Molecular basis of altered red blood cell membrane properties in Southeast Asian ovalocytosis: role of the mutant band 3 protein in band 3 oligomerization and retention by the membrane skeleton

Southeast Asian ovalocytosis (SAO) is an asymptomatic trait characterized by rigid, poorly deformable red cells that resist invasion by several strains of malaria parasites. The underlying molecular genetic defect involves simple heterozygous state for a mutant band 3 protein, which contains a deletion of amino acids 400 through 408, linked with a Lys 56-to-Glu substitution (band 3-Memphis polymorphism). To elucidate the contribution of the mutant SAO band 3 protein to increased SAO red blood cell (RBC) rigidity, we examined the participation of the mutant SAO band 3 protein in increased band 3 attachment to the skeleton and band 3 oligomerization. We found first that SAO RBC skeletons retained more band 3 than normal cells and that this increased retention preferentially involved the mutant SAO band 3 protein. Second, SAO RBCs contained a higher percentage of band 3 oligomer-ankyrin complexes than normal cells, and these oligomers were preferentially enriched by the mutant SAO protein. At the ultrastructural level, the increased oligomer formation of SAO RBCs was reflected by stacking of band 3-containing intramembrane particles (IMP) into longitudinal strands. The IMP stacking was not reversed by treating SAO RBCs in alkaline pH (pH 11), which is known to weaken ankyrin-band 3 interactions, or by removing the cytoplasmic domain of band 3 from SAO membranes with trypsin. Finally, we found that band 3 protein in intact SAO RBCs exhibited a markedly decreased rotational mobility, presumably reflecting the increased oligomerization and the membrane skeletal association of the SAO band 3 protein. We propose that the mutant SAO band 3 has an increased propensity to form oligomers, which appear as longitudinal strands of IMP and exhibit increased association with membrane skeleton. This band 3 oligomerization underlies the increase in membrane rigidity by precluding membrane skeletal extension, which is necessary for membrane deformation.     

Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes.

Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). We report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambda gt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. Their pI values overlapped in 2-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and showed the same response to delipidation. Either polypeptide reacted with and blocked the antiserum raised against the other polypeptide. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens.     

Exome Sequencing Reveals RAG1 Mutations in a Child with Autoimmunity and Sterile Chronic Multifocal Osteomyelitis Evolving into Disseminated Granulomatous Disease

We describe a boy who developed autoinflammatory (chronic sterile multifocal osteomyelitis) and autoimmune (autoimmune cytopenias; vitiligo) phenotypes who subsequently developed disseminated granulomatous disease. Whole exome sequencing revealed homozygous RAG1 mutations thus expanding the spectrum of combined immunodeficiency with autoimmunity and granuloma that can occur with RAG deficiency.     

Exotic and indigenous problem plants species used,by the Bapedi,to treat sexually transmitted infections in Limpopo Province,South Africa

Background

The ethnic usage of exotics and indigenous problem plants is a highly debated topic, as legislative requirements over-shadow their potential medicinal value, particularly to treat sexually transmitted infections (STIs). Limited information exists regarding their medicinal value among the Bapedi.

Objectives

To ascertain the importance of exotics and indigenous problem plants in the treatment of STIs, a major global primary health care challenge.

Methods

A field observation and semi-structured questionnaire focussing on species diversity, types of STIs treated and medicinal preparation as well as application was used to collect data from 34 traditional healers.

Results

Seven exotics and three indigenous problem species were identified. These species were used to treat four STIs; with Catharanthus roseus illustrating its dominance in the treatment of gonorrhoea. Some medicinal species used by Bapedi traditional healers have been validated through scientific research or through their extensive use by various cultures in South Africa and other parts of Africa. To the best of our knowledge Alternanthera pungens, Caesalpinia decapetala, Cinnamomum verum, and Citrullus lanatus are reported for the first time in the treatment of the investigated STIs.

Conclusions

Exotic and indigenous problem species constitute an important component of the STIs treatment protocol. Their utilization by Bapedi cautions against the narrow-minded approach of indiscriminate eradication, as these species can play a significant role in the primary health care needs of socio-economic vulnerable people.     

Different roles of nitric oxide synthase isoforms in cardiopulmonary resuscitation in pigs

The purpose of the present study was to identify the roles of the three nitric oxide synthase (NOS) isoforms on whole body ischemia-reperfusion injury during cardiopulmonary resuscitation (CPR) with periodic acceleration (pGz) in pigs. Thirty-two anesthetized pigs (27.6+/-3.4 kg) were monitored for hemodynamics and selected echocardiographic variables. Twenty minutes after NOS inhibition or placebo administration, ventricular fibrillation (VF) was induced and remained untreated for 3 min, followed by CPR with pGz for 15 min, plus 3 min of manual chest compressions and defibrillation attempt. Four groups were studied: (1) saline control; (2) L-NAME (non-selective NOS inhibitor); (3) aminoguanidine (inducible NOS inhibitor); (4) TRIM (neuronal NOS inhibitor). Return of spontaneous circulation (ROSC) to 180 min occurred in 6/8 controls, 4/8 L-NAME, 7/8 aminoguanidine, and 2/8 TRIM animals. The L-NAME group had significantly lower organ blood flow, impaired cardiac function, but higher vascular tone than control group. The aminoguanidine group had the highest organ blood flows and survival rate. Six out of eight TRIM treated animals had initial return of heartbeat; however, with impaired heart contractility and could not survive more than 20 min of ROSC. This study reveals the differential role of endogenous NO produced from the three NOS isoforms during pGz-CPR. Both endothelial and neuronal NOS derived NO show predominantly protective effects while inducible NOS derived NO plays a detrimental role in pGz-CPR. The present study has shown that cardiac arrest and resuscitation appears to be associated with a different expression of NOS isoforms which appear to affect resuscitation outcomes differently.     

Nitric oxide synthase isoform inhibition before whole body ischemia reperfusion in pigs: Vital or protective?

BACKGROUND: Nitric oxide (NO) is a critical regulator of vascular tone, and signal transduction. NO is produced via three unique synthases (NOS); endothelial (eNOS), and neuronal (nNOS) are both constitutively expressed and inducible (iNOS) produced primarily after stimulation. NO has been implicated during and after ischemia reperfusion injury as both a detrimental and cardioprotective mediator. Since cardiopulmonary resuscitation (CPR) in ventricular fibrillation (VF) is a model of whole body ischemia reperfusion injury, it provides an opportunity to assess the effects of NO from the three NOS isoforms. OBJECTIVE: To determine the differential role of nitric oxide synthase isoforms inhibition in ventricular fibrillation CPR and investigate whether inhibition of the NOS isoforms afford any cardioprotection in this model. METHODS: Thirty-two pigs, weight range 25-35 kg, were assigned to four groups of eight animals each. The animals were randomized to receive (1) N(G)-nitro-L-arginine methyl ester (LNAME), a non-selective endothelial nitric oxide synthase inhibitor, (2) 1-(2-trifluoromethylphenyl) imidazole (TRIM), a selective neuronal NOS inhibitor, (3) aminoguanidine (AMINOG), a selective inducible NOS inhibitor or (4) saline control (Control) in equal volumes, 30 min before induction of ventricular fibrillation (VF). After 3 min VF with no intervention, the animals received standard chest compressions using an automated chest compression device (Thumper) for 15 min. After 18 min of VF, single doses of vasopressin and bicarbonate were given and defibrillation attempted. Hemodynamics, regional blood flows, and echocardiography and were performed, before and after drug infusion, during CPR, and after return of spontaneous circulation (ROSC). RESULTS: ROSC for 3 h occurred in 5/8 (63%), 1/8 (13%), 0/8 (0%), and 6/8 (75%) in Control, LNAME, TRIM, and AMINOG treated animals, respectively. After infusion of LNAME, there was a significant increase from baseline in blood pressure [127+/-6 mmHg versus 169+/-3 mmHg, p<0.002] and coronary perfusion pressure [119+/-6 mmHg versus 149+/-6 mmHg, p<0.003]. During CPR, there were no differences among groups in hemodynamics or regional blood flow. In surviving animals, AMINOG had significantly better myocardial function (left ventricular ejection fraction, fractional shortening, and wall motion score index) than control or LNAME treated animals, and attenuated the post-resuscitation hyperemic response in heart and brain. CONCLUSIONS: Intact basal nNOS activity is vital for survival from whole body ischemia reperfusion injury. iNOS inhibition prior to ischemia reperfusion, protects myocardial function after ROSC and decreases myocardial and brain hyperemic response after ROSC.