Bruno Lucio Masala

Detection of globin chains by reversed-phase high-performance liquid chromatography.

Publisher Summary This chapter discusses the principal for the detection of globin chains by reversed-phase high-performance liquid chromatography (HPLC). Reversed-phase high-performance liquid chromatography (RP-HPLC) has greatly influenced and stimulated research in detection, quantitation, and identification of normal and abnormal globin chains in human newborn and adult red blood cells. In fact, RP-HPLC methodology has a number of advantages: it is fast and accurate, minute amounts of material are sufficient for each determination, complete automation is possible, and, most importantly, identification of electrophoretically silent mutant hemoglobins caused by neutral-to-neutral amino acid substitutions is feasible and reliable. It also greatly facilitates the separation and isolation of even microquantities of a mutant globin chain, and of globin proteolytic fragments, for subsequent characterization of the substitution within the abnormal peptide. This is possible by means of HPLC techniques for the determination of the amino acid composition at the picomole or subnanomole level, and microsequencing techniques, fast atom bombardment mass spectrometry (FAB mapping), electrospray mass spectrometry, and capillary electrophoresis. The chapter describes several different HPLC procedures. Some use cation-exchange systems whereas others use the reversed phase with different columns, such as Waters μBondapack C 18 , Merck LiChrospher 100 CH-8/2, Aquapore RP-300, and Partisil C 18 .

Detection of the common Hb F Sardinia [Aγ(E19)Ile → Thr]variant by isoelectric focusing in normal newborns and in adults affected by elevated fetal hemoglobin syndromes

A simple and rapid conventional isoelectric focusing technique for the detection of the silent Hb F Sardinia variant, containing the mutated A gamma T chain, is described. The method is based on thin-layer gels of shallow pH gradient (pH 6.7-7.7) and allows the direct detection of this rather common and widespread Hb variant at a screening level. 15-30 hemolysates from newborns and adults affected by elevated Hb F syndromes, both in the heterozygous and homozygous condition, could be examined simultaneously. The frequency of the A gamma T gene in Sardinian newborn (f = 0.175), in beta 0-thalassemia (f = 0.722), in beta (+)-thalassemia (f = 0.346), and in the non-deletional type of A gamma-HPFH (f = 0), as evaluated with this method, is in accordance with that previously reported by means of other methodologies.

The gamma globin chain heterogeneity of the Sardinian newborn baby.

The hemoglobin of 2048 newborn babies from Sardinia was analyzed by isoelectric focusing and polyacrylamide gel electrophoresis in order to determine the level of Hb F-Sardinia (with A gamma T) and the G gamma chain. Hb F-Sardinia values of 15.5 +/- 2.6% were present in the A gamma T heterozygote whereas 30.7 +/- 5.2% were present in the homozygote. The A gamma T gene frequency was 0.17. Most of the babies tested showed the normal G gamma level either in the absence of the A gamma T anomaly (69.6 +/- 4.1%), or in the presence of the anomaly in both the heterozygous state (70.9 +/- 4.8%) and the homozygous state (71.1 +/- 3.4%). Similar values were shown in nine homozygotes for beta-thalassemia discovered during the screening. Nine newborn babies (0.44%) showed particularly low G gamma levels (38.3 +/- 6.8%) whereas 18 newborn babies showed high G gamma levels (83.9 +/- 2.6%). The frequencies of the anomalies (0.0022 for the low G gamma and 0.0044 for the high G gamma) were the lowest observed in Caucasian and other ethnic groups. Data suggest the presence of abnormal gamma globin gene arrangements in the Sardinian population.

Disorders of the synthesis of human fetal hemoglobin

Fetal hemoglobin (HbF), the predominant hemoglobin in the fetus, is a mixture of two molecular species (α2Gγ2 and α2Aγ2) that differ only at position 136 reflecting the products of two nonallelic γ‐globin genes. At the time of birth, HbF accounts for ∼70% of the total Hb. The Gγ:Aγ globin ratio in the HbF of normal newborn is 70:30 whereas in the trace amounts of HbF that is found in the adult it reverses to 40:60 because of a γ‐ to β‐globin gene switch. Alterations of these ratios are indicative of a molecular defect at the level of the HbF synthesis. Qualitative hemoglobinopathies due to Gγ and Aγ chain structural variants, and quantitative hemoglobinopathies affecting the synthesis of HbF such as γ‐thalassemias, duplications, triplications, and even sextuplications of the γ‐globin genes, which may be detected in newborn blood lysates, have been described. Moreover, several pathological and nonpathological conditions affecting the β‐globin gene cluster, such as β‐thalassemia, sickle cell disease, δβ‐thalassemia, and hereditary persistence of HbF syndromes, are characterized by the continued synthesis of γ‐globin chains in the adult life. Studies of these natural mutants associated with increased synthesis of HbF in adult life have provided considerable insight into the understanding of the control of globin gene expression and Hb switching.

The origin and extinction of the large endemic Pleistocene mammals of Cyprus

Biogeographia - vol. XXI - 2000 (Pubblicato il 30 giugno 2000) Biogeografia deI|’Anatolia The origin and extinction of the large endemic Pleistocene mammals of Cyprus ELEFTHERIOS I-IAD]ISTERKOTIS*, BRUNO MASALA**, DAVID S. REESE *** *Gzzme and Fzzunzl Service, Ministry of Interior — Nicosia (Cyprus) “Department of Physiological Bioc/aemiczzl and Cell Sciences, Faculty of Biological Sciences, University of S[l55ll7’i - Sizssrzri (Italy) ***Depimment of/lntbropology, Field Museum 0fNzztzmzl Histmy — Chicago (USA) Key words: Fossils, hippopotamus, elephant, Cyprus, Pleistocene, extinction, Mediterranean SUMMARY All fossil terrestrial mammal sites on the island of Cyprus are dated from the Late Pleistocene period and consist almost exclusively of the remains of two terrestrial mammals, pygmy hippopotamus (P/J/mmzrios nzizzz/ms) and pygmy elephant (Elep/ms cypriater). Two theories exist on the arrival of these species on Cyprus. The first is that they arrived by a land bridge. This took place during the Pliocene about five to six million years ago when the Mediterranean sea was sealed at both ends clue to tectonic movements and its water evaporated creating a land bridge. However, there are no fossils dating from the Pliocene The second theory is that they arrived under circumstances described by the Island Sweepstakes model. The latter theory refers to cases in which animals may venture far from the coast, reach an isolated island from which they cannot return and are Forced to settle there. We reject the possibility that the Late Pleistocene mammals of Cyprus arrived by a land bridge, because during the Pleistocene such a bridge never existed. The cause of extinction of the earlier Cypriot endemic large mammals remain unclear. The discovery at the site ofAltrotiri /letokrenmos of the above pygmy mammal species together with man—made artefacts has been interpreted by some researchers as proof of a human role in the extinction of these mammals. Others rejected this view. We review the facts about this discovery and believe that humans did play a role in the final extinction of these species. THE ORIGIN OF THE LARGE PLEISTOCENE MAMMALS OF CYPRUS In Cyprus, Late Pleistocene fossil and subfossil sites consist almost exclusively of pygmy hippopotamus (P/annourios minzitus) (Forsyth Major, 1902; Bate 1906; Boekschoten and Sondaar, 1972; Houtekamer and Sondaar, 1979; Faure et al., 1983, Hadjisterkotis and Masala, 1995; Reese, 1975, 1989, 1992, 1995, 1996; Simmons, 1988a, b, 1989, 1991a,b,c, 1992, 1996; Swiny, 1988, 1995; Simmons and Reese, 1993; Simmons and Wigand, 1994) and pygmy elephant (Elepnczs Q)/priotes) (Bate, 19033, 1904a,b,c; Hadjisterkotis and Masala, 1995; Reese, 1995, 1996b; Simmons, 1988, 1989, 1991a,b,c, 1992, 1996). Both species were considerably smaller than their mainland counterparts, and most researchers believe that the dvvarfism was an evolutionary response both to the lack of predators and to the limited resources available (Sondaar, 1977; Azzaroli, 1982).

DNA polymorphisms in North Sardinian newborns and their linkage with abnormal γ globin gene arrangements and with βo-thalassemia

Fetal hemoglobin analysis and globin gene mapping have identified one type of βo-thalassemia and four different γ globin gene arrangements among newborn babies from the northern part of Sardinia. The βo-thalassemia with a nonsense mutation at codon 39 was found on two chromosomes, each with a distinct pattern of polymorphic restriction sites; one had the AγT (Aγ75 Ile → Thr) mutation, while the second did not. Four closely related haplotypes were identified for chromosomes with the AγT mutation. The γ-thalassemia heterozygosity with the —GAγ— hybrid gene fell into two categories. One apparently originated through crossing-over between mismatched chromosomes characterized by the most common haplotype, while the other had polymorphisms resembling those of a less frequently occurring chromosome. Chromosomes with the —Gγ—AGγ—Aγ— triplication had polymorphic sites to be expected for this condition, being complimentary to the —GAγ— thalassemias. Of the two additional γ globin gene variations the —Gγ—Gγ— arrangement was associated with the chromosome with the most commonly occurring haplotype, while the chromosome with the —Aγ—Aγ— arrangement had a haplotype characteristic for that with the AγT mutation, which identified an —Aγ—AγT— arrangement. The incidental discovery of a silent β-chain mutant, Hb Hamilton, with the Val → Ile substitution at position β11, in five newborns was also reported.

Kinetics of the ontogenic and reversible hemoglobin switching in the mouflon (Ovis musimon) and sheep × mouflon hybrid

1. Hemoglobin (Hb) switching in the perinatal life of wild mouflon (Ovis musimon) was characterized by the replacement of Hb F by 60% levels of Hb C, and subsequently of Hb C by Hb B. 2. The recently discovered Hb M variant was not replaced by Hb C; thus, Hb BM heterozygote newborns synthesized 30% Hb C at the expense of Hb B. 3. Hybrid B mouflon x B sheep synthesized only 5% Hb C at birth but were able to produce 30% Hb C in adult life following induced anemia. 4. Adult BB and BM mouflons, after the same extent of induced anemia, synthesized HB C levels similar to those produced at birth. The results indicate a mouflon beta-globin gene cluster arrangement similar to those of sheep and goat, the beta C gene having an intermediate expression. Results also suggest a selective disadvantage in hybrid animals.

Hb G-Philadelphia, or [α68(E17)Asn → Lys], in North Sardinia: detection by isoelectric focusing and identification by HPLC of tryptic peptides

11,129 individuals of the northern area of the island of Sardinia (7,717 newborns and 3,412 adults) were examined for the Hb G-Philadelphia variant [alpha 68(E17)Asn----Lys]. Hemolysates were analysed by isoelectric focusing, and the variant identified by reversed phase high performance liquid chromatography of tryptic peptides. A total of seven heterozygotes (1 in 1,589) were identified. This is probably the highest prevalence of this mutant so far described. Percentages of the variant (average 35.1 +/- 6.2%) were trimodally distributed with modes centering on 28, 36, and 42%, respectively. These values suggest that the G-Philadelphia allele occurs in Sardinians both on a single and on a double locus chromosome. The linkage with alpha-thalassemia may be the reason for the high frequency of the variant.

Evidence for the presence of two different β-globin chains in the hemoglobin of the river buffalo (Bubalus bubalis L.)

1. Hemoglobin (Hb) of the river buffalo (Bubalus bubalis L.) was studied by employing isoelectric focusing (IEF) in the 6.7-7.7 pH range and by IEF in ultra-narrow immobilized pH gradient (IPG) 7.1-7.5. 2. Three Hb BB phenotypes were identified which were characterized by sets of two or four Hbs with different isoelectric points. 3. These type were called BB, BsBs and BBs, the Bs phenotype showing Hbs with slightly slower mobility. 4. Analysis of constituent globin chains by acid urea polyacrylamide gel electrophoresis in the presence of Triton X-100, provided clear evidence of a novel polymorphism at the beta-globin level. 5. Titration curves of beta-globins from heterozygous Hb BBs indicated a single curve thus suggesting the absence of a net charge in all the pH field. 6. A neutral-to-neutral amino acid replacement probably differentiates the two beta-globins.

Complete nucleotide mtDNA sequence of Barbary sheep (Ammotragus lervia).

In this report we describe the complete sequence of the mtDNA genome of Ammotragus lervia (Barbary sheep or aoudad) as obtained by PCR and sequencing with primer walking using flanking sequences. The molecule is 16,530 base pairs in length, resulting similar to those of goat and sheep. The genome organization matches to those of other mammalian mitochondrial genomes. The phylogenetic position of the Ammotragus relative to twelve other mammalian species was assessed based on protein-coding sequences. A goat-aoudad split of 9.9–8.9 MYBP has been estimated, whereas the divergence ancestor/caprines was dated at 12.1–10.8 MYBP.