Human erythrocyte protein 4.2 deficiency associated with hemolytic anemia and a homozygous 40glutamic acid-->lysine substitution in the cytoplasmic domain of band 3 (band 3Montefiore)

Red blood cell (RBC) protein 4.2 deficiency is often associated with a moderate nonimmune hemolytic anemia, splenomegaly, and osmotically fragile RBCs resembling, but not identical to, hereditary spherocytosis (HS). In the Japanese type of protein 4.2 deficiency (protein 4.2Nippon), the anemia is associated with a point mutation in the protein 4.2 cDNA. In this report, we describe a patient with moderate and apparently episodic nonimmune hemolytic anemia with splenomegaly, spherocytosis, osmotically fragile RBCs, reduced whole cell deformability, and abnormally dense cells. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis of the proposita's RBC membrane proteins showed an 88% deficiency of protein 4.2 and a 30% deficiency of glyceraldehyde-3-phosphate dehydrogenase (band 6). Structural and molecular analyses of the proposita's protein 4.2 were normal. In contrast, limited tryptic digestion of the proposita's band 3 showed a homozygous abnormality in the cytoplasmic domain. Analysis of the pedigree disclosed six members who were heterozygotes for the band 3 structural abnormality and one member who was a normal homozygote. Direct sequence analysis of the abnormal band 3 tryptic peptide suggested that the structural abnormality resided at or near residue 40. Sequence analysis of the proposita's band 3 cDNA showed a 232G-->A mutation resulting in a 40glutamic acid-->lysine substitution (band 3Montefiore). Allele-specific oligonucleotide hybridization was used to probe for the mutation in the pedigree, showing that the proposita was homozygous, and the pedigree members who were heterozygous for the band 3 structural abnormality were also heterozygous for the band 3Montefiore mutation. The band 3Montefiore mutation was absent in 26 chromosomes from race-matched controls and in one pedigree member who did not express the band 3 structural abnormality. In coincidence with splenectomy, the proposita's anemia was largely corrected along with the disappearance of most spherocytes and considerable improvements of RBC osmotic fragility, whole cell deformability, and cell density. We conclude that this hereditary hemolytic anemia is associated with the homozygous state for band 3Montefiore (40glutamic acid-->lysine) and a decreased RBC membrane content of protein 4.2. We speculate that band 3 structural abnormalities can result in defective interactions with protein 4.2 and band 6, and in particular, that the region of band 3 containing 40glutamic acid is involved directly or indirectly in interactions with these proteins.     

Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease

The genetic defect in the p67phox-deficient form of chronic granulomatous disease (CGD) follows an autosomal recessive pattern of inheritance. When genomic DNA from normal individuals is digested with HindIII and probed with p67phox cDNA an allelic restriction fragment length polymorphism (RFLP) of 4.0 kb or 2.3 kb is detected. We cloned and characterized the p67phox gene using the cDNA and sequenced the exon/intron boundaries, mapping 16 exons on the 40-kb gene. The polymorphic region was then sequenced to identify the inheritance pattern of amniocentesis-derived fetal cells by genomic amplification. The proband, a 9-year-old female patient with p67phox-deficient CGD, and her phenotypically normal mother are homozygous for the RFLP marker, whereas the father and two brothers are heterozygous. The fetus was shown to be heterozygous as well, showing it had inherited at least one normal p67phox gene from the father and that it was predicted to have a normal phenotype. Cord blood samples at birth showed normal oxidative function. Amplification allows rapid detection of the inheritance pattern for fetal diagnosis in informative families. We report the genomic structure of p67phox and an amplification-based method for detection of the marker on chromosome 1q25, used here for prenatal diagnosis of CGD.     

Sickle erythrocyte-endothelial interactions in microcirculation: the role of von Willebrand factor and implications for vasoocclusion

To determine the role of von Willebrand factor (vWF) in adhesion of sickle (SS) erythrocytes in microvascular flow conditions, we have perfused the ex vivo mesocecum vasculature of the rat with desmopressin, an analogue of vasopressin that causes the release of endothelial vWF. Analysis of vWF in the venous effluent of the isolated vasculature showed mainly the presence of extra-large molecular weight forms characteristic of endothelial vWF, which in the presence of desmopressin showed an average increase of 54%. Also, desmopressin induced a significant increase in adhesion of washed oxygenated (oxy) unseparated SS erythrocytes, accompanied by a persistent microvascular obstruction and a pronounced increase in the peripheral resistance (PRU). In contrast, infusion of SS deformable discocytes (SS2) in desmopressin-perfused vasculature resulted in a significant adhesion but not in persistent vasoocclusion, showing that SS2 discocytes alone are not sufficient for microvascular obstruction. Furthermore, SS4 erythrocytes (dense discocytes and irreversibly sickled erythrocytes) caused a persistent microvascular blockage and a significantly higher PRU than SS2 discocytes. However, the increase in PRU for SS4 erythrocytes following desmopressin treatment was 50% less compared with a corresponding increase for SS2 discocytes over the control values, which showed a smaller effect of desmopressin on the hemodynamic behavior of SS4 dense erythrocytes. Incubation of desmopressin-treated vasculature with anti-vWF antibodies resulted in a pronounced decrease in adhesion and significantly improved hemodynamic behavior of SS cells. Also, in untreated vasculature, similarly incubated with anti-vWF antibodies, there was almost complete inhibition of adhesion. Under the described perfusion conditions, antibodies to fibronectin and thrombospondin, as well as incubation of SS erythrocytes with anti-vWF antibodies did not affect adhesion. These results are compatible with a model for SS vasoocclusion in which extra- large vWF-mediated adhesion of deformable SS erythrocytes is the first step followed by an accelerated entrapment of dense SS erythrocytes.     

A Medical Curriculum for all seasons - a dialogue

Ten years have now passed since the College of Medicine of the University of Malawi was opened. The College''s Curriculum is firmly based on community needs. We describe the genesis and implementation of the curriculum of the College of Medicine and how it has persisted over the past ten years. The challenges that have so far been met are outlined.     

Platelet-derived growth factor promotes polymorphonuclear leukocyte activation

The platelet-derived growth factor (PDGF) has several well defined important biologic activities. Platelet-derived growth factor is the major mitogen in human serum for cells of mesenchymal origins; it is a potent chemoattractant protein for human monocytes, neutrophils, fibroblasts, and smooth muscle cells; and has been implicated in transformation by simian sarcoma virus and perhaps in transformation by other agents as well. In this article, PDGF has been shown to stimulate activation of human peripheral blood neutrophils defined by loss of membrane associated calcium as reflected by loss of chlortetracycline fluorescence, release of superoxide anion and specific granule enzymes, and enhanced neutrophil adherence and aggregation. These responses occurred in a dose-dependent fashion at concentrations of PDGF between 10 ng/mL (0.4 nmol/L) and 40 ng/mL (1.5 nmol/L) and were comparable to effects obtained with optimal concentrations of fMLP and C5a. Degranulation induced by PDGF was selective for secondary (specific) granules and not primary (azurophil) granules. Platelet-derived growth factor thus is ideally suited for a pivotal role in attracting inflammatory cells locally and initiating neutrophil activation at sites of blood vessel injury. Platelet-derived growth factor or a closely related protein also may play an important role in attracting and activating neutrophils in association with inflammatory tumors.     

Molecular and cellular effects of antisickling concentrations of alkylureas

Alkylureas are capable of inhibiting sickling in vitro and the gelation of solutions of hemoglobin S at concentrations between 0.05 and 0.1 M with increasing effectiveness that is directly proportional to the length of the alkyl chain (butyl greater than propyl greater than ethyl greater than methyl). 6The inhibitory effect is independent of pH between 6.5 and 7.5 and is a process driven by entropy. The alkylureas at concentrations of 0.1 M have minimal effects on several erythrocyte functions. Oxygen equilibria, osmotic fragility, reduced glutathione content, and glutathione reductase activity are totally unaffected, while pyruvic kinase activity is decreased only by butylurea by about 20%, and glucose-6-phosphate dehydrogenase activity is decreased progressively to a maximum of 30% in direct proportion to the length of the alkyl chain. Alkylureas not only inhibit sickling but are also capable of desickling erythrocytes that have been maintained in the deoxygenated state. They have little effect on several erythrocyte functions at antisickling concentrations, but their toxicity must be evaluated before they can be examined as potential therapeutic agents for the treatment or prevention of acute episodes in sickle cell anemia.     

Signal transduction and the regulation of actin conformation during myeloid maturation: studies in HL60 cells

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl- L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F- actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F- actin at 48 to 72 hours of maturation. The PKC inhibitor 1-5- isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F- actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F- actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a "mature" response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.     

Associations of combining paid work and family care with gender-specific differences in depressive symptoms among older workers and the role of work characteristics

ObjectivesThis study aims to provide insight into (i) how the combination of paid work and family care is longitudinally associated with gender-related differences in depressive symptoms and (ii) the role of work characteristics in this association.MethodsData were derived from STREAM, a Dutch prospective cohort study of older workers aged 45–64 years. Respondents were included if they were employed in at least one measurement between 2015 and 2017 (N=12 447). Mixed-models were applied to disentangle between-person (BP) and within-person (WP) effects of family caregiving on depressive symptoms. Analyses were stratified by gender. Work characteristics (social support, autonomy, emotional and mental workload) were separately added to the multivariable models.ResultsFor older employees, family caregiving was positively associated with depressive symptoms between and within persons for both women [BP B=0.80, 95% confidence interval (CI) 0.52–1.08; WP B=0.32, 95% CI 0.08–0.56] and men (BP B=0.75, 95% CI 0.45–1.05; WP B=0.25, 95% CI 0.01–0.48). Social support at work reduced the adverse effect of family care on depressive symptoms for women (BP) and men (BP and WP). Emotional workload partly explained the effect of family care for both women and men (BP).ConclusionsThe longitudinal association between family care and mental health was similar for male and female employees. Resources at work (ie, social support) could protect caregiving employees against depressive symptoms. More research is needed regarding the relative impact of the care context compared to the work context of working family caregivers.