Clayton Natta

Low serum levels of carotenoids in sickle cell anemia

Serum carotenoids, tocopherols and retinol were analyzed in patients with sickle cell anemia (SCA) and in control subjects. The data show the following: the serum levels of the major carotenoids, alpha and beta carotene, cryptoxanthin, lycopene lutein, alpha tocopherol and retinol were significantly lower in SCA patients. These findings reflect an additional abnormality of the antioxidant system in SCA patients. Gamma tocopherol, on the other hand, was significantly elevated, consistent with the previously reported reciprocal relationship between serum alpha and gamma tocopherols. Thus, these data taken together with the earlier findings on the lower levels of plasma alpha tocopherol and ascorbic acid suggest that the entire antioxidant system may be compromised in SCA patients. This may contribute in part to the phenotypic expression of the condition.

Preferential Binding of βS Globin Chains Associated with Stroma in Sickle Cell Disorders

Sickle cell anemia (SS) is associated with abnormalities of the red cell membrane and decreased red cell deformability. The present study assesses globin chain binding to stroma in SS, sickle cell trait (AS), and nonsickling (AA) cells. The results indicate that there is preferential binding of newly synthesized beta(S) globin to red cell stroma in SS cells and preferential binding of beta(S) to stroma compared to beta(A) in AS cells. These studies show that beta(S) globin binding to stroma accompanies the membrane abnormalities in SS and AS patients.

Balanced globin chain synthesis in hereditary persistence of fetal hemoglobin.

In two black families with the hereditary persistence of fetal hemoglobin (HPFH) gene there are eight A-F heterozygotes and two double heterozygotes for sickle cell trait and HPFH. These patients are clinically asymptomatic and have homogeneous acid elution smears. Measurement of globin chain synthesis in peripheral blood demonstrates balanced production of a alpha and non-alpha (beta plus gamma) chains. In these patients, the balance is achieved by increased gamma globin production and increased activity of the remaining beta globin allele. In two patients, one A-F and the other S-F there is also balanced globin synthesis in the bone marrow. In a double heterozygote for HPFH and beta-thalassemia, anemia (Hb: 11.5 g/100 ml) is associated with a moderate degree of globin chain imbalance. There is a correlation between balanced globin chain synthesis and the absence of anemia in patients with HPFH.

Polyamines in sickle cell disease

Abstract Sickle cell anemia, a chronic hemolytic disease characterized by severe pain and premature death, affects to varying degrees 1% of the worlds black population. Although a number of biochemical alterations have previously been associated with this anemia (1), the recent report of a five- to tenfold elevation in blood polyamine levels (2) is especially noteworthy because the polyamines can alter the normal electrokinetic properties of the red blood cell membrane (3). These observations may help explain the abnormalities of the erthrocyte membrane and decreased red cell deformability associated with the sickling phenomenon. The principal polyamines present in eukaryotes are, repectively, the di-, tri-, and tetramines-putrescine, spermidine, and spermine. Their biological function, which is related to their unique charge distribution, is linked to cell growth, division, and differentiation (4). The polyamines also exert direct on membrane stability and permeability (5), as well as modifying the activity of membrane-bound enzymes including acetylcholinesterase (6) and Na, K-ATPase (7). In the present study it is shown that the erythrocyte stroma fraction, obtained from sickle cells, has associated with it abnormally large amounts of spermine. Stroma binding appears to be selective and not directly dependent on either plasma or erythrocyte lysate polyamine concentration.

Quantitation of human gamma globin genes and gamma globin mRNA with purified gamma globin complementary DNA.

Complementary DNA (cDNA) specific for gamma-globin nucleotide sequences has been prepared by hybridizing total cDNA made from cord blood messenger RNA (mRNA) as template to an excess of normal adult human globin mRNA and recovering the single-stranded cDNA from hydroxylapatite. The specificity of the gamma cDNA for gamma mRNA sequences is strongly supported by the hybridization of this cDNA at low Cot values (Co, concentration of RNA and t, time in seconds) to RNA samples containing large amounts of functional gamma globin mRNA and the lack of hybridization to RNA samples containing little, if any, gamma-globin mRNA. The absence of cross-hybridization of gamma cDNA with alpha, beta, and delta mRNAs is demonstrated by the complete hybridization of the gamma cDNA to mRNA samples completely lacking either alpha or beta and delta mRNA. An estimate of the number of gamma-globin genes in human cellular DNA was obtained by hybridization of purified gamma cDNA to DNA from spleen and white blood cells of normal and beta-thalassemia subjects and measurement of the percent of gamma cDNA hybridized at saturation. The results indicate that there are between one and two gamma-globin genes per total haploid gene DNA equivalent obtained from both normal and beta-thalassemia subjects. These values are consistent with genetic evidence for the presence of multiple gamma gene loci in human cells. The finding that the number of gamma-globin genes in beta-thalassemia DNA is similar to that in nonthalassemia DNA indicates that a deletion of gamma-globin genes cannot account for either the inadequate gamma-globin synthesis or indirectly for the decreased or absent beta-globin synthesis in beta-thalassemia cells.

Cold-induced accumulation of ribosomal subunits from human reticulocytes

Abstract These studies indicate that ribosomal subunits can be prepared in high yield from human reticulocytes previously incubated at 4°. These ribosomal sub-units are active in stimulating amino acid incorporation upon addition to cell-free systems.

Quantitation of human globin chain synthesis by cellulose acetate electrophoresis.

We have developed a method for the separation and quantitation of human alpha-, beta-, and gamma-globins utilizing cellulose acetate electrophoresis. The relative rates of synthesis of globin chains in reticulocytes in peripheral blood is determined by: (i) incubating intact cells with [35S]methionine; (ii) preparing globin from the hemolysates; (iii) performing electrophoresis of the globin on cellulose acetate strips; and (iv) autoradiography or direct determination of the radioactivity incorporated into each globin chain. The method is simple and rapid, requires only small amounts of hemolysate (30 micrograms of globin), and provides excellent resolution and reproducible quantitation of alpha-, beta A-, beta S-, and gamma-globin chains for up to 24 peripheral blood samples at one time. Measurements by this method in patients with thalassemia variants and sickle-cell disorders correlate well with analysis of the same samples by carboxymethyl cellulose chromatography. This methodology may permit more widespread analysis of globin synthesis in the thalassemia syndromes and may also be useful in the analysis of globins synthesized from human globin mRNA in cell-free systems.

A Family Study of IgG Subclasses in Sickle Cell Anemia

Three siblings with sickle cell anemia were studied immunologically and hematologically. Their patterns of Protein A‐Sepharose chromatography distribution showed considerable heterogeneity, particularly with respect to the IgG2 and IgG3 subclasses, even though their hematological make up was similar. An attempt was made to correlate their IgG2: IgG1 subclass ratios with their clinical history of recurrent bacterial infections, as well as a possible compensatory IgG3 heterogeneity.

Erythrocyte polyamines of normal individuals and patients with Sickle Cell Anemia (SCA)

Aging of human red cells (RBC) appears to occur as a function of the age of the subject and time which is spent by the erythrocyte in the circulation. In SCA, there is a decrease in the time RBCs circulate. The polyamines, putrescine, spermidine and spermine can alter the electrokinetic properties of red cells. As red cells age, there is a decrease in the surface charge and altered deformability. To assess the effect of time in the circulation on RBC polyamine content of control subjects, we analyzed levels of density fractionated cells of ten healthy donors (HbAA) and compared to levels of four SCA patients (HbSS), ages 26 to 38. Perchloric acid extracts of erythrocyte stroma were analyzed by amino acid analysis using O-phthaldehyde fluorescent detection. At age 50–60 in normal subjects there is an accelerated loss of both spermidine and spermine in the dense cells relative to reticulocytes. This may be related to loss of surface area of the erythrocyte. In the four SCA patients, the spermidine and spermine ratios were similar to that of young normals of the same age group. It is considered that a large fraction of the total polyamines is firmly bound to spectrin. Polyamines may exert a regulatory role in the RBC stroma.

EFFECT OF DBA ON HEMOGLOBIN SS CELLS AND HEMOGLOBIN BIOSYNTHESIS

The present studies have shown that 3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-6-butyric acid (DBA), which does not bind covalently with hemoglobin, inhibits the loss of potassium ions from hemoglobin SS cells, and inhibits the binding of newly synthesized β S and α chains to membranes from these cells. This is the first report of an antisickling agent which does not modify hemoglobin covalently but may affect membrane-cytoskeletal interactions.