Cosegregation of elastin-associated microfibrillar abnormalities with the Marfan phenotype in families.

The Marfan syndrome is a serious heritable connective-tissue disorder characterized primarily by ocular, cardiovascular, and musculoskeletal abnormalities but also involving multiple other tissues and organs of the body. Inherited as an autosomal dominant disorder, the etiology and pathogenesis of the Marfan syndrome are presently unknown. We have documented consistent apparent deficient content of elastin-associated microfibrillar fibers by indirect immunofluorescent (IF) studies of Marfan skin, as well as deficient accumulation of related fibrous materials in cultures of Marfan fibroblasts as compared with normal controls and patients with other heritable disorders of connective tissue. These data have suggested that abnormalities in the microfibrillar component of elastic-fiber systems may have a role in the etiology and pathogenesis of the Marfan syndrome. In the present study, we have analyzed the IF staining patterns of skin and fibroblast cultures from Marfan syndrome patients and normal first-degree relatives in nine Marfan kindreds. Three of these families had at least one affected individual in each of 2 generations, permitting intergenerational comparison of IF patterns. Six kindreds had one or more affected individuals in a single generation, making comparisons between siblings and/or parent-child possible. In all cases, IF abnormalities cosegregated with the Marfan phenotype and all nonaffected family members were normal. Within family groups containing more than one affected individual, the IF staining patterns were similar between affected patients. These data provide further confirmation of consistent and relatively specific deficiency of microfibrillar fibers in Marfan syndrome.

Effect of monosaccharide on dehydroascorbic acid uptake by placental membrane vesicles.

Dehydroascorbic acid (DHA), the oxidized form of vitamin C, is transported across the microvillous surface of the human placental syncytiotrophoblast by the D-glucose transporter. The existence of this mechanism suggests that maternal hyperglycemia may influence placental transfer of vitamin C. Therefore, we examined the effect of monosaccharides, equilibrated across the membrane, on the uptake of 0.5 mmol/L DHA by placental membrane vesicles. Relative to uptake in the absence of monosaccharide, the rate of DHA uptake was enhanced by up to 90% in the presence of 3-O-methyl-D-glucose equilibrated across the membrane. Comparable results were obtained with D-glucose and D-galactose. An inward-directed monosaccharide concentration gradient inhibited DHA uptake. However, with elevated equilibrium concentrations of monosaccharide, the magnitude of such uptake inhibition was reduced. Relative to DHA uptake at normal blood glucose concentrations (5 mmol/L), the results suggest that moderate maternal hyperglycemia does not alter, but that severe hypo- or hyperglycemia decreases, placental uptake of DHA from the maternal circulation.

Role of monosaccharide transporter in vitamin C uptake by placental membrane vesicles.

Dehydroascorbic acid (DHA), the reversibly oxidized form of vitamin C, was taken up much more rapidly than L-glucose into membrane vesicles prepared from the maternal face of the human placental syncytiotrophoblast. DHA uptake was sensitive to inhibition by cytochalasin B and was independent of a sodium concentration gradient. At equilibrium, the concentration of DHA in the vesicles did not exceed that of the medium. DHA and the D-glucose analogue, 3-O-methyl-D-glucose (3-O-MG) appeared to compete with one another for the transporter. The 3-O-MG and DHA inhibitory constants were indistinguishable. Vesicles loaded with a high concentration of 3-O-MG and suspended in low 3-O-MG displayed a marked, transitory enhancement of DHA, but not L-glucose uptake. These findings suggest that DHA is taken into the first cellular boundary of the placenta between maternal and fetal circulations by the sodium-independent monosaccharide transporter. In contrast to DHA, L-ascorbic acid, the reversibly reduced form of vitamin C, was taken into these vesicles much more slowly. This uptake was not affected by cytochalasin B nor by a sodium concentration gradient; it appeared to occur by simple diffusion.

Defective triggering of polymorphonuclear leukocyte oxidative metabolism by Legionella pneumophila toxin.

Preincubation of human polymorphonuclear leukocytes (PMNLs) with Legionella pneumophila toxin impaired activation of the superoxide-generating complex induced by latex particles and by the Ca++ ionophore A23187. The toxin had no effect, however, on activation of the complex induced by phorbol myristate acetate (PMA), concanavalin A, valinomycin, or bromolasalocid. The toxin prevented PMNL plasma membrane depolarization induced by A23187 but failed to influence the membrane depolarization induced by PMA. These observations indicate that Legionella pneumophila toxin selectively impairs activation of the phagocyte superoxide-generating complex without affecting the functional integrity of components of the complex.

Dehydroascorbate uptake as an in vitro biochemical marker of granulocyte differentiation.

We tested the hypothesis that the rate of cellular uptake of dehydroascorbate in cultures of developing granulocyte-macrophage progenitors in vitro would serve as a biochemical marker of neutrophil maturation. Suspension cultures of low-density, nonadherent, T-lymphocyte-depleted bone marrow cells from eight normal volunteers were cultured in medium containing 10% human placental conditioned medium and were harvested at intervals over 14 days. The harvested cells were tested for their ability to take up dehydroascorbate. Mean cellular uptake rate increased 12-fold by Day 10, at which time the cells had differentiated to neutrophils. Uptake increased by less than 2-fold in cells which had been induced to differentiate to mature mononuclear phagocytes with 12-O-tetradecanoylphorbol-13-acetate. Additional studies using HL-60 cells induced to differentiate with dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate support the view that a major increase in dehydroascorbate uptake in cultured granulopoietic progenitors is a manifestation of a neutrophil differentiation.

Effect of oxygen-dependent antimicrobial systems on Legionella pneumophila.

Legionella pneumophila was susceptible to the antimicrobial action of oxygen metabolites generated by both the myeloperoxidase-H(2)O(2)-halide and the xanthine oxidase systems.

The effects of Legionella pneumophila toxin on oxidative processes and bacterial killing of human polymorphonuclear leukocytes.

The effect of Legionella pneumophila toxin on selected functions of human polymorphonuclear leukocytes was investigated. Amounts of L. pneumophila toxin that had no effect on leukocyte viability or phagocytosis significantly decreased hexose monophosphate shunt activity and O2 consumption during phagocytosis and bacterial iodination and killing in a dose-dependent fashion. The mechanism of action of this toxin appears to be unique among bacterial products thus far studied.

Biphasic radiosensitization of human lymphocytes by diethyldithiocarbamate: possible involvement of superoxide dismutase.

A biphasic radiosensitization of human lymphocytes by diethyldithiocarbamate (DDC), a metal chelator, was observed. The first phase occurred at 10(-5) M and the second at 10(-3) MDDC. The biphasic radiosensitization coincided with the previously reported biphasic toxicity of DDC. Inhibition of superoxide dismutase (SOD) occurred only in the second phase, suggesting that it may be a contributing cause of this phase. The mechanism of the first radiosensitization phase is not known. The radiation survival curves indicated the presence of at least two lymphocyte populations differing in their radiosensitivity and representing 40 per cent and 60 per cent of the cells. Both cell populations were biphasically radiosensitized by DDC.

Hemolytic anemia due to pyruvate kinase deficiency: characterization of the enzymatic activity from eight patients.

We have studied the red cell pyruvate kinase (PK) variants from eight patients representing five families with pyruvate kinase deficiency-associated hemolytic anemia. The kinetic properties, electrophoretic mobilities, and immunological reactivity with anti-normal red cell pyruvate kinase were determined. The patients differ in the severity of their clinical condition and in the molecular properties of their red cell pyruvate kinase variants. The most seriously affected patient (PK Beaverton) has no electrophoretically demonstrable red cell isozymes. The activity present is due to the M2 isozyme, however red cell isozyme can be detected immunologically. PK Molalla and PK Lake Oswego are thermolabile variants with normal kinetic parameters. PK Molalla, in addition, has altered electrophoretic mobility. PK Multnomah and PK Milwaukie have decreased affinity for the substrate phosphoenolpyruvate, and PK Multnomah also has altered electrophoretic mobility. PK Coos Bay shows electrophoretic variation and a slightly decreased affinity for phosphoenolpyruvate consistent with an increased modulating effect of fructose-1,6-diphosphate.

Gene dosage: evidence for assignment of erythrocyte acid phosphatase locus to chromosome 2.

A child, trisomic for the distal short arm of chromosome 2 due to a familial 2/18 translocation, has elevated levels of activity of erythrocyte acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), 3.1.3.2] Ferguson-Smith et al. [(1973) Nature New Biol. 243, 271-274] previously had found decreased levels of activity and loss of expression of an erythrocyte acid phosphatase allele in a subject who lacked one of the two homologous regions containing the distal three bands of chromosome 2. They suggested that the locus for erythrocyte acid phosphatase is located on that segment. Our findings provide further evidence for this assignment and also suggest an in vivo gene dosage effect of this autosomal locus, which depends on both the type and number of alleles present.

Ascorbate and phagocyte function.

Scorbutic guinea pig neutrophils (PMN) were found to produce H2O2 and kill Staphylococcus aureus as well as control PMN, suggesting that ascorbate does not contribute significantly to phagocyte H2O2 production or bacterial killing. Total and reduced ascorbate contents of human PMN was observed to fall upon phagocytosis, whereas dehydroascorbate increased to a lesser extent. These observations are consistent with the view that ascorbate constitutes a functional part of the PMN's redox-active components and may thus function to protect cell constituents from denaturation by the oxidants produced during phagocytosis.

Uptake and reduction of oxidized and reduced ascorbate by human leukocytes.

Incubation of human leukocytes with dehydroascorbate (DHA) results in an increase in their reduced ascorbate (AA) content and hexose monophosphate shunt (HMS) activity, independent of oxygen supply. Incubation with AA induces these changes only in the presence of oxygen. The increase in HMS activity observed as cell AA increases by 1 micromol is the same during incubation with either DHA or AA. We propose that human leukocytes take up ascorbate as DHA (AA after oxidation to DHA) and reduce it promptly to AA, and that HMS stimulation upon incubation with either AA or DHA is a result of DHA reduction.