Patients with sickle cell anemia vary in the hematologic and clinical features of their disease, in part because of variability in the presence of linked and unlinked genes that modify the expression of the disease. The hemoglobin S gene is strongly linked to three different haplotypes of polymorphic endonuclease-restriction sites of the beta-like gene cluster (genes in the vicinity of the beta-globin gene)--one prevalent in Atlantic West Africa, another in central West Africa, and yet another in Bantu-speaking Africa (equatorial, East, and southern Africa). We have studied the differences in the hematologic characteristics of patients with sickle cell anemia from the first two geographical areas. We find that the Senegalese (Atlantic West Africa) patients have higher levels of hemoglobin F, a preponderance of G gamma chains in hemoglobin F, a lower proportion of very dense red cells, and a lower percentage of irreversibly sickled cells than those from Benin (central West Africa). We interpret these data to mean that the gamma-chain composition and the hemoglobin F level are haplotype linked and that the decrease in the percentage of dense cells and irreversibly sickled cells is secondary to the elevation in the hemoglobin F level. Patients with sickle cell anemia in the New World probably correspond to various combinations of these types, in addition to the still hematologically undefined haplotype associated with sickle cell anemia in the Bantu-speaking areas of Africa.

It is generally accepted that human 13-melanocyte-stimulating hormone (h.BMSH) does not normally exist in humans but was merely an artifactually generated 22-amino acid peptide corresponding to a lipotropin (LPH) fragment (residues 35-56). We examined whether the shorter 18-amino acid peptide hBMSH-(5-22) could be detected in some human tissues. MATERIALS AND METHODSTissue Collection. The following tumors were surgically obtained from five patients with ectopic ACTH syndrome: tumors E1-E4 were bronchial carcinoid tumors; tumor E5 was a thymic carcinoid tumor. Fragments of pituitary corticotropic adenomas from two patients with Nelson syndrome (N1, N2) were surgically obtained by the transsphenoidal approach. In all cases, macroscopically homogeneous tumor fragments with no evidence of local necrosis were quickly selected by the surgeon, immediately frozen in liquid nitrogen, and subsequently stored at -85°C. Two normal human pituitaries (P1, P2) and hypothalami (HT1, HT2) were obtained at autopsy 2-and 6-hr post-mortem from patients with no evidence of endocrine disease and no history of prior hormonal treatment. They were frozen and stored as indicated. Patients with ACTH-producing tumors had not been subjected to either radiotherapy or chemotherapy.Tissue Extraction. Tissue fragments were homogenized in ice-cold 5 M acetic acid (10 ml/g of tissue) containing 1 mM phenylmethylsulfonyl fluoride, by three to five 15-sec bursts in a PCU2 Polytron (Polytron, Elkhart, IN). The homogenates were centrifuged at 5000 x g for 30 min at 4°C, and the supernatants were lyophilized in multiple aliquots that were subsequently used for RIA and/or chromatographic studies.

Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb S, 06 Glu + Val) have been obtained from X-ray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Site-directed mutagenesis with expression of the Hb S 0 subunits in Escherichia coli provides the experimental tools to test these models. For H b S, the 06Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88. Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophobicity of the residue at the 06 position with Ile > Val > Ala. We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the 06 location. Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation. The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact. Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand. This is demonstrated by associating the 023 Val -+ Ile mutation at the axial contact with either the 06 Glu -+ Val or 06 Glu + Ile substitution in the same 0 subunit. In comparison to native H b S the solubility of these deoxyHb double mutants is decreased two-and fourfold, respectively.

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