Effects of heterotropic allosteric effectors on the equilibrium and kinetics of the reaction of a single ligand molecule with an alpha or beta subunit of deoxygenated HbA

Symmetrical FeZn hybrids of human HbA have been used to measure K(1)(alpha) and K(1)(beta), the dissociation constants for the binding of a single molecule of oxygen to unliganded HbA at an alpha subunit and at a beta subunit, respectively. The kinetic constants, l(1)'(alpha) and l(1)'(beta), for the combination of the first CO molecule to unliganded HbA at an alpha or a beta subunit, respectively, were also measured. Measurements were carried out between pH 6 and pH 8 in the presence and absence of inositol hexaphosphate (IHP). Both equilibrium constants exhibit a significant Bohr effect in the absence of IHP. The addition of IHP to a concentration of 0.1 mM increases both dissociation constants in a pH-dependent manner with the result that both Bohr effects are greatly reduced. These results require a negative thermodynamic linkage between the binding of a single oxygen at either an alpha or a beta subunit and the binding of IHP to the T quaternary structure of HbA. Although the beta hemes are relatively near the IHP binding site, a linkage between that site and the alpha hemes, such that the binding of a single oxygen molecule to the heme of one alpha subunit reduces the affinity of the T state for IHP, requires communication across the molecule. l(1)'(alpha) exhibits a very slight pH dependence, with a maximum variation of 20%, while l(1)'(beta) varies with pH three times as much. IHP has no effect on the pH dependence of either rate constant but reduces l(1)'(alpha) marginally, 20%, and l(1)'(beta) by 2-fold at all pH values.     

Deletion and linkage mapping of eight markers from the proximal short arm of chromosome 6

Eight chromosome 6p markers (MUT, D6S4, D6S5, D6S19, D6S29, PIM, HLA, and F13A) were regionally mapped using somatic cell hybrid deletion cell lines that retained different regions of chromosome 6p. New restriction fragment length polymorphisms were identified at the D6S5 and PIM loci using newly isolated genomic clones at these loci. Genetic linkage among the eight loci was determined using the 40 CEPH reference families. Linkage analyses showed that these loci are in one linkage group spanning 48 cM in males and 128 cM in females. Using both the deletion mapping data and multipoint linkage analyses, chromosomal order for these loci was determined as centromere-(MUT, D6S4)-(D6S5, D6S19)-(D6S29, PIM)-HLA-F13-A-telomere. Analyses of sex-specific recombination frequencies revealed a higher rate of recombination in females in the region between D6S4 and D6S29, while the recombination rate in males was higher for the interval between D6S29 and the HLA loci.     

Accumulation of creatine kinase mRNA during myogenesis: molecular cloning of a B-creatine kinase cDNA

Cytosolic creatine kinase isoenzymes MM, MB, and BB are assembled from M or B subunits which occur in different relative amounts in specific tissues. The accumulation of mRNAs encoding the M and B subunits was measured during myogenesis in culture. The relative concentration of the two mRNAs was determined by hybridization with a M-CK cDNA probe isolated previously and a B-CK cDNA probe, the cloning and characterization of which is reported here. The B-CK cDNA hybridizes specifically to a 1.6-kb mRNA found in brain and gizzard but not in adult skeletal muscle tissue. The M-CK cDNA hybridizes to a smaller mRNA 1.4-kb long which is specific to skeletal muscle. In culture, the B-CK mRNA is transiently induced and then declines to a low but detectable level.     

Haplotype analyses of haemoglobin C and haemoglobin S and the dynamics of the evolutionary response to malaria in Kassena-Nankana District of Ghana

Background

Haemoglobin S (HbS) and C (HbC) are variants of the HBB gene which both protect against malaria. It is not clear, however, how these two alleles have evolved in the West African countries where they co-exist at high frequencies. Here we use haplotypic signatures of selection to investigate the evolutionary history of the malaria-protective alleles HbS and HbC in the Kassena-Nankana District (KND) of Ghana.

Methodology/Principal Findings

The haplotypic structure of HbS and HbC alleles was investigated, by genotyping 56 SNPs around the HBB locus. We found that, in the KND population, both alleles reside on extended haplotypes (approximately 1.5 Mb for HbS and 650 Kb for HbC) that are significantly less diverse than those of the ancestral HbA allele. The extended haplotypes span a recombination hotspot that is known to exist in this region of the genome

Significance

Our findings show strong support for recent positive selection of both the HbS and HbC alleles and provide insights into how these two alleles have both evolved in the population of northern Ghana.     

Auxin herbicides induce H(2)O(2) overproduction and tissue damage in cleavers (Galium aparine L.)

The phytotoxic effects of auxin herbicides, including the quinoline carboxylic acids quinmerac and quinclorac, the benzoic acid dicamba and the pyridine carboxylic acid picloram, were studied in relation to changes in phytohormonal ethylene and abscisic acid (ABA) levels and the production of H(2)O(2) in cleavers (Galium aparine). When plants were root-treated with 10 microM quinmerac, ethylene synthesis was stimulated in the shoot tissue, accompanied by increases in immunoreactive levels of ABA and its precursor xanthoxal. It has been demonstrated that auxin herbicide-stimulated ethylene triggers ABA biosynthesis. The time-course and dose-response of ABA accumulation closely correlated with reductions in stomatal aperture and CO(2) assimilation and increased levels of hydrogen peroxide (H(2)O(2)), deoxyribonuclease (DNase) activity and chlorophyll loss. The latter parameters were used as sensitive indicators for the progression of tissue damage. On a shoot dry weight basis, DNase activity and H(2)O(2) levels increased up to 3-fold, relative to the control. Corresponding effects were obtained using auxin herbicides from the other chemical classes or when ABA was applied exogenously. It is hypothesized, that auxin herbicides stimulate H(2)O(2) generation which contributes to the induction of cell death in Galium leaves. This overproduction of H(2)O(2) could be triggered by the decline of photosynthetic activity, due to ABA-mediated stomatal closure.