Abnormal minor human erythrocyte membrane sialoglycoprotein (beta) in association with the rare blood-group antigen Webb (Wb).

Individuals whose erythrocytes are positive for the rare blood-group antigen Webb (Wb) have an altered form of the minor sialoglycoprotein beta (synonyms glycophorin C and glycoconnectin). This altered sialoglycoprotein beta (beta Wb) has an Mr about 2700 lower than that of normal sialoglycoprotein beta. Treatment of normal sialoglycoprotein beta with endo-beta-N-acetylglucosaminidase F decreased its Mr by about 3600, but similar treatment of sialoglycoprotein beta Wb had no effect. These results suggest the possibility that sialoglycoprotein beta Wb lacks the N-glycosidically linked oligosaccharide found on normal sialoglycoprotein beta.     

Membrane proteins in hereditary spherocytosis, elliptocytosis and stomatocytosis

Cell membrane proteins of patients with hereditary spherocytosis, elliptocytosis and stomatocytosis were analyzed by SDS acrylamide gel electrophoresis. From 7 patients with hereditary spherocytosis only one can be supposed to have a reduction of a band with a molecular weight of about 80,000 daltons. 3 members of a family with hereditary elliptocytosis and 1 patient with hereditary stomatocytosis did not differ from the donors. The question of the nature of membrane defect in haemolytic anaemias could not be answered by the results obtained.     

Prevalence of diabetes mellitus in individuals heterozygous and homozygous for sickle cell anemia

Colorimetric determinations of glycosylated Hb were carried out in a sample (n = 97) of sickle cell anemia patients, and in an age- and sex-matched group of individuals (n = 45) heterozygous for sickle cell anemia, from the Eastern Province of Saudi Arabia. A statistically significant increase in the value of glycosylated Hb was found in sickle cell trait (HbAS) group, when compared with those of sickle cell anemia (HbSS) and normal (HbAA) groups. Since glycosylated Hb is considered a valid indicator of long-term blood glucose, and assuming normal red blood cell survival in HbAS carriers, the increased value of glycosylated Hb may suggest that there exists a higher incidence of undiagnosed diabetes mellitus in individuals with heterozygous inheritance for sickle cell hemoglobin than homozygous sickle cell patients and normal individuals. The mechanism underlying this observation remains to be defined.     

Sperm size abnormalities in homozygous and heterozygous In(5)9Rk mice.

Sperm abnormalities were scored in In(5)9Rk homozygotes, heterozygotes and in the inbred strains, C57BL/6J, C3H/HeJ, DBA/2J and JUFaCt, used to produce the geterozygotes. The sperm abnormalities of the inversion heterozygotes were remarkable, about 40% were either double-headed or abnormally large in size. The double-headed sperm had head dimensions similar to those of normal-sized sperm, but the large sperm heads were 1.2 times longer and 1.3 times wider and presumably had twice the usual nuclear volume. This observation suggests that the anaphase bridge formed in inversion heterozygotes resulting from the paracentric inversion may constitute an impediment to cytokinesis and may lead to two genomes being packaged into a single sperm. If this is a general phenomenon, sperm morphology may provide a useful screen for paracentric inversions.     

A new human erythrocyte variant (Ph) containing an abnormal membrane sialoglycoprotein

1. A new human erythrocyte variant (Ph) is described. The variant contains an unusual sialic acid-rich glycoprotein in addition to the blood-group-MN([unk])- and blood-group-Ss(δ)-active sialoglycoproteins found in normal erythrocytes. 2. The unusual component Ph has an apparent mol.wt. of 32000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Ph component is not degraded during trypsin treatment of intact erythrocytes. 3. The Ph component was labelled by lacto-peroxidase-mediated radioiodination of intact erythrocytes and was found to be present in amounts approximately equimolar to α-sialoglycoprotein in the variant erythrocytes. 4. The Ph component had receptors for the lectins from Maclura aurantiaca (osage orange) and Triticum vulgaris (wheat-germ), but lacked a receptor for the Phaseolus vulgaris (red kidney bean) lectin, suggesting that it carries only O-linked oligosaccharides. 5. The presence of the Ph component in these erythrocytes does not correspond to any of the known blood-group-MNSs-related antigens examined. 6. We suggest that this component may be a hybrid polypeptide containing the N-terminal portion derived from normal δ-sialoglycoprotein, and the C-terminal portion from normal α-sialoglycoprotein, in a manner similar to the anti-Lepore haemoglobin.     

Rearrangements of the red-cell membrane glycophorin C (sialoglycoprotein beta) gene. A further study of alterations in the glycophorin C gene.

We have cloned portions of the glycophorin C (sialoglycoprotein beta) gene from individuals with red cells of normal, Gerbich and Yus phenotypes. The clones contain up to three exons of the glycophorin C gene (designated exons 2, 3 and 4). Analysis by restriction mapping and DNA sequencing confirmed that the deletions causing the Gerbich and Yus phenotypes are located entirely within the glycophorin C gene. Sequencing of the normal gene showed that not only do exon 2 and exon 3 have related DNA sequences, but also that both the 5' and 3' flanking intronic DNA sequences are almost identical. The two variant genes each lack a different exon: the Yus type gene lacks exon 2, whereas the Gerbich-type gene lacks exon 3. We suggest that the observed deletions are due to recombination between the regions of homologous intronic repeats. We also provide evidence that an unequal cross-over mechanism may be responsible for a number of observed glycophorin C gene rearrangements, including an insertion mutation in Lewis II (Lsa)-type red cells that has not previously been reported.     

Red cell membrane lipids in hemoglobinopathies

The complex mixture of lipids and proteins of the red blood cell membrane is well maintained during the life of the cell. Lipid analysis of the red cell reveals hundreds of phospholipid molecular species and cholesterol that differ with respect to their (polar) head group, and (apolar) side chains. These molecules move rapidly in the plane, as well as across the lipid bilayer. This dynamic movement is highly organized. In the plane of the bilayer, areas enriched in certain lipids accommodate protein structure and modulate function. While lipids move across the bilayer, the organization is highly asymmetric. Amino phospholipids are mainly found on the inside and choline containing phospholipids on the outside. Both the composition and organization of the red cell membrane is maintained throughout the life of the red cell by an intricate mechanism that involves enzymes, transporters and cytosolic factors. Key proteins that maintain red blood cell lipid organization have recently been identified. Alterations in these mechanisms, as the result of the globin mutations in sickle cell disease or thalassemia will lead to loss of membrane viability, apoptosis during erythropoiesis, early demise of the cell in the circulation, and when these cells are not removed appropriately their presence has pathologic consequences.     

The genetic conditions in heterozygous and homozygous populations of Drosophila. II. X-ray induced lethals in a homozygous and a heterozygous population

Strains of Drosophila melanogaster were made isogenic for their second chromosomes by means of the marker strain LCy/Pm. One of these strains was used as a founder for a homozygous experimental population (W). All other strains were mixed and established a heterozygous population (LKW). Both populations were free of lethals in the beginning with respect to their second chromosomes. After they had been exposed to an X-ray irradiation of 7000 r they contained about 26 per cent newly induced lethal chromosomes. Whereas in the heterozygous population the lethal frequency decreased rather fast to 10 per cent, that of the homozygous population remained rather constant at 25 per cent during a period of 135 days. After a year of continuation, however, both populations reached the same lethal frequency of about 10 per cent. Allelism tests carried out after 10 generations revealed that there was a highly heterotic lethal factor in the homozygous population. After excluding this heterotic lethal from the calculations, the lethal frequencies of the two populations remained significantly different. It was assumed that the relative mean fitness of lethal heterozygotes was generally higher in the homo-than in the heterozygous populations. The results indicate that homozygous populations are much more capable of incorporation new mutations than heterozygous.     

Hemoglobinopathies, G6PD deficiency, and hereditary elliptocytosis in Bahrain

The native population of Bahrain has a high prevalence of hemoglobinopathies and G6PD deficiency, probably as a result of past malarial endemism. We used the Biorad-Variant hemoglobin testing system for primary screening of hemoglobinopathies in 20,000 individuals. Hemoglobin abnormalities were detected in 7,206 (36.3%) cases.     

Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins

Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types.     

Differences in open field behavior between heterozygous and homozygous negative gilts for the RYR(1) gene

A test widely used to assess fear and novelty responses in domestic species is the open field. The aim of this study was to investigate the effect of RYR(1) genotype on open field behavior in growing pigs. The study subjected 15 heterozygous (Nn) and 15 RYR(1)-free (NN) gilts of 19 weeks of age to 3 replicates of an open field test 2 days apart from each other. The study measured the number of grid lines crossed and defecation score in the test arena. There was a significant individual correlation among the 3 replicates of the test, both for number of grid lines crossed and defecation score (p <.05). RYR(1) genotype had a significant effect on number of grid lines crossed, with NN gilts showing more overall activity than Nn gilts (p <.05). The study observed no significant differences in defecation score between genotypes. This result suggests that the RYR(1) genotype may have an effect on the appraisal of novelty. Thus, it would be interesting to take this factor into account when using this methodology to assess fear responses in pigs and in interpreting the results with respect to welfare.     

Amyloid beta(1-42) and its beta(25-35) fragment induce activation and membrane translocation of cytosolic phospholipase A2 in bovine retina capillary pericytes

We investigated changes in cytosolic phospholipase A(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) activities in bovine retina capillary pericytes after stimulation with 50 microM amyloid-beta (Abeta) (1-42) and its (25-35) fragment, over 24 h (mild, sublethal model of cell damage). In the presence of Abeta peptides, we found that cPLA(2) activity was increased and translocated from the cytosolic fraction to the membrane system, particularly in the nuclear region. Reversed-sequence Abeta(35-25) peptide did not stimulate or induce cPLA(2) translocation. Exposure to both Abeta peptides had no significant effect on cPLA(2) protein content as tested by Western immunoblot analysis. The addition of Abetas to quiescent pericytes was followed by phosphorylation of cPLA(2) and arachidonic acid release. Treatment with inhibitors (AACOCF(3), staurosporine and cycloheximide) resulted in a sharp decrease in basal and stimulated cPLA(2) activity. Inactivating effects of bromoenol lactone (BEL), inhibitor of iPLA(2), demonstrated that the stimulation of total PLA(2) activity by Abetas was mediated by both PLA(2) enzymes. Taken together with our previous observations that both Abeta peptides may induce hydrolysis of phosphatidylcholine, the present results provide evidence that this process is cooperatively mediated by cPLA(2) activation/translocation and iPLA(2) activation. The effect is very likely triggered by a mild prooxidant mechanism which was not able to divert the cell to degeneration. The data confirm the hypothesis that pericytes could be a target of potential vascular damage and reactivity during processes involving amyloid accumulation.     

Hyposensitivity to the amnesic effects of scopolamine or amyloid beta(25-35) peptide in heterozygous acetylcholinesterase knockout (AChE(+/-)) mice

We examined the sensitivity of AChE(+/-) mice to the amnesic effects of scopolamine and amyloid beta peptide. AChE(+/-) and AChE(+/+) littermates, tested at 5-9 weeks of age, failed to show any difference in locomotion, exploration and anxiety in the open-field test, or in-place learning in the water-maze. However, when treated with the muscarinic receptor antagonist scopolamine (0.5, 5mg/kg s.c.) 20min before each water-maze training session, learning impairments were observed at both doses in AChE(+/+) mice, but only at the highest dose in AChE(+/-) mice. The central injection of Abeta(25-35) peptide (9nmol) induced learning deficits only in AChE(+/+) but not in AChE(+/-) mice. Therefore, the hyper-activity of cholinergic systems in AChE(+/-) mice did not result in increased memory abilities, but prevented the deleterious effects of muscarinic blockade or amyloid toxicity.     

The membrane change in En(a-) human erythrocytes. Absence of the major erythrocyte sialoglycoprotein.

We investigated the membrane of En(a-) human erythrocytes as part of a study of the structure and biochemical function of the surface glycoproteins of the mammalian cell. 2. En(a-) erythrocytes were selected because they have more extensive changes at the cell surface than any other known erythrocyte variant. 3. Our results show that in En(a-) erythrocytes: (a) the major membrane sialoglycoprotein is lacking; (b) the other major membrane-penetrating glycoprotein (band 3) has an altered electrophoretic mobility. 4. The apparent clinical normality of En(a-) cells suggests that the change in band 3 may compensate for the loss of the membrane sialoglycoproteins. It is clear that a viable erythrocyte can exist despite the absence of one of its major surface components.     

Genetic variants of human red-cell membrane sialoglycoprotein beta. Study of the alterations occurring in the sialoglycoprotein-beta gene.

We have studied the DNA of individuals who express an altered sialoglycoprotein beta on their red cells by using Southern blotting with sialoglycoprotein-beta cDNA probes. Individuals of the Leach phenotype do not express any beta (sialoglycoprotein beta) or gamma (sialoglycoprotein gamma) on their red cells, and we show that about 7 kb of DNA, including the 3' end of the beta gene, is deleted in this DNA. Any protein product of this gene is likely to lack the membrane-associating domain of beta. We have also examined the DNA of two types of other individuals (Yus-type and Gerbich-type) who have red cells that lack beta and gamma, but contain abnormal sialoglycoproteins related to beta. These two types of DNA contain different internal deletions of about 6 kb in the beta gene. We suggest that these deletions result from the presence of two different sets of internal homology in the beta gene, and on this basis we propose structures for the abnormal Yus-type and Gerbich-type sialoglycoproteins which are consistent with the other evidence that is available. We provide evidence that beta and gamma are products of the same gene and suggest a possible mechanism for the origin of gamma based on leaky initiation of translation of beta mRNA.