Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Structural Features of Pregna-D′-pentaranes Determining Their Interaction with Three Rat Proteins

Competition of a number of progesterone 16,17-cycloalkane derivatives with ³H-labeled ligands for the binding sites of rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16,17-cyclopropanoprogesterone, and 16,17-cyclopent-3-enoprogesterone and the selective ligands for serum pentaranophilin are 6-methyl-16,17-cyclohexanopregna-1,4-diene-3,20-dione and 3-hydroxy-16,17-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D we studied decreased the affinity of ligands for all the three proteins. The substitution of the ⁵-3-hydroxy grouping for the ⁴-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3,4-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

Mapping the α-globin genes in HB J Mexico carriers

Summary the organization of the -globin genes was studied by restriction endonuclease mapping, in subjects carrying the variant Hb J Mexico. A subject homozygous for Hb J synthesized both Hb J (about 55%) and Hb A and had two loci per chromosome. His restriction site map was found to be identical to that obtained with a normal DNA, except for a mutant Bgl II site which was observed on the Hb J chromosome proximal to the 5-locus. We have also mapped the DNA of a compound heterozygote for Hb J and -thalassemia, who synthesizes 38% Hb J and we have found a single gene corresponding to a –^3.7 haplotype on one chromosome and two genes, respectively ^J and ^A, on the other.     

Study of amino acid formation during palmitate oxidation in rat brain mitochondria

The interrelation of palmitate oxidation with amino acid formation in rat brain mitochondria has been investigated in purified mitochondria of nonsynaptic origin by measuring the formation of aspartate, -ketoglutarate, and glutamate during palmitate oxidation, and also by assaying¹⁴C-products of [1-¹⁴C]palmitate oxidation. Oxidation of palmitate (or [1-¹⁴C]palmitate) resulted in the formation of aspartate (or¹⁴C-aspartate), and the oxidation was inhibited by aminooxyacetate (an inhibitor of transaminase), Palmitate oxidation also resulted in -ketoglutarate formation, which was sensitive to the effect of aminooxyacetate. Addition of NH₄Cl was found to increase¹⁴C-products and formation of -ketoglutarate, whereas glutamate formation was not increased unless the rate of palmitate oxidation was reduced by 50% by aminooxyacetate or -ketoglutarate was added exogenously. Exogenous -ketoglutarate was found to decrease¹⁴C-products, but not aspartate formation. These results indicated that palmitate oxidation was closely related to aspartate formation via aspartate aminotransferase. During palmitate oxidation without aminooxyacetate or added -ketoglutarate, however, -ketoglutarate was not available for glutamate formation via glutamate dehydrogenase. We discuss the possibility that this was because (a) oxidative decarboxylation of -ketoglutarate to form succinyl-CoA was favored over glutamate formation for the competition for -ketoglutarate in the same pool, and (b) the pool of -ketoglutarate produced in the aspartate aminotransferase reaction did not serve as substrate for glutamate formation.     

Subunit structures of multiple hemoglobins in carp

Three hemoglobin components in carp designated CI, CII, and CIII, were isolated by DEAE-Toyopearl ion-exchange chromatography. Constituent globin chains, ¹, ², ¹ and ², were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography with a reversed-phase column. Tryptic peptide mapping indicated that the -globin chains of the three hemoglobin components have slightly different structures. In addition, N-terminal amino acid sequence analysis indicated that the ¹-globin chain has a primary structure different from that of the ²-chain. A series of hybridization experiments between isolated hemoglobins, together with such structural properties of globin chains, suggested that the three hemoglobins have the following compositions: CI (^{1 2} ₂ ¹ ), CII (^{1 2 1 2}), and CIII (^{1 2} ₂ ² ). Hemoglobin CII was a hybrid between the two types each of - and -chain and could be constructed in vitro from two hemoglobin components CI and CIII.Abbreviations a-a amino acid - Hb hemoglobin - HPLC high performance liquid chromatography - P ₅₀ oxygen pressure at half saturation - PAGE polyacrylamide gel electrophoresis - TFA trifluoroacetic acid     

A new mouse T-cell receptorα chain variable region family

Sequence analysis of the rearranged T-cell receptor a chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribedV gene family. We have designated this familyV 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously describedV haplotypes:V ^a ,V ^b , andV ^c .     

T-cell repertoire in a strain of transgenic C57BL/6 mice with the HLA-DRA gene on the X-chromosome

We have established a strain of transgenic mice in which the HLA-DRA gene was integrated into the X-chromosome and the xenogeneic mixed isotype molecule, DRE^b, was expressed in a cell type-specific manner, although the transgenic DRA gene contained only 268 base pairs of the 5-flanking region. The DRE^b molecules expressed in the transgenic mice functioned as major histocompatibility complex (MHC) class II to select T-cell repertoire, and to stimulate mixed lymphocyte reaction. In female transgenic mice homozygous for HLA-DRA (DR-B6-F-homo) and male transgenic mice (DR-B6-M), DRE^b molecules were expressed in almost all of the MHC class II A^b-positive cells. In contrast, the expression of DRE^b molecules in female transgenic mice hemizygous for HLA-DRA (DR-B6-F-hemi) was found only in part of the A^b positive cells, and the proportion of cells expressing the DRE^b molecules varied due to random inactivation of one of the X-chromosomes. Clonal deletions of the T cells and mature thymocytes bearing Tcrb-V5 and Tcrb-V11, which are eliminated from the peripheral repertoire in mice expressing self-superantigen and MHC class II E molecules, were incomplete in DR-B6-F-hemi as compared with those in DR-B6-F-homo, and were correlated with the proportion of DRE^b-positive spleen cells. These observations suggested that the number of bone marrow-derived cells expressing DRE^b molecules was critical for clonal deletions of Tcrb-V5⁺ and Tcrb-V11⁺ T cells in the thymus.     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

Synthesis of Nα,Nδ-dicarbobenzoxy-L-ornithyl-β-alanine benzyl ester,a derivative of salty peptide,in 1,1,1-trichloroethane with polyethylene glycol-modified papain

Summary N,N-Dicarbobenzoxy-L-ornithyl--alanine benzyl ester, a derivative of salty peptide, was synthesized from N,N-dicarbobenzoxy-L-ornithine ethyl ester and -alanine benzyl ester in 1,1,1-trichloroethane using papain modified with polyethylene glycol. The peptide bond formation proceeded in a transparent organic solvent at room temperature and the product was obtained as precipitates from the reaction system.     

The hemoglobins of Artemia salina. V. Genetic variation in hemoglobin 1, hemoglobin 2, and hemoglobin 3

Electrophoretic mobilities of three hemoglobins (Hb1, Hb2, and Hb3) were studied in 15 populations of brine shrimps. Genetic segregation data support the model that Hb2 contains n -polypeptides and n -polypeptides; Hb1 contains 2n -polypeptides. Hb3 contains neither - nor -polypeptides. There is no evidence of linkage of and loci with each other or with the locus (or loci) which governs Hb3 or with the nonhomologous portion of the sex chromosomes. Hemoglobins of different populations may be hybridized in vitro by incubation at high temperature. Reversible dissociation to subunits which contain only one ( or ) polypeptide occurs at 40 C (for Hb1) and at 50 C (for Hb2).Supported by Grant HD-11445 from the National Institutes of Health.     

Phenylalanyl-tRNA synthetase fromE. coli MRE-600: Localization of the phenylalanine binding sites on the subunits by affinity reagents

The phenylalanine and the phenylalanyl-tRNA^Phe binding sites on the subunits of phenylalanyl-tRNA synthetase fromE.coli MRE-600 were localized using p-azidoanilidate of [¹⁴C]phenylalanine and N-bromoacetyl[¹⁴C]phenylalanyl-tRNA^Phe. The phenylalanine recognizing site was shown to be situated on the subunit of the enzyme in close proximity to the contact region of the and subunits and the phenylalanyl-tRNA^Phe recognizing site on the subunit. Transfer of the aminoacyl moiety from the subunit to the subunit of the enzyme was assumed to take place in the process of catalysis of the aminoacylation reaction.     

Site-specific lipophilic modification of interferon-alpha

Interferon- (IFN),⁴ a cytokine with modulatory activities on many cell types, is useful for treating many types of cancer and infectious diseases. This study investigates whether modification of a protein, using IFN as an example, with a lipophilic group can alter its distribution and kinetic properties in the body. Ser163 of IFN2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFN2a(S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFN2a was purified by gel filtration chromatography. The biological activities of IFN2a(S163C) and lipophilized IFN2a(S163C) were similar to that of IFN2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in Madin-Darby bovine kidney cells. Lipophilized IFN2a(S163C) bound to human serum albumin and cell membranes more readily than did IFN2a. Future experiments will investigate whether lipophilized IFN2a(S163C) has improved pharmacokinetic properties.     

Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit.

Summary The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in thetrp A gene. Screening of the resulting clones allowed selection of non-interactive mutant subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: 126 valine (GTG)glutamic acid (GAG) and 128 valine (GTT)aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as a66 asparagine (AAC)aspartic acid (GAC); 109lysine (AAA) arginine (AGA); 118 cysteine (TGC)arginine (CGC). Where possible, we individually assessed the importance of these residues in interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.     

Alpha-thalassemia in Papua New Guinea

Summary A study of the distribution of -thalassemia in Papua New Guinea (PNG) was carried out by DNA analysis. A total of 664 DNA samples were screened for -thalassemia 2 and -thalassemia 1 caused respectively by either deletion of one or both of the duplicated -globin genes. -Thalassemia 2 was detected in high frequencies in coastal and lowland regions where malaria has been holo- to hyperendemic but in low frequencies in non-malarious highland regions. The highest frequency was observed in the north coast of PNG. The distribution of -thalassemia 2 seems to be in accordance with other conditions such as ovalocytosis and G6PD deficiency which are also prevalent in this population, suggesting that they may interact in protection against malaria. However, it appears to be negatively correlated with -thalassemia and -thalassemia 1, the latter being extremely rare in this population. Analysis of the types and subtypes of the single -globin gene deletion revealed a predominance of the –^4.2 type in general, except in some regions in the south where the –^3.7 type is prevalent. The –^3.7 I subtype is the common form of the –^3.7 deletion in the PNG mainland. The –^3.7 III subtype, previously reported to be unique in Melanesians and Polynesians, was detected in an offshore island of PNG. However, this subtype is very rare in Melanesians from the PNG mainland.     

The α1 and α2 domains of H-2 class I molecules interact to form unique epitopes

Mouse class I antigens are the major targets of cytolytic T lymphocytes in both major histocompatibility complex (MHC)-restricted and allogeneic responses. Considerable evidence has recently accumulated demonstrating that MHC class I molecules encoded by genes whose 1 and 2 coding exons were interchanged are not recognized by T lymphocytes specific for parental class I products. Along with the loss of T -cell reactivity, there is a loss of recognition by some, but not all monoclonal antibodies. In this communication we report that the loss of reactivity by monoclonal antibodies is accompanied by the gain of new epitopes caused by the interaction of 1 and 2 domains. These epitopes are immunodominant. They are the major determinant recognized by polyclonal antisera raised by immunization with L cells transfected with exon-shuffled class I genes. Four new monoclonal antibodies have been produced which recognize at least two separate epitopes caused by the interaction of the 1^P and 2^d domains.     

Synthesis of the acceptor analog αFuc(1→2)αGal-O(CH2)7 CH3: A probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase

We report the chemical synthesis of Fuc(12)Gal-O(CH₂)₇CH₃ (1) an analog of the natural blood group (O)H disaccharide Fuc(12)Gal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog Gal(3)[Fuc(12)]Gal-O(CH₂)₇CH₃ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide Fuc(12)Gal-O(CH₂)₇CH₃ (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.