol present in the membrane in an apparent "futile cycle." Recent reports have proposed that this phosphorylation/dephosphorylation cycle is a significant consumer of adenosine-5'-triphosphate (ATP) in the erythrocyte. This study details two independent techniques for quantitating the ATP consumed by this phosphoinositide futile cycle.

Peripheral nerve changes following epineurial injection of saline and blood in rat sciatic nerve.

The effects of warm saline and heparinized autogeneic blood injection on a rat sciatic nerve were observed by histologic, histochemic, and CT planimetry. Demonstrable intraneural degenerative changes and epineurial cellular proliferation were present in both groups. Hemosiderin deposits were present in the epineurium and intraneurally only in the blood group.

Molecular forms of acetylcholinesterase in Hirschsprung's disease.

We describe changes in the levels of different molecular forms of acetylcholinesterase in four cases of Hirschsprung's disease linked to the transition from aganglionic to normal bowel. In addition changes in a control case with histologically normal bowel is reported. In all patients with Hirschsprung's disease there is a marked increase in the level of the tetrameric form of the enzyme in the aganglionic region.

Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase.

A method has been developed to quantitate megakaryocytopoiesis in culture by measuring acetylcholinesterase synthesized in vitro. Murine marrow cells, treated with diisopropylfluorosphosphate (DFP) to inactivate initial acetylcholinesterase (AchE) present in megakaryocytes and contaminating blood, were set up in Iscove's medium supplemented with 15% DFP-treated horse serum +/- pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) in 96-well microplates. Following the culture period, Triton X-100, dithiobisnitrobenzoic acid (DTNB), and acetylthiocholine iodide were added to each well.

A sensitive fluorimetric rate assay for biotinidase using a new derivative of biotin, biotinyl-6-aminoquinoline.

The previously reported method for the estimation of biotinidase (EC 3.5.1.

Glutathione-dependent protection against oxidative damage of the human red cell membrane.

Glutathione (GSH) dependent protection against oxidative damage of human red cell membrane was examined. An artificial system was used in which chloroform/methanol-extracted red cell lipids, in the form of liposomes, were subjected to attack by a peroxidation system consisting of ascorbate-Fe3+. Human erythrocytes contained a nondialyzable factor, completely inactivated by heating in a boiling water bath for 3 min, which showed GSH-dependent inhibition against lipid peroxidation and was devoid of GSH peroxidase activity.

Pyruvate carboxylase deficiency.

The causes of congenital lactic acidaemia are outlined. Isolated pyruvate carboxylase deficiency is reviewed in detail with a report of a recent case and a discussion of the biochemical consequences. Other causes of defective pyruvate carboxylation are described, particularly the combined carboxylase defects.

The effect of phlebotomy as a treatment of Fabry disease.

Senescent erythrocytes are considered a major source of the ceramide trihexoside which accumulates in Fabry disease patients. We have evaluated weekly phlebotomy as a method to reduce the catabolic load imposed by the senescence of erythrocytes and thereby ceramide trihexoside in one Fabry patient; no change was observed in either plasma or urinary level of ceramide trihexoside. The implications of this observation are discussed with regard to glycolipid metabolism in man.

Gaucher disease: a century of delineation and research. Enzyme replacement therapy: model and clinical studies.

A number of investigators have attempted to treat Gaucher disease with exogenous glucocerebrosidase. Although at times encouraging biochemical changes and suggestive alterations in organomegaly have been reported, overall, the results of enzyme replacement therapy must be judged to be a failure. In order to understand this lack of success with a promising treatment modality, four aspects of enzyme replacement therapy require examination: 1.

Antithrombin III does not have bound glucocerebroside.

Purified antithrombin III has been reported to have bound glucocerebroside, the major glycolipid of plasma. We have separated whole plasma by ultracentrifugation into lipoprotein-rich and lipid-deficient fractions and demonstrated that glucocerebroside and antithrombin III clearly separate into different fractions. Antithrombin III does not have glucocerebroside associated with it.

Studies on glutathione transport utilizing inside-out vesicles prepared from human erythrocytes.

Adenosine triphosphate-dependent glutathione transport was characterized using inside-out vesicles made from human erythrocytes. Kinetic analysis of the glutathione disulfide (GSSG) transport showed a biphasic Lineweaver-Burk plot as a function of GSSG concentration suggesting the operation of two different processes. One phase had a high affinity for GSSG and a low transport velocity.