The mutation G298A®Ala100Thr on th coding sequence of the Duffy antigen/chemokine receptor gene in non-caucasian Brazilians

Ala100Thr has been suggested to be a Caucasian genetic marker on the FY*B allele. As the Brazilian population has arisen from miscegenation among Portuguese, Africans, and Indians, this mutation could possibly be found in Euro- and Afro-Brazilians, or in Brazilian Indians. Fifty-three related individuals and a random sample of 100 subjects from the Brazilian population were investigated using the polymerase chain reaction and four restriction fragment length polymorphisms. Confirming the working hypothesis, among the related individuals three Afro-Brazilians (two of them a mother and daughter) and a woman of Amerindian descent had the Ala100Thr mutation on the FY*B allele. Five non-related Euro-Brazilians also carried the mutation. All nine individuals presented the Fy(a-b+) phenotype. We conclude that the Ala100Thr mutation can occur in populations other than Caucasians and that this mutation does not affect Duffy expression on red blood cells. Gene frequencies for this allele in the non-related individuals were in agreement with those of other populations. The Duffy frequencies of two Amerindian tribes were also investigated.

Electrophoretic variation of hair proteins

Hair follicle cells secrete a complex assortment of proteins that form the hair shaft, and can be classified into two major groups. The lowsulfur proteins are keratins that contribute to the backbone of intermediate filaments, and the high-sulfur proteins are associated with these filaments. In the present investigation we describe a comparative electrophoretic study of normal human hair proteins from 182 individuals, including some families. Hair proteins were extracted in urea buffer (pH 9.3), examined by 1O per cent polyacrylamide gel electrophoresis (pH 8.8) in the presence of sodium dodecyl sulfate and stained with Coomassie brilliant blue. Eighteen bands appeared and were reproducible in most individuals, with apparent molecular mass ranging from 10.0 to approximately 100 kDa. Based on the most prominent bands, an electrophoretic profile defined as the "frequent profile" was observed. This profile was observed in 180 individuais and consisted of 6 prominent bands, 4 of them of apparent molecular mass in the 407O-kDa range, which is characteristic of keratins (61.9 ñ 1.02, 58.5 ñ 1.21, 47.9 ñ 1.58, and 45.4 ñ 1.53 kDa), and 2 bands with lower molecular mass (18.9 ñ 0.75 and 13.7 ñ 0.91 kDa). In 2 samples from unrelated women, an additional band of 42.1 ñ 1.72 kDa appeared. The meaning of this variant is still under investigation.

An improved method for the isolation or erythrocyte antigen band-3

An improved method for isolation of human and Rhesus monkey band-3 separated by sodium dodecyl sulfate poliacrylamide gel electrophoresis (SDS-PAGE) is described. Purified band-3 was obtained from human hemoglobin-free ghosts (Hb-free ghosts) after SDS-PAGE by chemical elution + sonication (CE + S). The section of the gel corresponding to the antigen was cut out, mechanically disrupted and incubated in 1 per cent NaHCO3, containing 1 per cent SDS, for 2h, with shaking, at room temperature, followed by overnight incubation at 4ºC. The preparation was subsequently sonicated and clarified by centrifugation. Supernatants were dialyzed against distilled water, their protein contents were measured, and the presence of purified band-3 was demonstrated by SDS-PAGE. A calibration curve was developed for assay of CE+S material using densitometric evaluation of the protein profile on SDS-PAGE. An amount of 37.5 mg of Hb-free ghost grave 3.15 mg of purified band-3 after CE+S, corresponding to an 8.4 per cent yield. Rabbits were immunized with 50µg CE+S antigen. Serawere collected and assayed by Western blot analysis against its proteolytic fragments, which were obtained from packed red blood cells by treatment with protease type VI from Streptomyces griseus (1h at 37ºC), followed by extensive washing and hypotonic lysis. Specific antibodies recognized band-3 and its proteolytic fragments 60 and 63 kDa in human ghosts obtained from different blood donors, confirming the genetic polymorphism. Analogous serum obtained against the Rhesus monkey band-3 proteolytic fragment 63 kDa recognized the human antigen and its respective fragments. These results indicate the existence of similarities between these two species of band-3, suggesting the potential use of this technique in taxonomic and phylogenetic studies. Purification by CE+S is an efficient and rapid method for isolation of band-3 and its fragments with satisfactory yield and maintenance of both their immunogenic and antigenic properties