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Biomedical subjects

W P Winter

Publications and source records attributed to W P Winter.

At least 19 recordsLinked to original sources
PubMed2000

Automated oxyhemoglobin dissociation curve construction to assess sickle cell anemia therapy.

Oxyhemoglobin dissociation curves measure the most important function of red blood cells - the affinity for oxygen and its delivery to the tissues. This function may be deranged in sickle cell anemia and some other hemoglobinopathies. An automated oxyhemoglobin dissociation curve analyzer constructed dissociation curves in 55 patients with hemoglobinopathies and in 24 control subjects while maintaining constant temperature and pH. Sigmoid curves were converted to rectilinear ones using the Hill equation. Oxygen affinity of red cells was assessed by calculation of P50 (the PO2 at which hemoglobin is half saturated). Results revealed separation of oxyhemoglobin dissociation Hill plots according to phenotype but with wide variability. Mean oxygen affinity of fetal hemoglobin was greatest, whereas that of sickle hemoglobin was least. Other hemoglobins were intermediate. A positive correlation between decreased oxygen affinity and carboxyhemoglobin confirmed the decreased oxygen affinity of sickle hemoglobin and decreased oxygen affinity and increased diphosphoglycerate in red cells. Hill plots are less sensitive discriminators of oxygen affinity than traditional sigmoid dissociation curves and offer no particular advantage. Serial studies in a subset of three sickle cell anemia patients treated conservatively suggest automated oxyhemoglobin dissociation curves may be useful in assessment of effectiveness of newer therapies of sickle cell anemia after refinement of the method and studies of larger populations.

Adult↗
PubMed1994

A four base pair deletion 5' to the A gamma T gene is associated not only with decreased expression of the A gamma T-globin gene, but also of the G gamma-globin gene in cis.

A four base pair deletion 5' to A gamma T-globin gene at positions -222 to -225 has been reported to reduce the expression of this gene. To evaluate the prevalence and effect of this deletion, PCR-based methods were employed. The deletion had a gene frequency of 0.06 in a sample of African-American individuals with sickle cell trait, 0.18 in adult African-Americans with normal Hb AA, and 0.36 in caucasians. Seventy cord blood samples from African-American newborns with Hb AA were evaluated by both HPLC and PCR. The frequency of the A gamma T allele was 0.13. The A gamma T-globin chain was always present in a lower proportion than the A gamma I allele (70% of A gamma I), but the percentage of A gamma-globin was the same whether or not A gamma T was present. The total percentage of Hb F, however, was significantly lower in the group with the A gamma T allele (77.1% vs. 87.4%, P < 0.01). These results indicate that the four base pair deletion is not only associated with reduced expression of the A gamma T allele, but also of the G gamma allele in cis, further suggesting a possible role of this region in the modulation of the expression of the linked gamma-globin genes.

Base Sequence↗
PubMed1993

G gamma A gamma (beta+) hereditary persistence of fetal hemoglobin: the G gamma -158 C-->T mutation in cis to the -175 T-->C mutation of the A gamma-globin gene results in increased G gamma-globin synthesis.

Hereditary persistence of fetal hemoglobin (HPFH) can be generally classified into deletional and nondeletional forms. The family described in the present study has characteristics of both types of HPFH. The proband is a healthy 30-year-old black woman. Analysis of her hemoglobin revealed 40.4% HbS, 40.9% HbF (G gamma/A gamma ratio 0.53), 16.8% HbA, and 1.9% HbA2. All of her hematologic indices were normal, and the distribution of HbF in her red cells was pancellular. Family studies demonstrated that the proband has one chromosome 11 bearing the beta s-globin gene and the other bearing a G gamma A gamma (beta+) HPFH determinant in cis to the beta A-globin gene. Gene mapping studies of the region between the G gamma- and beta-globin genes were normal. However, when the A gamma and G gamma promoters were amplified by polymerase chain reaction (PCR) and sequenced, the A gamma promoter was found to have the T-->C mutation at -175, and the G gamma promoter region was found to have the C-->T mutation at -158. The -158 C-->T mutation has been associated with elevated G gamma levels and high HbF in hemolysis, although its role in causing these effects is unclear. The present study suggests that this mutation can also enhance G gamma-globin expression in cis to the -175 T-->C mutation in the absence of hemolysis. We suggest that the alteration of the A gamma gene octamer binding site by the -175 mutation, as well as the loss of a putative G gamma "silencer" caused by the -158 mutation may account for this phenotype. We propose calling these linked mutations the G gamma A gamma(beta+) HPFH.

Adult↗
PubMed1984

Interaction of hemoglobin S with anionic polysaccharides.

Our previous studies on the mechanism by which citrate agar electrophoresis separates hemoglobins led to the conclusion that hemoglobins bind to at least some sulfated polysaccharides. In the present report, we describe our deduction at the location of the binding site on the hemoglobin molecule. This led to the prediction, on theoretical grounds, that the anionic polysaccharides should possess anti-gelling actions toward hemoglobins and might be useful drug models. We have shown that anionic polysaccharides including heparin, lambda-carrageenan, dermatan sulfate, fucoidan, and agaropectin have anti-gelling activity. Evidence indicates that heparin can be introduced into red cells by synthetic lipid vesicles (liposomes) and that, once introduced, acts to block sickling. Because of the high solubility and low toxicity of the polysaccharides, we propose that these compounds deserve further study as potential anti-sickling agents.

Binding Sites↗
PubMed1983

Interaction of human hemoglobin and its variants with agar.

In citrate agar electrophoresis hemoglobin appears to bind reversibly to the sulfated polysaccharide agaropectin, a natural component of Difco Bacto-Agar. This complex migrates anodally, since the hemoglobin is only weakly positively charged at pH 6.2 whereas the carbohydrate carries a net negative charge. Electroendosmosis, on the other hand, proceeds in the cathodal direction. These opposing fluxes separate the hemoglobins in the order of their affinity for agaropectin. An agaropectin binding site was identified on hemoglobin by computer-assisted modeling, and the relation of the site to hemoglobin variants that exhibit abnormal citrate agar mobility was established. The citrate anion is postulated to function as a "counter ion." Preliminary evidence indicates that agaropectin has antigelling properties with respect to hemoglobin S.

Agar↗
PubMed1981

Freeze preservation of sickle erythrocytes.

The effect of short-term cryopreservation on metabolic, functional, and survival characteristics of erythrocytes from patients with sickle cell disease was examined. Post-thaw hemolysis of glycerolized sickle (SS) erythrocytes was greater (mean 12.9%) than in hemoglobin-AA cells (mean 4.7%). Freeze preservation had no apparent effect on red cell morphology, percent irreversibly sickled cells, and fetal hemoglobin content. There were modest reductions in ATP and 2,3-diphosphoglycerate in thawed, washed sickle erythrocytes (12.7% and 29.7%, respectively). However, the autologous survival of 51Cr-labelled SS red cells was not shortened by cryopreservation. The safety and efficacy of autotransfusion of cryopreserved red cells in alloimmunized sickle cell disease patients with anemic episodes unrelated to sickling need to be determined.

Adenosine Triphosphate↗
PubMed1981

Prevalence of alpha-chain variants at birth.

Hemoglobin electrophoresis of cord blood from 4,499 newborns was performed as part of a sickle cell disease detection program. Although the expected frequency of the genes for hemoglobins S and C were observed, six newborns (five kindreds) were heterozygous for an alpha G Hb. In four kindreds, the alpha chain variant was identified as Hb G-Philadelphia. In each case, heterozygosity for this Hb was manifested at birth by the presence of Hb F/G (alpha 2 G gamma 2), a slow Hb migrating between the positions of Hbs S and C on cellulose acetate. In some newborns, Hb G (alpha 2 G beta 2) was also detectable, so that these cord bloods had four hemoglobin components: F, F/G, A, and G. The prevalence of Hb G-Philadelphia in this population sample is higher than that usually reported for black Americans and may represent a founder effect. Comprehensive screening of cord blood hemoglobins should use electrophoresis on alkaline media as the primary testing procedure because it allows recognition of most variants, such as the D (G) hemoglobins. Reliable identification of Hbs S and C requires citrate agar electrophoresis as a confirmatory test.

Blood Protein Electrophoresis↗
PubMed1981

Use of citrate agar electrophoresis in evaluation of antisickling agents.

Hemoglobin S (Hb S), treated with antisickling agents which interfere with gel formation, has been found to migrate on citrate agar electrophoresis in a manner similar to Hb A or Hb F. This finding suggests a rapid and inexpensive method for screening potential antisickling compounds of the types which interfere with gelation. This investigation has also led to an improved understanding of the molecular basis of citrate agar electrophoresis and to the realization that agar itself may contain a natural antisickling agent.

Antisickling Agents↗
PubMed1978

Turnover of metallothioneins in rat liver.

Two electrophoretically distinguishable metallothioneins were isolated from the livers of Cd2+-treated rats and had thiol group/metal ratios of 3:1, a total metal content, in each of these proteins, of 3.6 atoms of Cd2+ + 2.4 atoms of Zn2+/molecule and 4.2 atoms of Cd2+ + 2.8 atoms of Zn2+/molecule and respective apoprotein mol.wts. of 5844 and 6251. Studies with 1 h pulse labels of [3H]cysteine, given after a single injection of ZnCl2 or CdCl2, showed that these metals stimulated radioactive isotope incorporation into the metallothioneins over the control value by 10- and 15-fold respectively. This stimulation was maximal at 4 h after a single CdCl2 injection and decreased to control values by 16 h, suggesting that either a translational event is responding to free intracellular Cd2+ or a short-lived mRNA is being produced or stabilized in response to the metal treatment. In rats chronically exposed to CdCl2, the metallothioneins increased to 0.2% of the liver wet weight from a control value of 2--4 mumol/kg of liver, with a maximum rate of accumulation of 2--3 mumol/h per kg of liver. The turnover of these proteins in control animals was 0.3--0.6 mumoles/h per kg of liver, measured by the rate of disappearance of 203Hg2+, which binds irreversibly to the metallothioneins. Pretreatment with CdCl2 completely stopped the rapid 203Hg turnover observed in untreated animals. Unlike CdCl2, treatment with ZnCl2 increased the concentration of metallothioneins to a new steady-state pool, 11 mumole/kg of liver, after 10 h. The increase in the zinc-thionein pool by exposure to ZnCl2 in vivo was determined to be primarily due to a stimulation of metallothionein biosynthesis.

Amino Acids↗
PubMed1976

Hemoglobin Wayne: a frameshift mutation detected in human hemoglobin alpha chains.

Hemoglobin Wayne is an alpha chain variant which manifests itself as two minor hemoglobin (Hb) components that migrate more rapidly than Hb A on electrophoresis at pH 8.6. It has been found in a child with Fanconi's anemia and in three generations of the child's family. Each of the minor components yields an alpha chain in which the carboxyl-terminal tripeptide sequence, Lys-Tyr-Arg, has been replaced by the octapeptide sequence Asx-Thr-Val-Lys-Leu-Glu-Pro-Arg. In alpha Wayne I, the slower of the pair, Asx is asparagine, whereas in alpha Wayne II it is aspartic acid. Comparison of the alpha Wayne sequences with the amino-acid sequences of alpha A and alpha Constant Spring leads to the conclusion that Hb Wayne I is the result of a -1 frameshift mutation in the alpha chain and that Hb Wayne II is formed secondarily by spontaneous deamidation of the new asparagine residue. A frameshift is consistent with a single mRNA base sequence for the last eight codons involved and supports the view of Clegg, Weatherall, and Milner [Nature (1971) 234, 337-341] that Hb Constant Spring is the result of a terminator mutation leading to translation of 31 codons not normally translated.

Amino Acid Sequence↗