Eugene F. Roth

Resistance to malaria in ankyrin and spectrin deficient mice

Summary Inbred mice carrying mutations in ankyrin and/or spectrin synthesis and assembly were studied for their ability to support the growth of the rodent malarias, Plasmodium chabaudi adami and P. berghei, in vivo. Mice carrying the nb/nb (normoblastosis) mutation which do not synthesize ankyrin and therefore also have a deficiency in membrane‐bound spectrin, were refractory to P. chabaudi adami, which invades mature erythrocytes and to P. berghei, which invades reticulocytes. Similarly, sph/sph mice which do not synthesize the α chain of spectrin but do synthesize ankyrin, were also resistant to both parasites. The heterozygote for the nb defect (nb/+) exhibited a diminution of parasitaemia. We conclude that the host cell spectrin may be necessary for the invasion and/or growth of rodent malarial parasites.

The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.

Plasmodium falciparum: Physiological interactions with the human sickle cell

Abstract The malaria parasite, Plasmodium falciparum, enhances the rate and extent of sickling of infected hemoglobin S heterozygous human erythrocytes. Upon sickling of the host cell, the parasite is killed. Parasite-free lysates of highly infected cells were analyzed to determine the mechanism by which sickling is enhanced. The intraerythrocytic pH of the infected cell was estimated to be 0.4 units below that of the uninfected cell, a difference which could result in a 20-fold increase in the extent of sickling under physiological conditions. Sickle-cell hemoglobin (HbS) heterozygous (AS) erythrocytes had decreased intracellular potassium after 24 hr of culture under conditions which cause sickling and parasite death. When infected AS cells were cultured in high-potassium medium under these conditions the parasites were protected. The medium did not prevent sickling but did maintain normal intracellular potassium levels. It is suggested that sequestration of trophozoite-infected AS cells in the venules leads to the sickling of the host cell, loss of erythrocytic potassium, and parasite death. The resulting attenuation of parasite multiplication would favor the survival of the HbS heterozygote and maintain the HbS gene at high frequencies in areas endemic for falciparum malaria.

Surface activity of hemoglobin S and other human hemoglobin variants

The kinetics of surface pressure change (deltapi vs. t isotherms) were determined for several single point mutations of the human hemoglobin system. It was observed that hemoglobin S and hemoglobin CHarlem (both containing beta6 Glu leads to Val substitutions) have a specific behavior at the water-air interface: their extent of surface pressure change is larger than for hemoglobin A, hemoglobin C and hemoglobin Korle Bu (beta73 Asp leads to Asn). In addition, hemoglobin S seems to occupy a larger area per molecule than hemoglobin A. The conformational requirements for this property, in addition to the beta6 Val substitution, appear to be the liganded state of the betas chain in the tetramer. Electrostatic, hydrogen bonding and hydrophobic interactions are involved in determining the surface activity of a hemoglobin molecule. The differences between the surface activity of oxyhemoglobin S and oxyhemoglobin A could be the basis for their differences in mechanical precipitability, although other factors may play a role.

The adaptation of Plasmodium falciparum to oxidative stress in G6PD deficient human erythrocytes

It has recently been found that growth of P. falciparum in human G6PD deficient red cells is impaired in vitro; however, the inhibition is overcome after two or three growth cycles. There is evidence to suggest that the parasite can produce its own G6PD enzyme which may compensate for the lack of host enzyme and could account for the resumption of normal growth in G6PD deficient host cells. It is unclear whether the parasite enzyme can enable the host cell to resist oxidative stress as normal cells do. To answer this question, P. falciparum was grown in vitro in: (a) normal red cells, (b) G6PD deficient red cells for one growth cycle only, (c) G6PD deficient cells for a minimum of five cycles. All groups were then challenged with acetylphenylhydrazine (APH) which served as an oxidative stress. Both G6PD (A ‐) and Mediterranean deficient types were studied. The results show a two‐fold increase in resistance to oxidative stress by parasites adapted to G6PD‐Mediterranean deficient host cells as compared to unadapted ones, but the parasite‐red cell system remains 4 times more sensitive to APH than normal infected cells. In parasitized G6PD (A ‐) red cells, evidence of adaptation could be seen in the growth curves, but no detectable increase in resistance to APH was found in adapted parasites. It is concluded that the role of the parasite G6PD is not likely to be mainly related to oxidative stress resistance and therefore other functions of this enzyme should be investigated.

Benign sickle cell anemia in Israeli-Arabs with high red cell 2,3 diphosphoglycerate.

Arabs living near the Sea of Galilee were found to be homozygous for hemoglobin S. Studies of solubility, mechanical precipitability, electrophoretic mobility on starch-gel and citrate agar media, minimum gelling concentration, and peptide mapping of the hemoglobin beta-chain confirmed complete identity of the hemoglobin with that found in Afro-American hemoglobin S homozygotes. A comparison of Arab Hb S homozygotes with Afro-American Hb S patients showed no significant differences in hemoglobin levels, red cell indices or morphology. Hb F averaged 4.4% in Arab patients. The 2,3 diphosphoglycerate levels were increased approximately twofold in Arabs, whereas in Afro-Americans, it was increased by only 7% in females and 20% in males.

Acridine Orange Potentiation of Actinomycin D Uptake and Activity

Acridine orange enhances the uptake of [3H]actinomycin D in disrupted and intact human lymphocytes, as measured by liquid scintillation and autoradiography. Proflavine, quinacrine, chloroquine, and ethidium bromide are not effective. In mice, acridine orange increases the capacity of actinomycin to reduce spleen weight. Type II acridine binding to DNA may be a prerequisite for actinomycin enhancement.

Potassium cyanate, an in vitro inhibitor of lymphocyte blast transformation without in vivo activity.

Summary Potassium cyanate (400 μg/ml) inhibited the blastogenesis of lymphocytes in response to PHA and the response of lymphocytes in mixed lymphocyte cultures. Cyanate administered to mice at a dose of 30 mg per kg per day did not delay allograft rejection. The authors wish to thank Dr. Sandra Nehlsen for help with the skin grafting.