Perinatal lethal form of Gaucher's disease presenting with hemosiderosis.

A term infant with hydrops fetalis presented with hypotonia, massive splenomegaly, renal failure, and severe hyperferritinemia. Multiple organ failure, myoclonus, and opisthotonus ensued and she died at 15 days of age. High rounded forehead, large open fontanel, and a small recessed chin led to initial premortem diagnosis of Zellweger syndrome, but her plasma profile of long chain fatty acid was normal. Her subsequent clinical course and findings of postmortem examinations were consistent with perinatal lethal form of Gaucher's disease (PLGD). The diagnosis was confirmed by deficiency of enzyme beta-glucocerebrosidase in white blood cells and in cultured fibroblasts. In addition to the crossover features of Zellweger phenotype, this infant exhibited a number of unusual features including, severe hyperferritinemia, rapid progression of splenomegaly, and absence of icthyosis.

In vitro susceptibility of Mucor ramosissimus Samutsevitsch, a rare fungal pathogen, to antimycotics.

The in vitro susceptibility of a rare fungal pathogen, Mucor ramosissimus Samutsevitsch, to antimycotics in clinical use was determined. Minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs) were (MIC/MFC, microgram/ml): amphotericin B, 3.125/25; miconazole, 6.25/6.25; clotrimazole, 25/25; fluconazole, 5-fluorocytosine, itraconazole, and ketoconazole, > or = 100/100. Our results show that amphotericin B and miconazole are the most effective antimycotics.

Membrane-bound hemoglobin as a marker of oxidative injury in adult and neonatal red blood cells.

A comparative study of the effect of hydrogen peroxide on adult and neonatal red blood cell (RBC) membrane protein composition has been carried out. The results indicate that (a) the native neonatal RBC membranes contain higher levels of membrane-bound hemoglobin (MBHb) than the adult RBC membranes. (b) The content of MBHb increases when RBCs are incubated with increasing concentrations of hydrogen peroxide (H2O2), more so in neonatal than in adult RBCs; however, neonatal RBC membrane proteins are less susceptible to H2O2 oxidation than adult ones. This could be attributed to the fact that Hb F, which is more susceptible to oxidation than Hb A, adds to the reduction potential of neonatal RBC (in which it is present in large amounts) and partially protects neonatal membrane proteins against oxidant stress compared to Hb A in adult RBC. (c) In both neonatal and adult RBCs, Spectrin 1 is relatively more susceptible to oxidant stress than spectrin 2, and spectrins in adult RBC are more labile for peroxidation than the spectrins in neonatal RBC. (d) Based on electrophoretic studies with and without reduction of membranes with mercaptoethanol, we have classified two types of MBHb: Type I is adsorbed to membrane by noncovalent interactions and Type II MBHb is chemically crosslinked to membrane components by disulfide bridges; the content of both these types increases when RBCs are incubated with increasing concentrations of H2O2. (e) Band 6 protein is present in higher amounts in neonatal than in adult RBC membranes. (f) Since the total content of MBHb increases linearly with the level of oxidant stress, we suggest that it could be used as a marker for oxygen radical-induced injury to tissues.

Interaction of p-aminobenzoic acid with erythrocyte membrane: photoaffinity labeling of the binding sites.

p-Aminobenzoic acid (PABA) was found to prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right-side-out membrane vesicles revealed a similar number of binding sites and Kd values for both normal and sickle cell membranes. A [14C]Azide analog of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface. The azide was found to covalently incorporate into the membrane components upon irradiation; 52-35% of the label was associated with the proteins and the remaining with the lipids. Electrophoretic analysis of photolabeled membranes revealed that the azide interacts mainly with Band 3 protein in the case of intact erythrocytes and right-side-out sealed vesicles; however, if unsealed ghosts are used, other membrane proteins besides Band 3 are photolabeled. PABA was found to inhibit both high and low affinity calcium-binding sites situated on either surface of the membrane apparently in a non-competitive manner. However, calcium binding stimulated by magnesium and ATP was only slightly affected. Calcium transport into inside-out vesicles was inhibited by PABA, but it did not affect the calcium ATPase activity.

Studies on the turnover of methionine oxidized alpha-1-protease inhibitor in rats.

Alpha-1-protease inhibitor (alpha-1-PI) is the major regulator of extracellular leukocyte elastase activity and can be rendered impotent against elastase by oxidation of a critical methionine, residue 358. Alpha-1-PI was isolated from rat plasma by affinity chromatography on Sepharose-bound anhydrochymotrypsin, DEAE-cellulose anion-exchange, and Sephadex G-150 gel filtration. The product was radiolabeled using non-oxidative conditions with Bolton-Hunter reagent, and an aliquot subsequently oxidized with N-chlorosuccinimide. Turnover studies in rats indicated that both native and oxidized alpha-1-PI had half-lives of 170 min. Using partially purified human neutrophil methionine sulfoxide-peptide reductase (Met(O)PR), it was demonstrated that oxidized product could be converted back "in vitro" to an active inhibitor of elastase. To assess whether oxidized alpha-1-PI underwent reduction "in vivo," methionine-oxidized rat inhibitor was injected into the rats, aliquots of plasma samples were withdrawan and passed through a Sepharose-bound anhydrochymotrypsin affinity resin, and bound functional alpha-1-PI was eluted with 0.1 M chymostatin. Radioactive counting of bound and unbound fractions indicated that reduction does not occur in vivo and suggested that, at least under homeostatic conditions, the Met(O)PR is confined to intracellular sites where it does not have access to the circulating protein.

Interaction of hemoglobin and its component alpha and beta chains with band 3 protein.

The equilibrium binding of hemoglobin to isolated band 3 protein exhibited positive cooperativity [Hill coefficient = 1.65 +/- 0.1; total number of binding sites at pH 6.6 in 5 mM sodium phosphate buffer = 32 500 +/- 940 pmol/mg; Ka = (3.0 +/- 0.5) X 10(5) M-1]. The binding was reversible and ionic in nature as the bound hemoglobin was readily displaced by KCl, ATP, and 2,3-diphosphoglycerate, the latter two being more effective than KCl on a molar basis. The ratio of the interaction of hemoglobin to band 3 protein per se was 1:1, whereas the band 3 preparation as a whole (protein + lipids) was 3:1. Saturating levels of glyceraldehyde-3-phosphate dehydrogenase blocked only 33% of the total binding sites which were localized at the cytoplasmic segment; the remaining 67% was localized in lipids by their extraction with acetone. Reconstitution of acetone-extracted band 3 with phospholipid liposomes indicated phosphatidylserine as the binding site. The positive cooperativity in binding to acetone-extracted band 3 was increased (Hill constant = 2.1 +/- 0.1) compared to the band 3 preparation. After separation of the alpha and beta chains of hemoglobin, only the alpha chain binds to band 3 with positive cooperativity to an extent of 45-50% of native hemoglobin with similar affinity. The binding capacity of p-(hydroxymercuri)benzoate (HMB) derivatives of hemoglobin and its alpha chain was less than that of native hemoglobin, whereas HMB-beta chain or beta chain did not bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic regulation of carotene biosynthesis in selected tomato strains: aspects of beta-carotene biosynthesis and B gene specificity.

A comparative qualitative and quantitative study of the carotene compositions of a high beta-carotene type mutant Priya Darshini-1 (PD-1), high lycopene type, Pusa Ruby (PR) and the F1 hybrid of these two strains was carried out. Increased amounts of beta-zeacarotene and gamma-carotene were realized in the PD-1 parent and in the F1 hybrid. This correlates the increased beta-zeacarotene and gamma-carotene synthesis with increased beta-carotene synthesis. Introduction of gene B (the gene that regulates beta-carotene biosynthesis) to a system lacking it induces considerable amount of beta-carotene synthesis at the expense of lycopene in the hybrid. Based on these observations a 'three point control' for the beta-carotene biosynthesis in tomato fruits is suggested with the gene B capable of promoting two different pathways in the high beta-carotene types. The probable sites of action of the modifier mOB (the gene that regulates expression of B gene) are indicated. Beta-carotene and xanthophyll contents of different parts of tomato plants of a PD-1 type and the PR type F2 segregates of the F1 hybrid (PR X PD-1)F1 have been studied. Results indicate that the action of gene B is highly specific and exclusively oriented towards the fruit and not expressed in any other parts of the tomato plant.

A simple TLC method for the determination of pro-vitamin A content of fruits and vegetables.

A simple and rapid method to isolate and estimate carotene compositions of tomatoes, carrots and green vegetables is described. The chief distinguishing features of this method are its simplicity in the preparation of TLC plates that avoids the requirements of the TLC applicator, and avoiding the meticulous conditions like saturation of chamber etc. employed in the routine TLC methods. By employing this method carotene compositions of tomatoes, carrots and the green vegetable, Amaranthus gangetica are determined. The time taken for the separation of various carotene bands of several samples is as short as 10 minutes. As many as 20 different samples could be analyzed at a time. The values obtained for the individual carotenoids by this TLC method are in very good agreement with those obtained after the column chromatographic separation of the same material. Saponification is not required for the extracts from tomatoes and carrots as it does not influence the individual values of carotenes. Hence in these cases, crude extracts can be directly analyzed by TLC. However, in case of the green vegetables (Amaranthus gangetica, for example) the value of beta-carotene after saponification was found to be twice the value obtained by TLC of the crude extract as such. The general applicability of this method in the separation of vitamin A alcohol from its esters, retinoic acid from retinoic acid anhydride etc. has been discussed. The method would be quite handy and of particular use to especially those carrying out the genetic studies with tomatoes and carrots. It could be employed for a quick and accurate determination of pro-vitamin A content of fruits and vegetables and is more economical than the standard TLC procedures.

Photoaffinity labeling of procaine-binding sites in normal and sickle cell membranes.

A photoaffinity probe, procaine azide, was employed to determine the sites of interaction of procaine in normal and sickle cell erythrocytes. Studies show that the number of binding sites and affinity of procaine to membranes derived from normal and sickled cell erythrocytes were similar, although procaine retards the in vitro formation of irreversibly sickled cells from cells, The results show that procaine azide, a photoaffinity analogue of procaine, is covalently incorporated into both protein (60--70%) and lipid (40--30%) components of the membrane. Sodium dodecyl sulfate-gel electrophoresis of the labeled ghosts show that procaine binds specifically to band 3 and periodic acid Schiff staining bands in membranes derived from labeled erythrocytes. Binding of procaine or covalent incorporation of procaine azide into membrane proteins does not affect the phosphate transport. Moreover, pre treatment of intact erythrocytes with 4,4-'diisothiocyano-2,2'-stilbene disulfonate, an anion transport inhibitor, did not affect either the binding or covalent incorporationof procaine azide into erythrocytes. These results indicate that the binding of procaine azide to Band 3 protein occurs at a locus different than that involved in anion translocation process.

Preparation, properties and metabolism of retinoic acid anhydride.

A new analogue of vitamin A, viz., retinoic acid anhydride was prepared, for the first time, by the action of thionyl chloride on retinoic acid in benzene containing pyridine. The amhydride was charcterised by its chromatographic properties, elemental analysis, ultraviolet absorption, infrared and nuclear magnetic resonance spectral characteristics. The compound could be readily hydrolysed to retinoic acid both by acid and alkali treatments and reduced by lithium aluminium hydride to vitamin A alcohol (retinol). The spectral changes with antimony trichloride reagent were similar to those observed for retinoic acid. The metabolism of retinoic acid anhydride was found to be similar to that of retinoic acic. When administered either orally or intraperitoneally, the compound promotes growth in vitamin A-deficient rats. Time-course experiments revealed that retinoic acid anhydride is converted into retinoic acid by non-enzymatic hydrolysis and thereby exerts its biological activity. The biopotency of the anhydride was found to be nearly the same as that of the acid. A new method of preparing esters of retinoic acid employing retinoic acid anhydride as an intermediate, has been described.