Difference between the Hb C variants in Brahman and in indigenous Southern African cattle breeds

Haemoglobin polymorphism in Brahman cattle and seven Southern African cattle breeds was investigated by means of starch gel electrophoresis. A difference was found between the migration rates of the Hb C of Brahman cattle and that of the indigenous Southern African cattle breeds, showing that these are actually two separate variants. We suggest that the faster migrating type, which occurs in Brahman cattle, should be called Hb C, while the slower migrating type of the Southern African breeds, should be called Hb I.
A comparison of the migration of the α and β chains of all the variants we encountered, including foetal haemoglobin, was carried out by means of starch gel electrophoresis in urea at acid and alkaline pH levels. Evidence was found of a difference in mobility of the non-a chains of haemoglobin A, B, C, I and F at different pH levels while no difference was detected in the migration rate of their respective α chains. These results confirm the theory that genetic variation is restricted to the non-α chain of bovine haemoglobin.
Progeny studies on 225 families confirm that the Hb I variant is allelic to Hb A and Hb B variants. The gene frequencies have been calculated on the basis that Hb I and Hb C are allelic at the β locus. The distribution of the different phenotypes is in accordance with this theory, assuming that the populations obey Hardy-Weinberg equilibrium.     

Hemoglobin Bali (bovine): βA18(Bl)Lys → his: One of the “missing links” between βA and βB of domestic cattle exists in the Bali cattle (Bovinae,Bos banteng)

The structure of the beta chain of adult bovine hemoglobin Bali of the Bali cattle was determined and compared to those of beta A, beta B, and other beta-chain variants of domestic cattle reported previously. The lysine residue at beta A18 was substituted by histidine in beta Bali18. This change requires two base substitutions at the codon and is also found in beta B18. The beta B chain differs from the beta A chain at residue Nos. 15, 18, and 119. It was concluded that a common ancestor of the beta B and beta Bali first diverged from the beta A chain through the Lys leads to His substitution. This fact indicates that the high degree of dimorphism of the beta A and beta B chains in Indian humped cattle is a result of its hybrid origin. An evolutionary tree for the bovine hemoglobin beta-chain variants was constructed based on parsimonious evolution and homology with related species.     

Haemoglobin Avicenna (beta 47 (CD6) Asp replaced by Ala). A new abnormal haemoglobin.

In a survey for abnormal haemoglobin variants in voluntary blood donors in Iran, a new variant was found in a young male who presented no clinical symptoms. It had the same electrophoretic mobility as haemoglobin D in alkaline buffers. Separation of the constituent polypeptide chains in acid urea buffer revealed it to be different from haemoglobin D previously found among Iranians. Analysis of its structure demonstrated a substitution to alanine (beta 47 Asp replaced by Ala) in the same residue as involved in haemoglobin G-Copenhagen (beta 47 Asp replaced by Asn).     

Aspartate aminotransferase of Escherichia coli: nucleotide sequence of the aspC gene

The nucleotide sequence of the aspartate aminotransferase [EC 2.6.1.1] structural gene, aspC, of Escherichia coli K-12 was determined. The coding region of the aspC gene contained 1,188 nucleotide residues and encoded 396 amino acid residues. The amino acid sequence deduced from the nucleotide sequence agreed perfectly with that of the protein recently determined for the aspartate aminotransferase of E. coli B (Kondo, K., Wakabayashi, S., Yagi, T., & Kagamiyama, H. (1984) Biochem. Biophys. Res. Commun. 122, 62-67).     

Familial Polycythaemia Caused by a New Haemoglobin Variant: Hb Heathrow, β 103 (G5) Phenylalanine Leucine

A new variant of haemoglobin A (Hb A) with a high affinity for oxygen has been found in an English family. Five members are affected and all are polycythaemic. This variant (Hb Heathrow) is the first of this class to be found in this country and has the same electrophoretic mobility as Hb A. It was discovered only by measuring the oxygen affinity of the patients'' red cells. This emphasizes the need for measuring the oxygen affinity of haemoglobin in patients with polycythaemia if other clinical and haematological features associated with polycythaemia rubra vera are absent.     

Evidence for the presence of alpha 1B-glycoprotein in mammalian sera: immunoblotting studies

1. A monospecific antiserum to pig alpha 1B-glycoprotein (PO2) was produced in rabbits and was used to search for homologues of alpha 1B in sera of 41 mammalian species belonging to seven orders. 2. Specific reactions were detected in the sera of representatives of Insectivora, Primates, Carnivora, Proboscidea, Perissodactyla and Artiodactyla. No cross-reactions were observed in the sera of two species of Rodentia (mouse, rat). 3. Cross-reactions in the sera of Erinaceus europaeus, Homo sapiens and Macaca mulatta were rather weak; this indicates a greater structural difference between the alpha 1 B of Insectivora and Primates and that of the other mammalian orders. 4. Electrophoretic patterns of alpha 1 B were, in most cases, heterogeneous, the most heterogeneous being in ruminants. 5. Evidence was obtained that the alpha 1 B of sheep is identical with the earlier described (Juneja and Gahne (1980) Anim. Blood Grps Biochem. Genet. 11, 81-92.) polymorphic post-transferrin (Ptf).     

Identification of proteins similar to Bothrops jararaca coagulation inhibitor (BjI) in the plasmas of Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus snakes

Bothrops jararaca coagulation inhibitor (BjI), a protein isolated from B. jararaca plasma, specifically inhibits the coagulant activity of thrombin. Our group previously identified proteins similar to BjI in the plasma of other snakes [Tanaka-Azevedo, A.M., Tanaka, A.S., Sano-Martins I.S., 2003. A new blood coagulation inhibitor from the snake Bothrops jararaca plasma: isolation and characterization. Biochem Biophys Res Commun 308, 706-712.]. In the present study, we analyzed the presence of BjI-like proteins in the plasmas of three different species of viperid snakes, Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus. These proteins exhibited 109 and/or 138 kDa and were immunologically related to BjI. They also inhibited the coagulant activity of thrombin, evaluated by the thrombin time test. These findings demonstrate the presence of proteins similar to BjI in these three species, although such inhibitor could not be observed in all samples of the specimens tested. Moreover, the presence of these proteins in the plasma is related to prolongation of thrombin time, implying a relationship between these proteins and their inhibitory coagulant activity upon thrombin. Our results suggest that BjI-like proteins are widely distributed among Crotalinae snakes found in Brazil.     

Analysis of genetic diversity of domestic cattle in East and Southeast Asia in terms of variations in restriction sites and sequences of mitochondrial DNA

There are three major groups of domestic cattle in East and Southeast Asia: European cattle, Zebu cattle, and Bali cattle. Ten restriction enzymes were used to analyze restriction site variants in the mitochondrial DNA (mtDNA) in 178 individuals belonging to these three groups of cattle. The results indicate that each of the three groups has mtDNA with a specific haplotype. The sequence of the mitochondrial gene for cytochrome b in representative haplotypes of Zebu and Bali cattle was determined and was compared with that of European cattle in the literature. We calculated 51 pairwise nucleotide sequence differences between European and Zebu cattle and 91 between European and Bali cattle. Our results suggest that ancestral populations of Asiatic domestic cattle may have diverged into two lineages—Bali and European plus Zebu—more than 3 million years ago, and then the European and Zebu groups diverged more than 1 million years or so before domestication occurred.The sequences of mitochondrial genes for cytochrome b from Zebu cattle, Bali cattle and Water buffalo will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases, with the following accession numbers: D34635, D34636 and D34637.     

Nomenclature of lipoxins and related compounds derived from arachidonic acid and eicosapentaenoic acid

Oxygenated derivates of arachidonic acid and eicosapentaenoic acid which contain conjugated tetraene structures and are non-cyclized C20 carboxylic acids were first isolated and characterized from human and porcine leukocytes (Serhan, C.N. et al, 1984, Biochem. Biophys. Res. Commun. 118, 943-949; Wong, P.Y.-K., et al, 1985, Biochem. Biophys. Res. Commun. 126, 765-775). The trivial names lipoxins and lipoxenes have been introduced for compounds belonging to each of these series. Here, we propose that tetraene-containing compounds derived from arachidonic acid be denoted as lipoxins (LX) of the four series (i.e. lipoxin A4 or LXA4 and lipoxin B4 or LXB4) and those derived from eicosapentaenoic be termed lipoxins of the five series (i.e. lipoxin A5 or LXA5 and lipoxin B5 or LXB5).     

Molecular mechanics and dynamics calculations on (dA)10.(dT)10 incorporating distance constraints derived from NMR relaxation measurements

Structural constraints derived from proton NMR relaxation measurements on poly(dA).poly(dT) in the form of interproton separations and orientation have been combined with molecular mechanics and annealed molecular dynamics calculations to derive a model for the solution-state structure of this molecule. Three different possible starting configurations, including the standard A and B forms of Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, 1506-1509] and the heteronomous (H) structure [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155], were examined. Both the B- and H-DNA structures converged to the same B-like structure (approximately C2'-endo conformation on both the A and T sugars, glycosidic bond torsional angle of 63-73 degrees) with the same energies and average helical parameters that gave good fits of the NMR relaxation rates. This model also accounts for the experimental observation [Behling, R. W., & Kearns, D. R. (1986) Biochemistry 25, 3335-3346] that the AH2 proton interacts more strongly with the H1' sugar proton on the T strand than on the A strand. Although the helix repeat angle (39 degrees) is larger than that for standard B-DNA (36 degrees), this does not result in a significantly smaller minor groove, as monitored by the interstrand P-P separation. Calculations starting with the A-DNA structure lead to a very high energy structure that gave a poorer fit of the NMR data.     

Search for genetic variation in the blood of Norwegian dairy goats reveals a new polymorphism at the HbβA locus

A total of 150 blood samples tested for serum albumin and transferrin and for red cell carbonic anhydrase, phosphoglucomutase, phosphogluconate dehydrogenase, phosphohexose isomerase, nucleoside phosphorylase, acid phosphatase, 'X'-protein and potassium concentration only showed variation at the 'X' protein and nucleoside phosphorylase loci. Isoelectric focusing over pH range 6-8 showed 145 samples to be of haemoglobin type A and 5 type AD. The haemoglobin A type was resolved into further types by separation over pH 6.9-7.5 in Immobiline polyacrylamide gels. A 2- or 4-band pattern was present in 136 of the samples; a genetic hypothesis based on four or more different haemoglobin A variants is proposed. 14 samples had a 3-, 5- or 6-band pattern. It is assumed that these are heterozygous for a variant of the II alpha gene.     

Major effect of retinal short-chain dehydrogenase reductase (RDHE2) on bovine fat colour

Beef with yellow fat is considered undesirable by consumers in most European and Asian markets. β-Carotene is the major carotenoid deposited in the adipose tissue and milk fat of cattle (Bos taurus), which can result in the yellowness. The effects of retinal short-chain dehydrogenase reductase (RDHE2) and β, β-carotene 9',10-dioxygenase (BCO2) were considered jointly as major candidate genes for causing the yellow fat colour, based on their genomic locations in the fat colour quantitative trait loci (QTL) and their roles in the metabolism of β-carotene. In a secondary pathway, BCO2 cleaves β-carotene into retinoic acid, the most potent form of vitamin A. RDHE2 converts trans-retinol to trans-retinal, a less active form of vitamin A. We evaluated the effects of two amino acid variants of the RDHE2 gene (V6A and V33A) along with a mutation in the BCO2 gene that results in a stop codon (W80X) in seven cattle populations. The RDHE2 V6A genotype affected several fat colour traits but the size of the effect varied in the populations studied. The genotype effect of the RDHE2 V33A variant was observed only in New Zealand samples of unknown breed. In general, the individual effects of RDHE2 V6A and V33A SNPs genotypes were greater in the random New Zealand samples than in samples from pedigreed Jersey-Limousin backcross progeny, accounting for 8-17 % of the variance in one population. Epistasis between the BCO2 W80X and RDHE2 variants was observed, and in some populations this explained more of the variation than the effects of the individual RDHE2 variants.     

Genetic variation in the blood of llamas, Llama glama, and alpacas, Llama pacos

Blood samples of llamas and alpacas were typed using haemolytic, electrophoretic and isoelectric focusing procedures to assay polymorphism at 13 loci. Blood group variation was assessed using six antibody specificities produced by allo- and heteroimmunizations. Two red cell factors (A and B) behave as autosomal, codominant alleles at a closed A locus. The other four factors (C, D, E and F) behave as autosomal, dominant traits. Biochemical variation was found for red cell enzymes catalase, phosphogluconate dehydrogenase, glucose phosphate isomerase and for plasma proteins transferrin and post-albumin. No variants were found for haemoglobin, phosphoglucomutase and albumin. Estimates of probability of exclusion were 0.883 for llamas and 0.681 for alpacas, which are adequate initial levels of efficacy for purposes of parentage verification. Preliminary estimate of Nei's genetic distance measure (D) suggests that llamas and alpacas are more likely related as subspecies than as separate species.     

The distribution of serum protein and enzyme groups among the Batak of Samosir Island (Sumatra,Indonesia)

Summary 188 blood samples from Batak of Samosir Island (Sumatra, Indonesia) have been studied for electrophoretic variants of haemoglobin, 14 red cell enzyme and 5 serum protein systems. The acid phosphatase, 6 PGD, PGM₁ and ADA enzyme systems are polymorphic, and a single AK 2-1 person was detected. Polymorphism is present in the haptoglobin, transferrin and protease inhibitor systems. Two variant alleles, Tf ^Dchi and Tf ^B are present in the transferrin system, but the B variant has not been identified. Similarly, 3 persons with caeruloplasmin variants were found, but also these variants have not been identified. No abnormal haemoglobins were detected. All other systems revealed the presence of only normal phenotypes.

---

Zusammenfassung 188 Blutproben des Batak-Stammes von Samosir Island (Sumatra, Indonesien) wurden auf elektrophoretische Varianten des Hämoglobins in 14 Erythrocyten-enzym-und 5 Serumprotein-Systemen untersucht. Die Saure Phosphatase, 6GPD, PGM₁ und ADA-Systeme sind polymorph, und eine einzige AK 2-1-Person wurde gefunden. In den Haptoglobin-, Transferrin- und Pi-Systemen treten Polymorphismen auf. Zwei abweichende Allele, Tf ^Dchi und Tf ^B, sind im Transferrin-System zu finden, aber die B-Variante ist nicht bestimmt worden. Ebenso wurden 3 Personen mit Ceruloplasmin-Varianten gefunden, doch auch diese Varianten wurden nicht identifiziert. Keine abnormen Hämoglobine wurden gefunden. Alle anderen Systeme wiesen nur normale Phenotypen auf.

     

Monitoring mobility in the early steps of unfolding: the case of oxidized cytochrome b(5) in the presence of 2 M guanidinium chloride

A Model-Free analysis of the (15)N relaxation properties of oxidized cytochrome b(5), a heme-containing electron-transfer protein, has been performed in 2 M guanidinium chloride (GdmCl), i.e., just before the heme is released by the action of denaturant. This analysis provides information on the mobility in the nano- to picosecond time range. A parallel study on the motions in the milli- to microsecond time scale has also been performed by analyzing rotating-frame (15)N relaxation rates. The protein contains a 60:40 ratio of two conformers (A and B) differing for the rotation of the heme group around the alpha-gamma meso axis. The effect of denaturant has been followed for both species, and the mobility properties have been compared with the analogous information in the absence of denaturant. To complete the picture, we also performed (15)N relaxation measurements and the Model-Free analysis of the native B form, whereas data on the A form [Dangi, B., Sarma, S., Yan, C., Banville, D. L., Guiles, R. D. (1998) J. Phys. Chem. B 102, 8201-8208], as well as rotating-frame measurements for both native forms [Banci, L., Bertini, I., Cavazza, C., Felli, I. C., Koulougliotis, D. (1998) Biochemistry 37, 12320-12330; Arnesano, F., Banci, L., Bertini, I., Felli, I. C., Koulougliotis, D. (1999) Eur. J. Biochem. 260, 347-354], are already available in the literature. It is found that GdmCl tends to increase the internal mobility, although some residues are rigidified on both time scales. In the milli- to microsecond time scale, the tendency to increased mobility is reflected in a decrease in the tau(ex) values rather than in the number of residues experiencing conformational equilibria. In the nano- to picosecond time scale, the tendency to increased mobility is indicated by an overall decrease in the S(2) values. Color pictures are reported to visually show these effects. On the fast time scale, the B form is more mobile than the A form, reflecting the different stability with respect to unfolding. The increase in mobility upon addition of denaturant largely occurs around the heme pocket, which facilitates the release of the heme. The relevance of the internal motions with respect to the early steps of the unfolding process is also analyzed and discussed.     

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.     

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.     

Gd(-) Gifu and Gd(-) Fukuoka. Two new variants of glucose-6-phosphate dehydrogenase found in Japan

Summary Two new glucose-6-phosphate dehydrogenase (G6PD) variants were discovered in Japan. The first, found in a 9-year-old male, was associated with chronic hemolysis and hemolytic crises after upper respiratory infections. The enzyme activity of the variant was 2.9% of normal. The patient's G6PD showed an increased utilization of substrate analogue, deamino-NADP, and thermal instability. The second variant occurred in a 7-year-old male with druginduced hemolysis. The main enzymatic characteristics were reduced enzyme activity, being 6.4% of normal, faster-thannormal anodal electrophoretic mobility, slightly high Michaelis constant for glucose-6-phosphate, thermal instability, and biphasic pH optima. Enzymatic properties of these variants allowed each to be distinguished from previously reported variants. The first variant was designated Gd (-) Gifu and the other, Gd (-) Fukuoka.     

Hepatitis C Virus Variants with Decreased Sensitivity to Direct-Acting Antivirals (DAAs) Were Rarely Observed in DAA-Naive Patients prior to Treatment

The prevalence of naturally occurring hepatitis C virus (HCV) variants that are less sensitive to direct-acting antiviral (DAA) inhibitors has not been fully characterized. We used population sequence analysis to assess the frequency of such variants in plasma samples from 3,447 DAA-naive patients with genotype 1 HCV. In general, HCV variants with lower-level resistance (3- to 25-fold increased 50% inhibitor concentration [IC₅₀]) to telaprevir were observed as the dominant species in 0 to 3% of patients, depending on the specific variant, whereas higher-level resistant variants (>25-fold-increased IC₅₀) were not observed. Specific variants resistant to NS5A inhibitors were predominant in up to 6% of patients. Most variants resistant to nucleo(s/t)ide active-site NS5B polymerase inhibitors were not observed, whereas variants resistant to non-nucleoside allosteric inhibitors were observed in up to 18% of patients. The presence of DAA-resistant variants in NS5A, NS5B, or NS3 (including telaprevir-resistant variants), in baseline samples of treatment-naive patients receiving a telaprevir-based regimen in phase 3 studies did not affect the sustained viral response (SVR). Treatment-naive patients with viral populations containing the telaprevir-resistant variants NS3 V36M, T54S, or R155K at baseline achieved a 74% SVR rate, whereas patients with no resistant variants detected prior to treatment achieved a 76% SVR rate. The effect of specific resistant variant frequency on response to various DAA treatments in different patient populations, including interferon nonresponders, should be further studied.