Distributions of hemoglobins A and S among erythrocytes of heterozygotes.

The recently developed capability to separate and quantify each of several proteins concurrently in single red cells presents an opportunity to test for biological variations in intercellular distribution of a protein as well as the extent of correlation between quantities of gene products derived from a single cell genome. In this preliminary study, erythrocytes from 30 sickle trait subjects were subjected to single cell electrophoresis and the resulting hemoglobin electropherograms were scanned by a recording densitometer. There was found to be heterogeneity among subjects in the form of the intercellular distribution of Hb S fraction, as tested by g statistics for skewness and kurtosis. Additionally, in all subjects there was statistically significant correlation between relative quantities of cellular Hb A and Hb S as measured concurrently in the same cell. These observations provide a basis for future research on the hypothesis that the form of the distribution of hemoglobin among erythrocytes is a heritable variable.

An erythrocyte membrane antigen associated with X-linked muscular dystrophy.

Goat antiserum was prepared against erythrocyte membranes obtained from a man who had X-linked muscular dystrophy. When cross-adsorbed with control membranes and tested on double immunodiffusion plates or by binding to intact erythrocytes suspended in thin-layer agar plates, this antiserum discriminated between membranes from phenotypically normal controls and dystrophic men, and between controls and obligate female dystrophy carriers. It also identified two populations among a small sample of women at risk for being dystrophy carriers.

Relative proportions of human hemoglobins A, S, C in individual erythrocytes of heterozygotes.

Inherited electrophoretic variants of hemoglobin in individual erythrocytes were separated by electrophoresis in ultrathin acid agar gels. The electropherograms were stained under a hood with hemoglobin specific chromogen, benzidine. Photomicrographs of the electropherograms were scanned with a recording densitometer. Relative quantities of the respective hemoglobins in individual erythrocytes were estimated by weighing the areas under the densitograph curves. The findings confirm that the intracellular proportions of Hb A and Hb S or Hb A and Hb C in heterozygotes are of heterogeneous distributions within a given population of cells. The tool of single cell electrophoresis may be useful for investigating relative quantities of other electrophoretically separable protein variants in individual erythrocytes.

Heritable variation in erythrocyte membranes of mice with muscular dystrophy.

Several physical and chemical characteristics of erythrocyte membranes from dystrophic mice differ from those of controls. In this study it is postulated that there is a heritable antigenic characteristic in erythrocyte membranes of dystrophic mice that is associated with muscular dystrophy. Accordingly, antisera were prepared in goats against erythrocyte membranes from dystrophic mice. These antisera discriminated between membranes from control mice and membranes from three different strains of dystrophic mice. Preliminary data on carrier detection are consistent with the hypothesis that the antigen is manifest in membranes of carriers.

Chromatographic fractionation of anti-sickling agents in Fagara xanthoxyloides.

Fractionation of an aqueous extract of root bark from Fagara xanthoxyloides by column chromatography on DEAE-A-50, utilizing an elution gradient of pH 7.5-5.0, yielded five fractions. All fractions reversed metabisulfite-induced sickling in vitro of erythrocytes homozygous for hemoglobin S. Only the fraction containing the least anti-sickling activity also contained alkaloids. Thin-layer chromatography on silica gel and aluminum oxide revealed further molecular heterogeneity within two of the fractions.

Identification of inherited protein variants in individual erythrocytes.

Inherited electrophoretic variations of hemoglobin, carbonic anhydrase, and glucose-6-phosphate dehydrogenase in individual erythrocytes were separated by electrophoresis in ultrathin agar gels. By staining the electropherograms with specific fluorescein-conjugated antibodies against hemoglobins, relative proportions of two hemoglobins within individual erythrocytes can be estimated. The findings suggested that the intracellular proportions of HbA and HbS in heterozygotes are heterogeneous within a given population of cells. By this method cells containing hemoglobin F (F cells) as well as a minor variant of hemoglobin F were identified. This tool potentially offers an approach to monitoring distribution of inherited variants in individual erythrocytes for a large number of proteins.

Identification of hemoglobins in single erythrocytes by electrophoresis.

We present here a method for electrophoretic identification of hemoglobin variants in many individual erythrocytes simultaneously, which is made possible by instruments developed in our laboratory. The system spaces and aligns the erythrocytes along a common origin and prevents 'doubling' of cells. Unambiguous separation of different hemoglobins (Hbs) occurred from single erythrocytes, such as Hbs A, C, F. Staged micrographs showed that hemoglobins of differing mobility from a single cell arise from a single point source of hemoglobin. In principle this capability is applicable to other proteins and could facilitate investigation at a cellular level into a variety of questions pertaining to gene action.

Biological variation in the heterogeneous distribution of haemoglobin F among erythrocytes.

The development of highly specific fluorescent labelled antibodies against haemoglobin F presented an opportunity to investigate variables which might influence distribution of this haemoglobin among individual erythrocytes. Earlier investigations revealed heterogeneous distribution within healthy individuals and in individuals with sickle cell disease and other haemoglobinopathies. The current study demonstrates quantitatively that there are normal biological determinants of variability in the frequency of F-containing erythrocytes in individuals who have a haemoglobin A electrophoretic phenotype. It also is shown that the frequency of F-containing erythrocytes during the early recovery phase of sickle cell crisis is significantly higher than when there is no recent history of crisis. The mean quantity of ahemoglobin F per F-containing erythrocyte appears to be lower after a crisis than in individuals without recent history of crisis. This suggests that following a crisis there may either be biochemical constraints on haemoglobin F synthesis per erythrocyte precursor cell or else there is limited opportunity for selective removal of low F-containing erythrocytes from the circulation.

Genetic aspects of quantitative variation in the carbonic anhydrases of the pig-tailed macaque, Macaca nemestrina. I. Response to thyroxine.

Effects of thyroxine on incorporation of L-serine-C14 into four carbonic anhydrase isozymes (CA II, CA Ia, CA Ib, CA Ic) and hemoglobin were quantified in reticulocytes of Macaca nemestrian in vitro. Response to thyroxine differed significantly between CA Ia and two allelic variants (CA Ib and CA Ic) and the nonallelic isozyme (CA II). The effects of thyroxine on serine incorporation into hemoglobin and three of the carbonic anhydrase isozymes were shown to be nonlinear with thyroxine concentration.

Identification of specific hemoglobins within individual erythrocytes.

The identification of specific antibodies for a variety of inherited hemoglobin variants presents the opportunity to develop sensitive immunochemical measurement techniques for such variants. In this study antibodies specific for human hemoglobins A1 and S were isolated from immune rabbit serum. These were labeled with either fluoroscein isothiocyanate or tetramethylrhodamine isothiocyanate. Erythrocytes suspended in agar were incubated with these labeled antibodies and hemoglobins A1, S, and F were identified within individual cells by fluorescence microscopy. This capability offers potential for more specific investigation into heterogenous distribution of specific hemoglobins within an erythroycte or bone marrow cell population and for developing a sensitive technique applicable to antenatal diagnosis of specific hemoglobinopathies.