Carbonic anhydrase-I polymorphism in a Philippine aboriginal population.

Polymorphism of carbonic anhydrase-I (CA1) was found by electrophoresis in an aboriginal group of Mindanao, Philippines, with a remarkably high frequency of variant types. The frequency of the variant allele was estimated at .256. The variant isozyme designated CA1 3Negrito (CA1 3N) is electrophoretically indistinguishable from the "Guam" variant and may be regarded as a potential anthropological marker in the Western Pacific.     

Amino acid substitution and chemical characterization of a Japanese variant of carbonic anhydrase I: CA I Hiroshima-1 (86 Asp → Gly)

An electrophoretic variant of red cell carbonic anhydrase I, designated CA I Hiroshima-1, has been observed in 12 apparently unrelated individuals during a survey of 13,019 individuals from the cities of Hiroshima and Nagasaki, Japan. Analyses of tryptic and chymotryptic peptide patterns of this CA I variant purified from 8 of the 12 individuals revealed the same altered peptides in each case. Examination of the amino acid sequence of an altered tryptic peptide purified from one of the variants showed that the aspartic acid residue at position 86 was replaced by a glycine residue. Thermostability studies demonstrated that all samples of CA I Hiroshima-1 were less stable than normal CA I. The specific esterase (p-nitrophenyl acetate) activities of the normal and variant CA I isozymes were essentially the same. The difference spectra of the normal and variant enzymes were essentially the same. The isoelectric focusing patterns of CA I Hiroshima-1 showed a different pattern of minor bands to those produced by normal CA I. The relative amounts of the normal and variant enzymes purified from single heterozygous individuals were similar.This work was supported by U.S. Public Health Service grant GM-24681 and U.S. Department of Energy contract 2828 (to Dr. J. V. Neel).     

Comparative in vivo and in vitro study of the cytogenetic effects of 153Sm-EDTMP in lymphocytes of patients with bone metastases.

153Sm-EDTMP is a radiopharmaceutical used in nuclear medicine for relief of metastatic bone pain with promising results, but there are few studies about the effects of 153Sm-EDTMP in human cells. This study was conducted for the evaluation of the cytogenetic effects of 153Sm-EDTMP in blood lymphocytes from patients with bone metastases (without previous radio or chemotherapy), using the chromosome aberration technique. The degree of cytological damage found in in vivo blood cells of patients was compared with those found in in vitro in an adjusted dose-response curve. Blood samples were collected before and 1 hr after the administration of 153Sm-EDTMP(about 42.31 MBq/kg). The frequency of structural chromosome aberration per cell observed in 1 hr samples (0.054+/-0.035 CA/cell) was higher than basal ones (0.031+/-0.026 CA/cell), although this difference was not statistically significant (p= 0.101). For in vitro assay, blood samples were exposed to different concentrations of 153Sm-EDTMP, during 1 hr (0.37-1.11 MBq/ml). An increase in the frequency of chromosome aberration per cell as a function of the radioactive concentration was found. The data were adjusted by linear regression model (Y= 3.52+/-2.24 x 10(-2) + 11.15+/-3.46 x 10(-2) X). The frequency of aberration/cell found in vivo was 0.054 and for the same activity in vitro was 0.098, this difference being statistically significant (p = 0.02). This result may be related to blood clearance, osteoblastic activity and individual variability. For a more accurate analysis, the study of more donors is necessary.     

Malate dehydrogenase types in the Asian-Pacific area,and a description of new phenotypes

A survey of more than 21 000 haemolysates from blood samples collected in various parts of south and southeast Asia, Australasia and the Western Pacific and examined in this laboratory has revealed several new alleles controlling variants of sMDH; in addition, further information has been provided on the distribution of sMDH3 in New Guinea. Two of the variant alleles, sMDH3 and sMDH6, achieve polymorphic frequency in various populations. sMDH3 is widely distributed in New Guinea, with highest frequencies in the Eastern Highlands. The pattern of its distribution suggests the mutant arose originally in a Papuan-speaking population. So far, sMDH6 has been detected only in Micronesians from a number of islands in the Carolines. A single example of another new variant, sMDH 5-1, and two examples of a slow variant, sMDH 7-1, were detected in samples from Iran and Singapore, respectively. No examples of mMDH variants were found in a total of 652 placental extracts from Papua New Guinea and Australia.     

Amino acid sequences of neurotoxic protein variants from the venom of Centruroides sculpturatus Ewing

The venom from the scorpion, Centruroides sculpturatus Ewing (range, Southwestern United States), was fractionated into ten protein zones by chromatography on CM-cellulose. Further purification of three of these zones on DEAE Sephadex in ammonium acetate developers yielded three principal neurotoxins designated variants 1, 2, and 3. Variants 1 and 3 each consist of 65 amino acid residues, variant 2 is composed of 66 residues, and each variant is a single polypeptide chain crosslinked by four disulfide bridges. The three variants have lysine at the amino terminus, serine at the carboxyl terminus, and their sequences exhibit a high degree of homology. The complete structure of variant 2 was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. The sequences of most of the tryptic peptides of variants 1 and 3 were determined, and the peptides were aligned by comparison with the homologous peptides in variant 2. The results show that there are 4 differences in sequence between variants 2 and 3 and 9 differences between variants 1 and 2. Variants 1 and 3 differ from each other at 5 positions in their sequences. These differences between the three protein variants are found in the amino terminal one-third of the molecules except for the deletion of one seryl residue at the carboxyl terminal of variants 1 and 3.     

Population genetic studies of the Philippine Negritos. I. A pilot survey of red cell enzyme and serum protein groups

Electrophoretic surveys of red cell enzyme and serum protein systems representing 21 genetic loci were carried out on 129 blood samples of the Negritos of Pampanga, Central Luzon, the Philippines. Nine (out of 16) red cell enzyme loci and four (out of five) serum protein loci showed polymorphic variation. Low frequencies of ACP 1A, GPTs1, ESD2, and Hp1, and a markedly high frequency of PGM12 were contrasted to those in non-Negrito Filipinos. Variant ESD phenotypes with a slowly migrating isozyme occurred in high frequency. The new allele designated as ESD3Negrito (ESD3N) had a frequency of .10 +/- .019. In AK, a variant phenotype indistinguishable from AK 2-1 was observed in 14% of the sample. In the Gc system, a fast migrating variant was discovered in high frequency which was distinct from Gc Ab and Gc J. The variant allele, denoted GcNegrito (GcN), had a frequency of .21 +/- .025. A relatively high degree of allelic diversity in the Negrito sample was also suggested by the average heterozygosity for 21 loci screened (.165), which is compared to that of the Japanese population (.140).     

Comparable Fitness and Transmissibility between Oseltamivir-Resistant Pandemic 2009 and Seasonal H1N1 Influenza Viruses with the H275Y Neuraminidase Mutation

Limited antiviral compounds are available for the control of influenza, and the emergence of resistant variants would further narrow the options for defense. The H275Y neuraminidase (NA) mutation, which confers resistance to oseltamivir carboxylate, has been identified among the seasonal H1N1 and 2009 pandemic influenza viruses; however, those H275Y resistant variants demonstrated distinct epidemiological outcomes in humans. Specifically, dominance of the H275Y variant over the oseltamivir-sensitive viruses was only reported for a seasonal H1N1 variant during 2008-2009. Here, we systematically analyze the effect of the H275Y NA mutation on viral fitness and transmissibility of A(H1N1)pdm09 and seasonal H1N1 influenza viruses. The NA genes from A(H1N1)pdm09 A/California/04/09 (CA04), seasonal H1N1 A/New Caledonia/20/1999 (NewCal), and A/Brisbane/59/2007 (Brisbane) were individually introduced into the genetic background of CA04. The H275Y mutation led to reduced NA enzyme activity, an increased K_m for 3′-sialylactose or 6′-sialylactose, and decreased infectivity in mucin-secreting human airway epithelial cells compared to the oseltamivir-sensitive wild-type counterparts. Attenuated pathogenicity in both RG-CA04^NA-H275Y and RG-CA04 × Brisbane^NA-H275Y viruses was observed in ferrets compared to RG-CA04 virus, although the transmissibility was minimally affected. In parallel experiments using recombinant Brisbane viruses differing by hemagglutinin and NA, comparable direct contact and respiratory droplet transmissibilities were observed among RG-NewCal^HA,NA, RG-NewCal^HA,NA-H275Y, RG-Brisbane^HA,NA-H275Y, and RG-NewCal^HA × Brisbane^NA-H275Y viruses. Our results demonstrate that, despite the H275Y mutation leading to a minor reduction in viral fitness, the transmission potentials of three different antigenic strains carrying this mutation were comparable in the naïve ferret model.     

Pyridoxine effects on ornithine ketoacid transaminase activity in fibroblasts from carriers of two forms of gyrate atrophy of the choroid and retina.

Gyrate atrophy of the choroid and retina that is due to ornithine ketoacid transaminase (OKT) deficiency is an autosomal recessive disorder. Fibroblasts from heterozygotes for the pyridoxine-responsive variant as well as those for the pyridoxine-nonresponsive variant contain intermediate levels of OKT activity. These two variants can be distinguished by the in vitro responsiveness of OKT activity to pyridoxal phosphate (PLP) stimulation. The ratios of OKT activity at 0.04 mM PLP compared with activity at 0 mM PLP were, respectively, lowest for controls (1.18 +/- 0.18; N = 12), intermediate for pyridoxine-nonresponsive heterozygotes (1.43 +/- 0.26; N = 5), and highest for pyridoxine-responsive heterozygotes (2.20 +/- 0.14; N = 3).     

Human catecholamine sulfotransferase (SULT1A3) pharmacogenetics: functional genetic polymorphism

Sulfotransferase (SULT) 1A3 catalyzes the sulfate conjugation of catecholamines and structurally related drugs. As a step toward studies of the possible contribution of inherited variation in SULT1A3 to the pathophysiology of human disease and/or variation in response to drugs related to catecholamines, we have resequenced all seven coding exons, three upstream non-coding exons, exon-intron splice junctions and the 5'-flanking region of SULT1A3 using DNA samples from 60 African-American (AA) and 60 Caucasian-American (CA) subjects. Eight single nucleotide polymorphisms (SNPs) were observed in AA and five in CA subjects, including one non-synonymous cSNP (Lys234Asn) that was observed only in AA subjects with an allele frequency of 4.2%. This change in amino acid sequence resulted in only 28 +/- 4.5% (mean +/- SEM) of the enzyme activity of the wild-type (WT) sequence after transient expression in COS-1 cells, with a parallel decrease (54 +/- 2.2% of WT) in level of SULT1A3 immunoreactive protein. Substrate kinetic studies failed to show significant differences in apparent Km values of the two allozymes for either dopamine (10.5 versus 10.2 micro m for WT and variant, respectively) or the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate (0.114 versus 0.122 micro m, respectively). The decrease in level of immunoreactive protein in response to this single change in amino acid sequence was due, at least in part, to accelerated SULT1A3 degradation through a proteasome-mediated process. These observations raise the possibility of ethnic-specific inherited alterations in catecholamine sulfation in humans.     

Characterization of ACP1TIC-1, an electrophoretic variant of erythrocyte acid phosphatase restricted to the Ticuna Indians of central Amazonas.

A new variant of erythrocyte acid phosphatase, designated ACP1TIC-1, is characterized by a more cathodal electrophoretic mobility than any of the common polymorphic phenotypes, both in the presence and absence of tricarboxylic acids. Individuals of the ACP1TIC-1 phenotype have a level of enzyme activity (4.8 +/- 0.1 mumol/g hemoglobin per min) similar to individuals of the ACP1A phenotype, although no differences in Km values were observed or is the extent of phosphate inhibition different between the ACP1TIC-1 and the ACP1B variants. The thermostability of the enzyme is less than that observed for any of the common variants. The TIC-1 variant is activated by adenine and inhibited by folic acid to the same extent as the type-A enzyme, while the stimulation of the activity of the TIC-1 enzyme by hypoxanthine and the inhibition of it by uric acid is similar to that for the B enzyme. Thus, the TIC-1 variant has a unique combination of kinetic properties, seeming to be a hybrid of A-type and B-type characteristics.     

Characterization of a new variant of human red cell carbonic anhydrase I,CA if London (Glu-102→Lys)

A new inherited variant of red cell carbonic anhydrase I (CA I), designated CA If London, was discovered during a survey of 1615 individuals from London, England. No electrophoretic variants of the other isozyme of carbonic anhydrase CA II, were observed in the same survey. Sequence analysis of a lysine-blocked tryptic peptide believed to contain the amino acid substitution in CA If showed that the glutamyl residue at position 102 had been substituted by a lysyl residue. This substitution results in a net increase of two positive charges in the mutant enzyme. Densitometric scanning of the electrophoretically separated forms in the variant hemolysate indicates that the levels of the normal and variant enzymes are approximately equal.Supported in part by U.S. Public Health Service grant GM15,419.     

Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes. II. Variant DK1: evidence for a discrete CTL recognition region

Multiple amino acid sequence differences distinguish individual HLA antigens. Those residues important in immune recognition events have not been defined. Recent studies have identified HLA-A2 structural variants that, although serologically indistinguishable from other HLA-A2 antigens, are recognized poorly, if at all, by HLA-A2-restricted, influenza virus-immune, or HLA-A2-specific alloimmune CTL. In this study we utilize double-label tryptic peptide comparisons performed by both reverse-phase HPLC and cation exchange chromatography, in conjunction with conventional and microsequence analysis, to characterize the HLA-A2 heavy chains derived from variant DK1. We detect a single tryptic peptide that distinguishes DK1 HLA-A2 from the predominant HLA-A2 heavy chain species. This peptide spans residues 147 to 157 in the second heavy chain domain, and carries substitutions at positions 149, 152, and 156. Residues in this segment of the polypeptide are also altered in another HLA-A2 variant, as well as one H-2Kb mutant. Thus, this segment appears to be critical in forming determinants important in CTL recognition of class I antigens in general. On the basis of these and other results, we suggest that in contrast to recognition by alloantibodies, a discrete region of class I antigens may be crucial for CTL recognition.     

Evolutionary variants of the human immunodeficiency virus type 1 V3 region characterized by using a heteroduplex tracking assay.

Syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) are evolutionary variants that are associated with rapid CD4+ cell loss and rapid disease progression. The heteroduplex tracking assay (HTA) was used to detect evolutionary V3 variants by amplifying the V3 sequences from viral RNA derived from 50 samples of patient plasma. For this V3-specific HTA (V3-HTA), heteroduplexes were formed between the patient V3 sequences and a probe with the subtype B consensus V3 sequence. Evolution was then measured by divergence from the consensus. The presence of evolutionary variants was correlated with SI detection data on the same samples from the MT-2 cell culture assay. Evolutionary variants were correlated with the SI phenotype in 88% of the samples, and 96% of the SI samples contained evolutionary variants. In most cases the evolutionary V3 variants represented discrete clonal outgrowths of virus. Sequence analysis of the six discordant samples that did not show this correlation indicated that three non-syncytium-inducing (NSI) samples had V3 sequences that had evolved away from the consensus sequence but not toward an SI genotype. A fourth sample showed little evolution away from the consensus but was SI, which indicates that not all SI variants require basic substitutions in V3. The other two samples had SI-like genotypes and NSI phenotypes, suggesting that V3-HTA was able to detect SI emergence in these samples in the absence of their detection in vitro. V3-HTA was also used to confirm SI variant selection in MT-2 cells and to examine the possibility of variant selection during virus culture in peripheral blood cells.     

Biochemical characterization of the human carbonic anhydrase variant CA ih Hiroshima

Summary Some biochemical properties of a new red cell human carbonic anhydrase variant, CA Ih Hiroshima, have been determined. Evidence is presented that the amino acid substitution in the Japanese variant is not the same as the previously characterized CA Ic variant from Guam of similar electrophoretic mobility. Based on a comparison with the normal CA I isoenzyme, a proposal for the site of the amino acid substitution is presented.     

Characterization of glucose-6-phosphate dehydrogenase variants in the Sudan--including GdKhartoum, a hyperactive slow variant.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was characterized in blood samples of 94 male subjects in Sudan having deficient and non-deficient electrophoretic variants. They comprised 44 GdB, 17 GdA, 19 GdB-, 11 GdA- and 3 nondeficient (GdKhartoum) variants. Biochemical characteristics including enzyme activity, electrophoretic mobility, Km for glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP), heat stability and pH optimum of all the common and deficient variants were consistent with the reported characteristics of these variants. The GdKhartoum variant had 90% mobility in TEB buffer and 100% in phosphate buffer, 120% activity, Km of 130 +/- 49 microns for G6P and 0.8 +/- 0.2 microns for NADP, lowered thermostability and an optimum pH of 7.6. This variant was not inhibited by 15 mM maleic acid, 10 mM iodoacetate and dehydro-iso-androsterone. All other variants were inhibited by dehydro-iso-androsterone but uninhibited by maleic acid and iodoacetate.     

The role of the internal hydrogen bond network in first-order protein electron transfer between Saccharomyces cerevisiae iso-1-cytochrome c and bovine microsomal cytochrome b5.

An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Asn52, E0 = 270 mV, kex = 0.3 s-1), Asn52----Ala (E0 = 240 mV, kex = 0.6 s-1), Asn52----Ile (E0 = 220 mV, kex = 1.0 s-1). The first-order rates are compared with that of a fourth variant Phe82----Gly which has been measured previously (E0 = 220 mV, kex = 0.7 s-1). An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both delta G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.     

Cyclophilin A-induced alterations of human immunodeficiency virus type 1 CA protein in vitro.

Cyclophilin A (CyP A), a cellular chaperone with cis-trans prolyl isomerase activity, is required for postassembly events in human immunodeficiency virus type 1 (HIV-1) replication. The requirement for CyP A maps to sequences in the capsid (CA) domain of the structural precursor, Gag. To determine the effects of interaction with CyP A on capsid (CA) protein structure, the binding interaction was investigated in vitro, using recombinant HIV-1 CA protein (which forms oligomers in solution) and human CyP A. The CA and CyP A proteins interacted to form a complex which was detected predominantly as a heterodimer on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Complex formation exhibited a pH optimum of 5. The CA protein in the complex was exclusively in a conformation whereby the N terminus was blocked to Edman degradation. This finding was unexpected since CyP A binds to the central region of the CA protein (residues 85 to 93). Examination of CA protein incubated with CyP A for alterations in structure indicated that CyP A preferentially interacted with the subpopulation of trypsin-susceptible subunits in the oligomers and significantly reduced their sensitivity to proteolysis. Like CA-CyP A complex formation, conversion to trypsin resistance also exhibited a pH optimum of 5. Both complex formation and the changes in tryptic susceptibility were only partially inhibited by cyclosporin A (CsA). This appeared to be due to a CA subpopulation able to bind CyP A despite the presence of CsA. Our results identify specific tryptic sites both proximal and distal to the CyP A binding region that are altered by CyP A binding and/or by CyP A's prolyl isomerase activity. Comparison with the X-ray structure of a complex containing CyP A bound to an amino-terminal fragment of the CA protein (CA1-151) (T.R. Gamble et al., Cell 87:1285-1294, 1996) indicates that the tryptic sites that become inaccessible are among the same residues that lose a significant amount of accessible surface area through CA-CA subunit contacts made in the presence of CyP A.     

Fructosamine in human and bovine semen.

We detected the presence of fructosamine in human and bovine semen. In seminal plasma of healthy normozoospermic men (N = 17) fructosamine was found in 53% of the cases (fru+). In fru+ semen samples the concentration of fructosamine was (mean +/- S.E.M., N = 9) 0.45 +/- 0.09 mmol/L and varied from 0.15 to 0.75 mmol/L. It was 3-12 times lower than in blood serum of healthy men. In semen of infertile men (N = 57) fructosamine was present only in 21% of the cases and its concentration was lower than in fertile men i.e. (mean +/- S.E.M., N = 12) 0.27 +/- 0.007 mmol/L. In bulls (N = 98) fructosamine was found in semen of 82% of animals. In fru+ semen samples the concentration of fructosamine was (mean +/- S.E.M., N = 80) 0.77 +/- 0.12 mmol/L and varied from 0.30 to 1.15 mmol/L. We did not find any correlation between the concentration of fructosamine on one hand, and that of fructose and glucose on the other hand, in either human or bull semen. The difference in the frequency of fructosamine appearance in semen of fertile and infertile men suggests that fructosamine may be in some way involved in the process of fertilisation.     

Chemical characterization of a new Japanese variant of carbonic anhydrase I,CA INagasaki 1 (76 Arg→Gln)

A new inherited variant of carbonic anhydrase I (CA I), designated CA I_{Nagasaki 1} (CA I_{NGS 1}), was discovered during a survey of hemolysates from 5852 individuals from the cities of Hiroshima and Nagasaki in Japan. Analysis of the amino acid composition of a tryptic peptide from the CA I_{NGS 1} variant indicated that a glutaminyl residue was substituted for an arginyl residue at position 76. Heat degradation studies showed that the CA I_{NGS 1} variant was less stable than normal CA I. The CO₂ hydrase and esterase activities of the normal and variant carbonic anhydrases I, as well as the relative amounts of the two enzymes in heterozygotes, were similar.This work was supported in part by Contract E(11-1)-1552 with the Energy Research and Development Administration, Washington, D.C. (to J. V. Neel), and by U.S. Public Health Service Grant GM-24681.