Studies on red cell carbonic anhydrase types in Korean cattle

Red cell carbonic anhydrase types in Korean cattle were investigated by means of starch gel electrophoresis.
A carbonic anhydrase zone (X) located ahead zone F was found in 12 out of 502 Korean cattle.
The distribution of CA phenotypes in 490 Korean cattle was CA FS type 92 and CA SS type 398. The CA FF type has not yet been recognized in Korean cattle. The gene frequencies were CA^F– 0.094 and CA^F = 0.906. No statistically significant differences were observed between gene frequencies in cattle from six different Korea provinces.     

Haemoglobin variants in cattle

From a comparative survey of reports on cattle Hb variants it is concluded that prior to the present communication the occurrence of at least seven different adult Hb molecules have been reported.
In addition comes the finding of a new Hb variant called HbG which is reported in this study. This was found in three animals among 101 East African Zebu cattle. It migrated more slowly than any cattle Hb variant previously reported and made up 20% of total Hb.
The gene frequencies of the East African Zebu population were: Hb^A = 0.52, Hb^B = 0.32, Hb^c = 0.14 and Hb^G = 0.01.     

Genetic variants and signatures of selective sweep of Hanwoo population (Korean native cattle)

Although there have been many studies of native Korean cattle, Hanwoo, there have been no selective sweep studies in these animals. This study was performed to characterize genetic variation and identify selective signatures. We sequencedthe genomes of 12 cattle, and identified 15125420 SNPs, 1768114 INDELs, and 3445 CNVs. The SNPs, INDELs, and CNVs were similarly distributed throughout the genome, and highly variable regions were shown to contain the BoLA family and GPR180, which are related to adaptive immunity. We also identified the domestication footprints of the Hanwoo population by searching for selective sweep signatures, which revealed the RCN2 gene related to BPV resistance. The results of this study may contribute to genetic improvement of the Hanwoo population in Korea. [BMB Reports 2013; 46(7):346-351]     

Advanced analytical methods for hemoglobin variants

Hemoglobin variants are the protein mutations most often encountered in the clinical scene. They have been useful for developing methods to analyze mutant proteins because of their size and ease of collection in large amounts. Improvements in analytical methods have been directed toward higher resolution in electrophoresis and shorter elution times in chromatography. More importantly, in the last 20 years, hemoglobin variants have been used in the development of mass spectrometric strategies for analyzing protein mutations. This approach consists of a series of steps: measurement of the molecular mass of globins to detect or confirm the presence of mutations, peptide mass mapping or peptide mass fingerprinting of an enzymatic digest to identify mutated peptides, and tandem mass spectrometry to determine or confirm the site and type of mutation. The mass spectrometric strategy has enabled rapid analysis and demonstrated a superb ability to detect a number of hemoglobin variants, particularly those without a change in electrophoretic or chromatographic properties. Even with the recent advances in DNA analysis, protein analysis is still essential, because post-translational modifications following amino acid substitutions can occur including N-terminal acetylation, deamidation and oxidation-mediated processes.     

Duffy blood group and hemoglobin variants

Summary Data on a sample of 809 Afro-Americans indicated that there is no association between Duffy null (a^-, b^-) blood type and sickle cell trait. The results further rule out close linkage as an alternative hypothesis to explain the reported association between these loci in areas where falciparum and vivax malaria are endemic and indicate that, even if the two loci are independent or loosely linked, direct evidence of the selection favoring AS Fy^-Fy^- individuals must come from populations where mixed malaria infections occur. Stratification, as an explanation for the reported association, is also discussed.     

Genetic variants of hemoglobin in canine erythrocytes*

Genetic polymorphism of hemoglobin was found in the erythrocytes of dogs of seven Japanese native breeds by using starch gel electrophoresis. Analysis of parentage records of the dogs revealed that the phenotypic variation of hemoglobin is controlled by one autosomal locus with two codominant alleles, Hb^A and Hb^B. The allele Hb^A occurred only in Japanese native breeds except Shikoku. The frequency of Hb^A in the Japanese breeds was low and 0.08. All the dogs belonging to 25 European breeds and 5 oriental origin (except Japan) breeds examined in this experiments had the homozygous genotype constitution Hb^B/Hb^B.     

Studies on the bacterial hemoglobin fromVitreoscilla

Summary Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO₂. EPR spectra of whole cells ofVitreoscilla exhibited a variety of intense signals at low and high magnetic field, theg-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction ofVitreoscilla showed the expected resonances for VtmetHb and FeSOD.Abbreviations A absorbance - DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EPR electron paramagnetic resonance - HiPIP high-potential iron protein - SDS sodium dodecyl sulfate - SOD superoxide dismutase - VtHb Vitreoscilla hemoglobin - VtmetHb oxidizedVitreoscilla hemoglobin - VtHbO₂ oxygenatedVitreoscilla hemoglobin     

Detection of hemoglobin variants in erythrocytes by flow cytometry

BACKGROUND: With the emergence of fetal hemoglobin (Hb F)stimulating agents as potential treatments for sickle-cell disease and thalassemias, procedures to monitor the effect of these agents on Hb F levels in individuals will be needed. We developed a rapid procedure that detects fetal hemoglobin in erythrocytes (F cells) using a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody against Hb F. METHODS: Ten microliters of washed blood was fixed in formaldehyde and glutaraldehyde, then permeabilized in a Triton X-100/PBS solution containing a FITC-labeled monoclonal antibody to Hb F. The blood was analyzed by flow cytometry to determine the percentage of F cells. RESULTS: Nearly 200 Hb F-containing samples were analyzed by this protocol and demonstrated good correlation to percent Hb F results determined by high pressure liquid chromatography (HPLC). In addition, a number of samples were fixed and permeabilized using this method as well as a previously-described method that uses dimethyl 3,3'dithiobispropionimadate (DTBP) as a fixative as well as a different anti-Hb F monoclonal. Good correlation (r = 0.96, r2 = 0.93, P<0.001) was observed between the two protocols. CONCLUSIONS: This procedure is easy, reproducible, and gives accurate F cell results. It can be used to measure a wide range of F cell percentages and may also be used to dual-stain Hb F along with other hemoglobin variants and erythrocyte surface antigens.