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R F Branda

Publications and source records attributed to R F Branda.

At least 55 records · Page 3Linked to original sources
PubMed1983

Oxygenation alters red cell folate levels.

Folate activity is consistently higher in deoxygenated than oxygenated human blood as measured by both radioassay and Lactobacillus casei growth. Red cell, but not serum, folate levels are rapidly mutable: when fully oxygenated, the mean red cell folate level of 15 samples was 489 ng/ml; after deoxygenation the mean rose to 553 ng/ml; following re-oxygenation the mean returned to 488 ng/ml. Transport of folate across the red cell membrane apparently does not contribute to these changes in folate levels, since agents known to inhibit permeation (N-ethyl-maleimide, p-chlormercuriphenyl sulfonic acid and ethacrynic acid) do not affect the rise with deoxygenation. Moreover, new synthesis of folate compounds seems unlikely, since the changes are observed in energy-depleted cells. Instead, direct binding of folate to haemoglobin is suggested by experiments in which the rise with deoxygenation was not seen in red cells exposed to carbon monoxide or treated with cyanate. Similar changes also occur in vivo: canine red cell, but not serum folate levels are significantly higher (P less than 0.05) in samples collected from five different veins compared with blood from the paired artery (carotid-jugular, renal, mesenteric, splenic, femoral) of four animals. Thus red cell folate levels are predictably influenced by the levels of oxygenation of the erythrocyte both in vitro and in vivo, possibly by reversibly binding to haemoglobin.

Animals↗
PubMed1983

Retinoic acid treatment of acute promyelocytic leukemia: in vitro and in vivo observations.

We describe in vitro studies and a therapeutic trial of retinoic acid (RA) in a patient with acute promyelocytic leukemia (APL) refractory to chemotherapy. Bone marrow promyelocytes from the patient, prior to RA, matured morphologically in liquid culture with RA (97% maturing myeloid cells compared with 26% in control cultures at 7 days). RA-cultured cells displayed leukocyte alkaline phosphatase activity and cytoplasmic maturation (by electron microscopy). Retinoic-acid-treated cells, compared to controls, demonstrated increased functional maturation, with phagocytosis of opsonized zymosan (90% versus 10%) and production of superoxide (measured by nitroblue tetrazolium reduction) in response to phorbol ester, opsonized zymosan, or the chemotaxin F-met-leu-phe. There was no evidence of active proliferation in the cultures. RA-treated cells continued to show 15;17 chromosomal translocation after 7 days in culture. The patient was treated with oral 13-cis-retinoic acid (100 mg/sq m/day) for 13 days. During that time, the peripheral white blood count rose from 300 cu mm to 6,700 cu mm, and the maturing myeloid cell count rose from 54 cu mm to 3,800 cu mm. Bone marrow maturing cells increased from 1.8% to 8.0%. Despite the increasing number of maturing myeloid cells, the patient died on day 13 from disseminated candidiasis. These data confirm that RA induces maturation of leukemic promyelocytes in vitro and suggest that similar maturation is achievable in vivo. We suggest that oral retinoic acid may be a useful adjunct in the treatment of APL.

Adult↗
PubMed1982

Effects of pH on 5-methyltetrahydrofolic acid transport in human erythrocytes.

We measured the effects of external pH on influx, efflux and net steady-state levels for 14CH3H4PteGlu1. Initial rates of uptake were inversely proportional to external pH. Lowering the pH of the suspending medium increased influx by enhancing the affinity of the carrier: the apparent Km values at pH 6, 7.2 and 8.1 were 0.14, 0.25 and 0.63 microM respectively. In contrast, Vmax was independent of external pH. Efflux rate constants at pH 6, 7.5 and 8.5 were 0.034, 0.059 and 0.067/min respectively. consequently, lowering the external pH increased steady-state levels of 14CH3H4PteGlu1.

Biological Transport↗
PubMed1982

Protein A adsorption of acute myelogenous leukemia serum induces in vitro blast lysis.

We studied cytotoxic activity of acute myelogenous leukemia (AML) sera for AML blasts before and after immunoadsorption with Staphylococcus aureus, Cowan I (SAC), which contains protein A. We found in vitro that incubation with treated AML sera reduced viability to 42.7% of control (p less than 0.01) for autologous and 21.0% of control (p less than 0.01) for allogeneic blasts. Normal peripheral blood cells were not killed by treated AML sera. Wood 46 strain of Staphylococcus aureus, which does not contain protein A, did not significantly reduce AML blast viability (94.8%, p greater than 0.4), while Sepharose-bound protein A reduced viability to 63.8% (p less than 0.01). Cytotoxicity does not appear to be complement-mediated, byt cytotoxic activity is trypsin-sensitive and is contained in the immunoglobulin fraction. This model for study of the tumoricidal action of protein A adsorption should be useful for predicting utility of plasma adsorption as a therapeutic adjunct in the future.

Cytotoxicity, Immunologic↗
PubMed1981

Transport of 5-methyltetrahydrofolic acid in erythrocytes from various mammalian species.

Uptake of 5-methyltetrahydrofolic acid, and serum and red cell folate levels were measured in peripheral blood samples from seven mammalian species. Velocity of uptake of 5-methyltetrahydrofolic acid, at a concentration of 1 microM, increased from 0.001 pmole/10(9) red cells/minute (guinea pig) to 0.7 pmole/10(9) red cells/minute (pig) in the sequence: guinea pig less than sheep less than dog less than man less than rabbit less than rat less than pig. Michaelis constants (Km) were similar for human, dog and sheep erythrocytes (0.5--0.7 microM) but higher in red cells from rabbit, rat and pig (1.7--3.5 microM). Serum and red cell folate levels were comparable in human and dog samples; both levels were higher in rabbit and rat blood. In contrast, red cell folate levels were much lower in pig, sheep and guinea pig peripheral blood. These studies provide evidence that folate compounds are transported by an erythrocyte membrane carrier which is separate from the known permeation system for anions, since the species sequence for the latter is entirely different. They also suggest that some mammalian species, such as the dog, may be more relevant to man for studies of blood folate metabolism than several other species.

Aging↗
PubMed1981

Inhibition of 5-methyltetrahydrofolic acid transport by amphipathic drugs.

Numerous chemically unrelated drugs after the membrane transport of folate compounds. To investigate drug structure-activity relationships, we measured the effect of amphipathic drugs (that is, compounds with polar-apolar character) on 5-methyltetrahydrofolic acid permeability of human erythrocytes. All drugs tested were inhibitory, but only compounds that exist at least partially in the anionic form were highly active. Ethacrynic acid, sulfinpyrazone, phenylbutazone, sulfasalazine, and furosemide were effective transport inhibitors in micromolar concentrations. In contrast, compounds that are capable of forming cations at physiologic pH, such as chlorpromazine, procaine, tetracaine, and papaverine, were inhibitory only in millimolar concentrations or caused hemolysis before major inhibition was seen. Inhibitory activity correlated with drug dissociation constant (r = 0.87). A double-reciprocal plot analysis of drug effect on 5-methyltetrahydrofolic acid transport showed changes in both Km and Vmax (indicating a mixture of competitive and noncompetitive inhibition) by ethacrynic acid, sulfasalazine, and phlorizin. Inhibitory activity of a series of eight phenoxyacetic derivatives, including ethacrynic acid, correlated highly with measurements of liposolubility (r = 0.87) but only weakly with the Hammet substituent constant (r = 0.56). These results suggest that the effect of amphipathic drugs on 5-methyltetrahydrofolic acid transport is influenced by drug pKa and by the presence of hydrophobic substituents, but is relatively independent of electron-attracting groups.

Adult↗
PubMed1980

Anion channel blockade: effects upon erythrocyte membrane calcium response.

The influx of small amounts of calcium into normal human red cells adversely affects cellular metabolism, shape, ion and water content, and deformability. Because restriction of cation (potassium) efflux moderates these deleterious effects of Ca++ influx, we investigated the possibility that inhibition of anion permeability might be similarly beneficial. Human red cells were treated with 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), which reacts preferentially with external sites on the transmembrane protein, band 3, and specifically inhibits inorganic anion transport. DIDS-treated and control cells were then exposed to the ionophore A23187 and Ca++. We find that DIDS treatment blocks Ca++-induced K+ and water loss and diminishes changes in osmotic fragility and cellular elasticity. Furthermore, DIDS reduces the "trapping" of hemoglobin by red cell membranes and increases the apparent affinity of the cell membrane for glyceraldehyde-3-phosphate dehydrogenase.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1979

Impaired bone marrow lymphocyte proliferation in acute myelogenous leukemia.

Bone marrow cells were cultured in liquid media with and without phytohemagglutinin (PHA) to test bone marrow lymphocyte response during the remission period of acute myelogenous leukemia (AML). Mitogen-stimulated cultures from nine leukemic patients while in complete remission showed high percentages of morphologically transformed lymphocytes, but proliferation of these cells was significantly decreased (p less than 0.02) in cultures from six patients who subsequently relapsed (mean remission duration 10 months) and died. In contrast, lymphocyte proliferation in cultures from three AML patients with long remission (greater than 30 months) was comparable to controls. Parallel cultures without added PHA showed a progressive decrease in total viable cell number with time, but an increasing percentage of macrophages in both control and AML cultures. These studies suggest that bone marrow lymphocyte proliferation, but not morphologic transformation, is impaired in some patients with AML, and that identification of this defect may be of prognostic value.

Adult↗
PubMed1979

Evidence for transfer of folate compounds by a specialized erythrocyte membrane system.

Transfer of folate compounds was studied in human erythrocytes. At steady state, the measured distribution ratio of 5-CH3-H4-folate in erythrocyte suspensions exceeded the ratio predicted from the chloride distribution ratio by a factor of 1.58, suggesting that human erythrocytes concentrate folate. Because folate compounds are anionic at physiologic pH, we investigated the possibility that transport occurs via the inorganic anion channel associated with the predominant integral membrane protein, band 3. Erythrocyte uptake of 5-CH3-H4-folate was decreased (60% to 80%) by several known inhibitors of anion transport--pyridoxal phosphate, dipyridamole, phlorizin, and SITS. However, unlike the inorganic anion transfer system, 5-CH3-H4-folate uptake was only slightly decreased by DIDS; was reduced 50% to 70% by the sulfhydryl reagents NEM, PMB, and pCMBS; and was not affected by the proteolytic enzymes trypsin, chymotrypsin, and pronase. These studies suggest that folate compounds are transported by a specialized carrier system, independent of the inorganic anion channel, which contains sulfhydryl and amino groups. In contrast to 5-CH3-H4-folate transfer, uptake of pteroylglutamic acid was either unaffected or somewhat increased by these membrane modifications. This result indicates that the human erythrocyte transports the reduced and oxidized forms of the vitamin by entirely separate mechanisms.

4-Chloromercuribenzenesulfonate↗
PubMed1979

Hereditary dyserythropoiesis with abnormal membrane folate transport.

Dyserythropoiesis, which morphologically and serologically resembles congenital dyserythropoietic anemia type III but is not accompanied by anemia, is described in a young man. In addition to striking gigantism and multinuclearity of erythroid precursors, electron microscopy revealed widening of nuclear pores, nuclear clefts, and cytoplasmic inclusions. Membrane transport of 5-methyltetrahydrofolate by the patient's red cells was markedly reduced; total uptake, uptake velocity, and maximal velocity of uptake were all significantly less than in controls. In contrast, red cell uptake of pteroylglutamic acid was normal. Bone marrow cells in culture also showed decreased 5-methyltetrahydrofolate uptake, as well as very low thymidine incorporation. Because folate uptake by mitogen-stimulated lymphocytes was normal, the defect apparently does not involve all cell lines. These results suggest that a specific membrane defect, affecting the carrier system for reduced folate compounds, is present in this patient's erythrocytes, and perhaps, their bone marrow precursors.

Adult↗
PubMed1978

Skin color and nutrient photolysis: an evolutionary hypothesis.

Human populations native to areas of intense sunlight tend to be heavily melanized. Previous explanations for this relationship have invoked only weak selective pressures. To test the hypothesis that dark pigmentation may protect against photolysis of crucial light-sensitive vitamins and metabolites by ultraviolet light, folate was used as a model. It was found that exposure of human plasma in vitro to simulated strong sunlight causes 30 to 50 percent loss of folate within 60 minutes. Furthermore, light-skinned patients exposed to ultraviolet light for dermatologic disorders have abnormally low serum folate concentrations, suggesting that photolysis may also occur in vivo. Deficiency of folate, which occurs in many marginally nourished populations, causes severe anemia, fetal wastage, frank infertility, and maternal mortality. Prevention of ultraviolet photolysis of folate and other light sensitive nutrients by dark skin may be sufficient explanation for the maintenance of this characteristic in human groups indigenous to regions of intense solar radiation.

Biological Evolution↗
PubMed1978

Plasma exchange in the treatment of fulminant idiopathic (autoimmune) thrombocytopenic purpura.

Rapid plasma exchange in a young man with idiopathic (autoimmune) thrombocytopenic purpura and life-threatening haemorrhage acutely increased the platelet-count and enhanced haemostasis. The patient had not responded to steroids, and massive intra-abdominal bleeding had developed after splenectomy, despite numerous platelet transfusions. After plasma exchange, his haemoglobin concentration became normal without further transfusion and his abdominal girth decreased. Therefore emergency reoperation because of intra-abdominal bleeding was no longer required. A cumulative-sums plot of his platelet-count confirmed that plasmapheresis on 2 occasions was followed by a pronounced rise in mean platelet-count. Neither prednisone nor vincristine therapy altered the slope of the plot. The high initial anti-platelet antibody concentration in this patient fell after plasma exchange. These findings indicate that plasmapheresis accelerated clearance of circulating anti-platelet factors in fulminant idiopathic thrombocytopenic purpura.

Adult↗
PubMed1978

Folate-induced remission in aplastic anemia with familial defect of cellular folate uptake.

Severe aplastic anemia developed in a young man with an extensive family history of leukemia, pancytopenia, and neutropenia. Megaloblastic changes became evident, and treatment with high doses of folic acid resulted in striking clinical improvement. However, red-cell folate levels remained persistently low despite high serum folate levels. A defect in cellular folate uptake was suspected, and, indeed, uptake of 5-14CH3-H4-folate by stimulated lymphocytes and by bone-marrow cells from the patient was significantly reduced (P less than 0.05 as compared to normal cells. Further characterization of folate metabolism showed that intestinal absorption of the vitamin, membrane transport of 5-14CH3-H4-folate by mature red cells, folate utilization in the conversion of deoxyuridylate to thymidylate and polyglutamate formation were all normal. At least five other family members manifest decreased uptake of 5-14CH3-H4-folate by stimulated lymphocytes. These studies suggest that a genetically induced abnormality of folate uptake contributed to this patient's severe, but reversible, aplasia.

Adult↗
PubMed1978

The mechanism of 5-methyltetrahydrofolate transport by human erythrocytes.

The mechanism involved in 5-methyltetrahydrofolate uptake by human cells is poorly understood. To more clearly elucidate this physiologically important process, transport of the vitamin was studied in human erythrocytes. 5-methyltetrahydrofolate uptake was found to increase with reticulocytosis, but measurable incorporation occurred in erythrocyte suspensions depleted of reticulocytes, leukocytes, and platelets, indicating uptake by mature erythrocytes. Incubation of erythrocytes with increasing concentrations of [(14)C]5-methyltetrahydrofolate resulted in increasing uptake but decreasing percentage incorporation, consistent with saturation of a carrier system. Both influx and efflux phases of uptake were temperature dependent, with almost no transport at 4 degrees C. Uptake of [(14)C]5-methytetrahydrofolate was effectively inhibited by unlabeled 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate, but not by pteroylglutamic acid. Prior incubation with 5-formyltetrahydrofolate increased uptake of [(14)C]5-methyltetrahydrofolate, and extracellular 5-formyltetrahydrofolate enhanced efflux of [(14)C]5-methyltetrahydrofolate. Nearly total depletion of ATP increased uptake of [(14)C]5-methyltetrahydrofolate, but efflux was unchanged. Column chromatography of membrane-free hemolysate after incubation with [(14)C]5-methyltetrahydrofolate showed 95% of radioactivity corresponded to marker radioisotope, and no other peak was noted. Thus peripheral erythrocytes incorporate 5-methyltetrahydrofolate by a saturable, temperature-dependent, substrate-specific process which is influenced by counter-transport. This mechanism is qualitatively similar to the carrier-mediated transport of folate compounds previously described in other cell types. Therefore, human erythrocytes should be useful for detailed characterization of this membrane carrier system.

Anemia, Aplastic↗