Background: Zinc and copper are physiologically antagonists and abnormal levels of both of them are known associated with neurodevelopmental disorders. Data in this area are lacking in Congolese children.

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li(+) and (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH(4)Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li(+) substitution or bath addition of 5 mM NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.

Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.

HEREDITARY spherocytosis is a heterogeneous disorder characterized by hemolytic anemia, spheroidal red cells, and increased osmotic fragility of erythrocytes. The majority of the patients have an a...

We have previously shown that irreversibly sickled cells (ISCs) form ISC-shaped ghosts and ISC-shaped membrane skeletons which suggests that an alteration in the membrane skeleton of the ISC may be responsible for its abnormal shape. In the present studies we observe that ISC-shaped ghosts become round in hypertonic media (>400 mM NaCl), hypotonic media (10 to 50 mM NaCl), and isotonic NaCl containing small amounts of Zn 2+ (0.1 to 0.5 mM). This ISC-shape reversal is time and temperature dependent and does not correlate with the elution or proteolysis of any membrane protein(s) detectable on SDS-gels. Conditions which promote ISC ghosts reversal also inhibit the formation of spectrin-actin complex(es). This suggests that ISCs may be stabilized by abnormal interactions between the spectrin and/or actin components of the membrane skeleton and that disruption of these bonds may allow the skeleton to resume a normal shape.

To determine whether splenic “polishing” and “pitting” of RBC includes removal of agglomerated membrane proteins as well as membrane lipids and intracellular debris we examined the RBC membrane proteins of normal and splenectomized patients. RBC ghosts were dissolved in sodium dodecyl sulfate (SDS) and chromatographed on Sepharose 2B (exclusion limit=40×106). Membranes from splenectomized patients contained an excluded macromolecular species not present in “normosplenic” individuals. This colorless material formed insoluble fibrils on removal of SDS and was proteinaceous as judged from amino acid analysis and ultraviolet spectroscopy. It comprised 4.2±1.3% (range 2.4-6.1%) of the total membrane protein. Equal proportions were present in membranes from older (more dense) and younger (less dense) cells. When freeze-thawed ghosts were banded on a sucrose density gradient, this material remained with the membrane fraction, indicating it was not a particulars cytoplasmic contaminant. Reduction partially disaggregated the complex and revealed spectrin (a high molecular weight RBC membrane protein thought to be involved in red cell shape maintenance), and at least one other unidentified protein component on SDS-gel electrophoresis. These studies suggest the spleen normally “polishes” or “pits” from RBC membranes an aggregated complex of RBC membrane proteins. Thus RBC membrane proteins join lipids and intracellular particles as targets for splenic cleansing.

ApoLP-Gln-II is one of the major protein constituents of human plasma high density lipoproteins (HDL). This protein and its two CNBr fragments, C-III (carboxyl-terminal) and C-IV (amino-terminal) were tested for ability to bind phosphatidyl choline by the formation of lipid-protein complexes of density 1.063 to 1.210 gm/ml, by changes induced in circular dichroism, and by the inhibition of the reactivation of delipidated mitochondrial β-hydroxybutyric dehydrogenase. In all three of these experimental procedures, C-III but not C-IV retained the ability to bind phosphatidyl choline. These findings suggest that the phospholipid binding site(s) of apoLP-Gln-II may be localized in the carboxyl-terminal portion of the molecule.

The ultraviolet fluorescence emission spectrum of bovine serum albumin was altered when free fatty acid (FFA) was added to the protein. This occurred when FFA was taken up from aqueous soap solutions, n-heptane solutions, rat epididymal fat pads, or fat emulsions undergoing enzymatic hydrolysis. As the molar ratio of FFA to albumin increased, the maximum emission was shifted to a shorter wave length and the fluorescence intensity was decreased. The blue shift did not exceed 7 mμ, and the maximum reduction in fluorescence intensity was less than 50% even when the molar ratio of FFA to albumin was high. Qualitatively similar results were obtained with FFA of 6–22 C atoms, but smaller effects occurred when FFA of less than 10 C atoms were used. FFA produced little or no change in the fluorescence intensity when the pH was less than 5.5 or greater than 10. FFA produced changes in the fluorescence spectrum of porcine albumin but had little effect on that of human, rabbit, equine or canine albumins.