Hemoglobin Beta Chain Research Articles

RYH: A minimal peptidic sequence obtained from beta-chain hemoglobin exhibiting an antimicrobial activity

Bovine hemoglobin is an animal protein described as source of bioactive peptides. Enzymatic hydrolysis of this protein results into some peptides exhibiting antimicrobial activity against Gram-positive and Gram-negative bacteria. In this study, a family of peptides from the beta chain (beta-114-145 derived peptides) obtained by peptic hydrolysis of bovine hemoglobin, was purified by reverse-phase HPLC and characterized by different analytical techniques (mass spectrometry, circular dichroism). The minimum inhibitory concentration was determined to show the antimicrobial activity of these peptides. Four bacterial strains were used: two Gram-negative (Escherichia coli and Salmonella Enteritidis) and two Gram-positive strains (Listeria innocua and Micrococcus luteus). The effect of these peptides on artificial membrane was also measured. Our findings showed that the peptide β114-145 and its peptic derivatives contain the RYH sequence. The most antimicrobial peptide is the RYH peptide which was the shortest one.

Recent Insights on the Medicinal Chemistry of Sickle Cell Disease

Sickle Cell Disease (SCD) is one of the most prevalent hematological diseases in the world. SCD is a genetic disease characterized by punctual mutation that basis on the exchange of glutamic acid to valine in a beta chain of hemoglobin. In deoxygenated state, the interaction among the beta chains leads to hemoglobin polymerization carrying out to deformation of cytoskeleton structure of red blood cells to a sickle shape. Currently, the treatment is performed with the antineoplasic drug hydroxyurea. This review summarizes current knowledge about possible targets and the approaches to discover new compounds to treat the SCD symptoms. Drug design based on therapeutical application and molecular modifications strategies will be discussed.

A toxicogenomic approach for identifying biomarkers for myelosuppressive anemia in rats

Myelosuppressive anemia is a serious side effect associated with several drugs. Thus, there is an increasing demand for sensitive biomarkers for the early detection of myelosuppressive anemia during toxicological studies. We applied a toxicogenomic approach to identify useful biomarker genes reflecting myelosuppressive anemia in the rat liver. Expression of the hemoglobin beta chain complex ( Hbb), aminolevulinic acid synthase 2 ( Alas2), and cell division cycle 25 homolog B ( Cdc25b) genes changed as a result of anemia induced by the myelosuppressive agents linezolid, cisplatin, and carboplatin, suggesting that these genes may be suitable biomarkers. Moreover, evaluation of perfused and unperfused livers indicated that changes in the expression of these genes originate in circulating reticulocytes in the liver. Erythroid differentiation-associated changes in expression of the Hbb, Alas2, and Cdc25b genes were confirmed in vitro using Friend leukemia cells. In conclusion, our current research provides novel evidence that gene expression in circulating reticulocytes contained in the liver changes dramatically under myelosuppressive conditions. While further large-scale validation studies are needed, our results indicate that the genes we identified might be useful biomarkers for the sensitive detection of myelosuppressive anemia in rats.

Glutathionyl Hemoglobin Has Potential as a Biomarker of Iron Deficiency Anemia

AbstractAbstract 3210 Introduction:Iron deficiency anemia (IDA) is a major health problem worldwide. Although a clinical diagnosis is relatively simple, specific laboratory markers of IDA are lacking especially in the setting of inflammation. Ferritin, the current standard to define IDA is an acute phase protein that is non-specifically elevated during inflammation. Serum transferrin receptor level measurements although available are not yet standardized as a clinical tool. Serum hepcidin is a recently developed novel marker that is currently neither available nor standardized sufficiently. Furthermore, such assays require instrumentation, technical sophistication, and are expensive. Objectives:We sought to identify novel markers of IDA using mass spectrometry based proteomics. Identifying such markers could yield targets that once validated could serve cost effective point of care assays to detect iron deficiency anemia. Since there is evidence for oxidative damage mediated by reactive oxygen species in IDA, as a first step, we characterized and quantified posttranslational oxidative modifications of hemoglobin and tested their utility as biomarkers. Patients and Methods:We prospectively enrolled patients with IDA (defined as ferritin <12ng/ml in the presence of normal CRP and/or a bone marrow aspirate with “0” iron stores) and healthy controls (n = 23 and 15 respectively). Patients with diabetes, cardiovascular disease, renal disease, cerebrovascular disease and liver disease were excluded as these are conditions associated with preexisting oxidant stress. Erythrocytes from the blood of IDA patients and controls were isolated by centrifugation, washed in 0.9% saline, and lysed in distilled water to yield intracellular hemoglobin. Hemoglobin was then either studied further as an intact molecule or after digestion with trypsin. We used matrix assisted laser desorption ionization (MALDI - TOF) mass spectrometry to identify oxidative modifications of tryptic digested hemoglobin. We used electro spray ionization (ESI) mass spectrometry to identify and semiquantitate oxidative hemoglobin modifications by methods previously established and published by others and ourselves. Results:Using a combination of mass spectrometric methods, we identified 4 oxidative modifications of hemoglobin in patients with IDA and healthy controls (Table 1). Interestingly, a non enzymatic posttranslational modification of hemoglobin, glutathionyl hemoglobin, was found to be significantly increased in IDA patients compared with healthy controls (Glutathionyl hemoglobin % of beta chain; mean ± SD 0.169 ± 0.096 vs 0.077 ± 0.037; p = 0.001). Markers of oxidative stress (reduced RBC glutathione) were lower in IDA compared to healthy controls but the difference was not significant (mean ± SD 0.92 ± 0.53 vs 1.08 ± 0.52 mmol/L; p = 0.54). Glutathionyl hemoglobin levels correlated inversely with serum ferritin (Spearman rho -0.485; p < 0.05).Table 1Oxidative modifications of specific amino acid residues on tryptic digested peptides of alpha and beta chains of hemoglobin obtained from healthy controls and patients with iron deficiency anemia. AA, amino acidMALDI analysisAA modifiedControl(n = 15)IDA (n = 23)alpha chain 1071 oxidation to 1087Methionine15/1522/23alpha chain 1529 oxidation to 1545Tyrosine15/1517/23Beta chain 1274 oxidation to 1290Tryptophan15/1523/23Beta chain 2060 oxidation to 2076Methionine15/1523/23 Conclusions:Using two distinct proteomic methods, we identified oxidative posttranslational modifications of hemoglobin in IDA and healthy controls. Glutathionyl hemoglobin, an established marker of oxidative stress was elevated in patients with IDA and correlated inversely with serum ferritin. Overall, these findings suggest that glutathionyl hemoglobin has potential as a biomarker of IDA. Disclosures:No relevant conflicts of interest to declare.

Allylation of Intraerythrocytic Hemoglobin by Raw Garlic Extracts

Recent studies have shown that deoxygenated human red blood cells (RBCs) converted garlic-derived polysulfides into hydrogen sulfide, which in turn produced vasorelaxation in aortic ring preparations. The vasoactivity was proposed to occur via glucose- and thiol-dependent acellular reactions. In the present study, we investigated the interaction of garlic extracts with human deoxygenated RBCs and its effect on intracellular hemoglobin molecules. The results showed that garlic extract covalently modified intraerythrocytic deoxygenated hemoglobin. The modification identified consisted of an addition of 71 atomic mass units, suggesting allylation of the cysteine residues. Consistently, purified human deoxyhemoglobin reacted with chemically pure diallyl disulfide, showing the same modification as garlic extracts. Tandem mass spectrometry analysis demonstrated that garlic extract and diallyl disulfide modified hemoglobin's beta-chain at cysteine-93 (beta-93C) or cysteine-112 (beta-112C). These results indicate that garlic-derived organic disulfides as well as pure diallyl disulfide must permeate the RBC membrane and modified deoxyhemoglobin at beta-93C or beta-112C. Although the physiological role of the reported garlic extract-induced allyl modification on human hemoglobin warrants further study, the results indicate that constituents of natural products, such as those from garlic extract, modify intracellular proteins.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides

BackgroundHemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins.ResultsAn aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'.ConclusionsBmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

Hydrophobicity Patterns and Biological Adaptation: An Exemplary Case from Fish Hemoglobins

The dissection of phylogenetic and environmental components in biological evolution is one of the main themes of general biology. Here we propose an approach to this theme relying upon the comparison between a phylogenetic oriented metrics spanning the hemoglobin beta chains of different fishes and a more physiologically oriented metrics defining the same sequences in terms of the dynamical features of their hydrophobic distributions. By analyzing the set of sequences more similar to the Gadus morhua (Atlantic cod) hemoglobin beta chain, we were able to give a proof of concept of the possibility to discriminate the phylogenetic and environmental (evolutive convergence) components by the comparative analysis of the Clustal W (phylogenetics first) and Recurrence Quantification Analysis (physiology first) metrics in which the sequences were embedded. The use of a molecular system like hemoglobin playing a crucial role in fishes adaptation to environmental cues allowed us to span different levels of biological variability by means of the same paradigm. Starting from the reconstruction of the general taxonomy of vertebrate groups we went down to the exploitation of the peculiar role played by Met55Val and Lys62Ala polymorphisms in the beta1 hemoglobin chain of the Atlantic cod able to influence the geographical distribution of its various stocks.

Beta-thalassemia

Beta-thalassemias are a group of hereditary blood disorders characterized by anomalies in the synthesis of the beta chains of hemoglobin resulting in variable phenotypes ranging from severe anemia to clinically asymptomatic individuals. The total annual incidence of symptomatic individuals is estimated at 1 in 100,000 throughout the world and 1 in 10,000 people in the European Union. Three main forms have been described: thalassemia major, thalassemia intermedia and thalassemia minor. Individuals with thalassemia major usually present within the first two years of life with severe anemia, requiring regular red blood cell (RBC) transfusions. Findings in untreated or poorly transfused individuals with thalassemia major, as seen in some developing countries, are growth retardation, pallor, jaundice, poor musculature, hepatosplenomegaly, leg ulcers, development of masses from extramedullary hematopoiesis, and skeletal changes that result from expansion of the bone marrow. Regular transfusion therapy leads to iron overload-related complications including endocrine complication (growth retardation, failure of sexual maturation, diabetes mellitus, and insufficiency of the parathyroid, thyroid, pituitary, and less commonly, adrenal glands), dilated myocardiopathy, liver fibrosis and cirrhosis). Patients with thalassemia intermedia present later in life with moderate anemia and do not require regular transfusions. Main clinical features in these patients are hypertrophy of erythroid marrow with medullary and extramedullary hematopoiesis and its complications (osteoporosis, masses of erythropoietic tissue that primarily affect the spleen, liver, lymph nodes, chest and spine, and bone deformities and typical facial changes), gallstones, painful leg ulcers and increased predisposition to thrombosis. Thalassemia minor is clinically asymptomatic but some subjects may have moderate anemia. Beta-thalassemias are caused by point mutations or, more rarely, deletions in the beta globin gene on chromosome 11, leading to reduced (beta+) or absent (beta0) synthesis of the beta chains of hemoglobin (Hb). Transmission is autosomal recessive; however, dominant mutations have also been reported. Diagnosis of thalassemia is based on hematologic and molecular genetic testing. Differential diagnosis is usually straightforward but may include genetic sideroblastic anemias, congenital dyserythropoietic anemias, and other conditions with high levels of HbF (such as juvenile myelomonocytic leukemia and aplastic anemia). Genetic counseling is recommended and prenatal diagnosis may be offered. Treatment of thalassemia major includes regular RBC transfusions, iron chelation and management of secondary complications of iron overload. In some circumstances, spleen removal may be required. Bone marrow transplantation remains the only definitive cure currently available. Individuals with thalassemia intermedia may require splenectomy, folic acid supplementation, treatment of extramedullary erythropoietic masses and leg ulcers, prevention and therapy of thromboembolic events. Prognosis for individuals with beta-thalassemia has improved substantially in the last 20 years following recent medical advances in transfusion, iron chelation and bone marrow transplantation therapy. However, cardiac disease remains the main cause of death in patients with iron overload.

Molecular Cloning and Characterization of AdultSparus aurataHemoglobin Genes

Among Teleosts, Sparus aurata occupies a prominent place in the gastronomic and economic fields of the Mediterranean basin and other geographic districts. The knowledge of its molecular structures and functional features, such as hemoglobin, may be helpful to understand the adaptive biochemical mechanisms that allow this fish to live under extreme conditions, including fish farming. In Sparus aurata red blood cells two different alpha and one beta hemoglobin genes have been identified. The alpha1 gene codifies a putative protein of 144 amino acids, the alpha2 gene produces a protein of 143 amino acids, and the beta gene encodes a chain of 148 amino acids. Comparative analysis of various hemoglobins indicates that allosteric regulation can be modified by the substitution of one or a few key residues. The comparison of the percentage sequence differences for alpha and beta chains in fishes indicates that evolutionary relationships between different species may be helpful to understand the mechanisms of their differentiation from other vertebrates. Hemoglobin alpha and beta chains of about 50 teleostean temperate and Antarctic fishes were analyzed to build phylogenetic trees using different algorithms: the neighbor-joining method, the maximum likelihood approach, and the Bayesian inference computation. Sparus aurata alpha chains are positioned in a paraphyletic cluster, which includes the same subunit of Chrysophrys auratus and Seriola quinqueradiata, whereas the beta chain is on an homophyletic branch with that of Chrysophrys auratus. Therefore, the phylogenetic approach suggests that both Sparus aurata hemoglobin alpha genes are paralogues and may have derived from a duplication event.

Haemoglobinopathy and sickle cell disease

Sickle cell disease (SCD), first described in the early twentieth century, is an inherited haemoglobinopathy resulting from a mutation on chromosome 11. The mutation causes an amino acid substitution on the b-globin subunit of normal adult haemoglobin A, resulting in the formation of haemoglobin S. Haemoglobin S is biochemically unstable and can precipitate out of solution when in the deoxygenated state, forming the pointed, slightly curved ‘sickle cells’ (Fig. 1). The greater the proportion of haemoglobin S in the cell, the greater is the propensity to sickle. The heterozygous carrier state or sickle cell trait results in the production of both haemoglobin A and S (usually 30–40% HbS), which has a predominantly benign clinical picture as the cells only sickle under extraordinary physiological conditions. Sickle cell trait provides some protection against the consequences of Plasmodium falciparum malaria. The homozygous state (with near 100% HbS) results in SCD. This is a debilitating disease characterized by chronic haemolytic anaemia, recurrent intermittent vaso-occlusion and severe pain, and progressive organ damage and early death. Survival is rare beyond the fifth decade. The HbS gene is found primarily in Africa and south-west Asia, but in light of population migration, is becoming a worldwide phenomenon. The thalassaemias are a group of hereditary anaemias caused by defective synthesis of the alpha chain (alpha thalassaemias) or the beta chain (beta thalassaemias) of haemoglobin. Heterozygotes have mild anaemia, whereas homozygotes have severe anaemia. Unbalanced synthesis of the alpha and beta chains leads to unstable haemoglobin and early red cell death, usually in the marrow. There may be a compensatory extramedullary haematopoiesis. Haemoglobin

Sickle cell disease: Selected aspects of pathophysiology

Sickle cell disease (SCD), a genetically-determined pathology due to an amino acid substitution (i.e., valine for glutamic acid) on the beta-chain of hemoglobin, is characterized by abnormal blood rheology and periods of painful vascular occlusive crises. Sickle cell trait (SCT) is a typically benign variant in which only one beta chain is affected by the mutation. Although both SCD and SCT have been the subject of numerous studies, information related to neurological function and transfusion therapy is still incomplete: an overview of these areas is presented. An initial section provides pertinent background information on the pathology and clinical significance of these diseases. The roles of three factors in the clinical manifestations of the diseases are then discussed: hypoxia, autonomic nervous system regulation and blood rheology. The possibility of a causal relationship between these three factors and sudden death is also examined. It is concluded that further studies in these specific areas are warranted. It is anticipated that the outcome of such research is likely to provide valuable insights into the pathophysiology of SCD and SCT and will lead to improved clinical management and enhanced quality of life.

Cysteine 93 of Hemoglobin Beta Chain Is the Major Target of Oxidation During Red Blood Cell Storage.

Abstract 4040Poster Board III-976Red blood cells (RBC) stored for prolonged time periods under blood banking conditions are known to accumulate irreversible damage that ultimately affects their viability and function. Among the consequences of this RBC storage lesion are enhanced hemolysis and oxidative stress after transfusion. Stored RBCs accumulate reactive oxygen species (ROS) and surface-associated hemichrome, a partially denatured form of oxidized hemoglobin. In this work, we examined whether the ROS created during RBC storage lead to characteristic oxidative modifications to hemoglobin (Hb) and RBC membrane proteins.To characterize Hb modifications leading to hemichrome formation, we oxidized human hemoglobin A with varied concentrations of hydrogen peroxide (H2O2), an oxidant relevant to RBC storage. The reaction was quenched with excess methionine and the protein digested with trypsin. The resulting peptides were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Methionine and cysteine residues are the major targets of oxidation. Oxidation of methionine to methionine sulfoxide and cysteine to cysteine sulfonic/sulfinic acids is accompanied by a mass shift related to the covalent modifications of each residue, which are readily detected by LC-ESI-MS/MS. We found that HBβ-Cys-93 was oxidized to a maximum of approximately 70% after treatment with 200 αM H2O2. This cysteine occurs adjacent to the FeII-coordinating His-92 and is thus exposed to the heme-FeII center. In contrast, HBβ-Cys-112 and HBα-Cys-104, which are buried and located away from the heme-FeII center, were oxidized to only 2% and 0%, respectively. The enhanced oxidation of the heme-FeII proximal HBβ-Cys-93 suggests that ROS are generated through a FeII-dependent mechanism such as Fenton's reaction. Oxidation of each of the three Hb methionines reached 20% upon treatment with 200 μM H2O2. Among these Met residues, HBα-Met-32 is located near the heme-FeII, center, while both Hbα-Met-76 and HBβ-Met-55 are near the protein/solvent interface. Thus, Cys and Met oxidations in Hb appear to be dependent on proximity to either the heme-FeII center or the protein/solvent interface.To test whether these Hb residues are also sensitive to oxidation during storage we acquired 3 RBC units stored for 3 days and 3 RBC units stored for 45 days, from the Puget Sound Blood Center. The cytosolic Hb was extracted by hypotonic lysis and digested with trypsin prior to LC-ESI-MS/MS analysis. We observed that oxidation of HBβ-Cys-93 was dramatically increased from 10% on day 3 to 29% on day 45 (p < 0.02). HBα-Met-32 was also oxidized to a small extent (5% on day 3 to 7.5 % on day 45, p<0.04). The two buried Cys residues were unoxidized, whereas two surface proximal Met residues were oxidized, but not in a significantly storage-time-dependent manner. These data implicate the redox active heme-FeII center in enhanced ROS production and oxidative stress in stored RBCs.Our observations suggest that HBβ-Cys-93 oxidation is a specific and sensitive marker for oxidative stress during storage. Moreover, we have correlated these progressive oxidations with increases in microvesiculation and hemolysis, storage-time-dependent changes that have been well documented. Future studies will evaluate the storage-time-dependence in other oxidative and post-translational modifications to RBC membrane proteins and correlate these findings with functional consequences such as microvesiculation and hemolysis. Disclosures:No relevant conflicts of interest to declare.

Effect of pH on Structural Changes in Perch Hemoglobin that Can Alter Redox Stability and Heme Affinity

pH can be manipulated to alter the oxidative stability of fish-based foods during storage. X-ray diffraction was used to investigate the ability of reduced pH to cause structural changes in fish hemoglobins that lead to enhanced oxidative degradation. Decreasing pH from 8.0 to 6.3 and 5.7 created a large channel for solvent entry into the heme crevice of perch hemoglobin beta chains. The proton-induced opening of this channel occurred between site CD3 and the heme-6-propionate. Solvent entry into the heme crevice can enhance metHb formation and hemin loss, processes that accelerate lipid oxidation. Reduced pH also decreased the distance between Ile at E11 in one of the alpha chains and the ligand above the heme iron atom. This sterically displaces O2 and protonated O2 which increases metHb formation. These studies demonstrate that pH reduction causes structural changes in perch hemoglobin which increase oxidative degradation of the heme pigment.

Orthodontic treatment of patients with sickle-cell anemia.

Sickle-cell anemia is a genetic blood disease characterized by a hemoglobin gene mutation. The genetic failure is basically constituted by replacement of the hemoglobin beta chain in the sixth position so that the amino acid valine is encoded instead of glutamic acid. As a result, the erythrocytes have their normal biconcave discoid shape distorted, generally presenting a sicklelike shape, which reduces both their plasticity and lifetime. Because a complete blood supply is so important during application of both intraoral and extraoral forces, this article addresses the general and oral aspects associated with sickle-cell anemia. This will guide the clinician regarding such patients who seek orthodontic treatment by making references to literature on multidisciplinary management.

Molecular tunneling in heme proteins

At low temperatures the rebinding of carbon monoxide to heme proteins, after photodissociation by a laser flash, occurs via quantum-mechanical tunneling. Quantum-mechanical tunneling and the classical over-the-barrier process, which dominates at higher temperatures, are both described by an energy spectrum. The barrier widths can be determined from the energy dependence of the tunneling rates. Rebinding of carbon monoxide to the beta chain of hemoglobin is treated in detail, and we find that in this case the barrier width d(E) depends on the barrier height and is given by d(E) = 0.05 nm ×(E/Epeak)1.5, with Epeak = 4.0 kj/mole, Epeak being the peak of the energy spectrum. Data on rebinding of carbon monoxide to the octapeptide formed by proteolytic digestion of cytochrome c are presented. In this system an additional fast component to the rebinding process is observed. The absorption spectrum and the approximately temperature independent behavior of this fast component suggest quantum-mechanical tunneling from an excited state.

Burrow characteristics of the co-existing sibling species Mus booduga and Mus terricolor and the genetic basis of adaptation to hypoxic/hypercapnic stress.

BackgroundThe co-existing, sibling species Mus booduga and Mus terricolor show a difference in site-preference for burrows. The former build them in flat portion of the fields while the latter make burrows in earthen mounds raised for holding water in cultivated fields. In northern India which experiences great variation in climatic condition between summer and winter, M. booduga burrows have an average depth of 41 cm, as against 30 cm in southern India with less climatic fluctuation.M. terricolor burrows are about 20 cm deep everywhere. The three chromosomal species M. terricolor I, II and III have identical burrows, including location of the nest which is situated at the highest position. In contrast, in M. booduga burrows, the nest is at the lowest position.ResultsThe nest chamber of M. booduga is located at greater depth than the nest chamber of M. terricolor. Also, in the burrows of M. booduga the exchange of air takes place only from one side (top surface) in contrast to the burrows of M. terricolor where air exchange is through three sides. Hence, M. booduga lives in relatively more hypoxic and hypercapnic conditions than M. terricolor.We observed the fixation of alternative alleles in M. booduga and M. terricolor at Superoxide dismutase-1 (Sod-1), Transferrin (Trf) and Hemoglobin beta chain (Hbb) loci. All the three are directly or indirectly dependent on oxygen concentration for function. In addition to these, there are differences in burrow patterns and site-preference for burrows suggesting difference in probable adaptive strategy in these co-existing sibling species.ConclusionThe burrow structure and depth of nest of the chromosomal species M. terricolor I, II and III are same everywhere probably due to the recency of their evolutionary divergence. Moreover, there is lack of competition for the well-adapted 'microhabitats' since they are non-overlapping in distribution. However, the co-existing sibling species M. booduga and M. terricolor exhibit mutual "exclusion" of the 'microhabitats' for burrow construction. Thus, location, structure and depth of the burrows might have been the contributory factors for selection of alternative alleles at three loci Sod-1, Trf and Hbb, which reflect difference in probable adaptive strategy in M. booduga and M. terricolor.

Analysis of protein posttranslational modifications by mass spectrometry: With special reference to haemoglobin

Mass spectrometry provides a convenient platform for the study of different protein post translational modifications from clinical specimen. Analysis of different post translational modifications of hemoglobin like glycation and glutathionylation can provide useful information on the disease progression and the possible outcome of therapies. In the present study, we have addressed post translational modifications of hemoglobin like glutathionylation and glycation in relation to diabetes and chronic renal failure. We found that both alpha and beta chains of human hemoglobin are glycated irrespective of the extent of glycemia as evidenced by a mass increment of 162 Da. The phenomenon of glutathionylation was observed with only the beta globin chain of hemoglobin probably due to the presence of an accessible cysteine residue indicated by a mass increment of 305 Da. Also, the extent of gltuathionylation observed in the CRF patients could correlate with the severity of the oxidative stress owing to renal replacement therapies like dialysis and transplantation.

Sickle Cell Patients Have Exagerated Autonomic Response to Transient Hypoxia

Sickle cell anemia (SCA) is a devastating disorder that results from a single amino acid substitution in the beta chain of hemoglobin causing polymerization of hemoglobin S when oxygen is removed. This causes normally flexible sickle red cells (SRBC) to become rigid, obstruct vasculature resulting in ischemic organ damage and decreased longevity. Anything which decreases microvasculature flow will promote the sickling process. Inflammation and adhesion of cellular elements to the vessel wall are known to increase the probability of vasoocclusive crisis (VOC). However, the precise event that triggers the cascade called “crisis” is not known. Microvascular flow changes rapidly in response to autonomic signals which can be assessed by measurement of heart rate variability (HRV). These autonomic signals may be the trigger that causes regional decrease in flow and initiates the events resulting in crisis.We have established a model of induced hypoxia in human subjects that was designed to mimic the transient hypoxia occuring naturally during sleep. Calibrated tidal volume, O2 saturation, and electrocardiogram, were recorded up to 200 times per second using a LifeShirt physiological monitoring garment and tissue oxygenation and microvascular blood flow was assessed by laser Doppler flowmetry or magnetic resonance imaging. The report focuses on safety and on HRV results. Subjects breathed 5 breaths of 100% N2 twice separated by a 5 to 10 minte recovery period on up to 4 separate days per subject. 15 SCA had two hypoxic exposures on 38 days.Subjects were contacted 1, 12, and 24 hrs and 7 to 14 days later and symptom questionnaires completed. On only 4 occasions, subjects reported mild transient sicklelike pain that required no or non-narcotic treatment within 24 hours of hypoxia and was deemed possibly related to the hypoxic exposure. About sixty percent of the exposures were associated with lightheadedness lasting 10 to 15 seconds at the nadir of the SpO2. The drop in SpO2 was greater in the SCA patients (p<.05) and lasted 15 to 20 seconds. However, when we used the subjects' individually measured oxyhemoglobin saturation curve to calculate change in pO2, there was no difference between SCA and normals.Using a novel algorithm we developed which allows second to second comparison of autonomic nervous system (ANS) balance to change in SaO2, we found that the high frequency component of HRV representing parasympathetic (HFP) and low frequency component representing mixed sympathetic activity (LFP) were significantly different between SCA and control (p<.001). SCA patients have a dramatic loss of parasympathetic signal in response to transient hypoxia resulting in significant loss of heart rate variability. These data suggest than SCA patients have a greatly amplified autonomic nervous system response, at least to hypoxia. Since these same ANS signals are also responsible for control of regional microvascular blood flow, it is reasonable to speculate that this hyperactive ANS response leads to regional drop in perfusion which, on a background of hyper-adhesiveness, nitric oxide depletion, inflammation, and dehydration, triggers the sickling cascade. It is important to note that loss of HRV is a powerful predicator of sudden death in several other settings of vascular disease and that 15 to 20% of SCA deaths are otherwise unexplained sudden deaths.These data demonstrate thattransient hypoxia can be safely induced in SCA subjects and used to study the relation between hypoxia and physiological responses andSCA patients have a marked abnormality in autonomic nervous system regulation in response to transient hypoxia that likely plays a role in the pathophysiology of this disorder.

Efficient Erythroid Differentiation of a PGD-Derived Human Pluripotent Stem Cell Line Affected with Sickle Cell Hemoglobinopathy

The technology of preimplantation genetic diagnosis (PGD) screens IVF-derived cleavage-stage embryos or oocytes from parents with inherited disorders, and is routinely used to avoid births with severe genetic disorders. More than one hundred testable genetic conditions, including severe hematologic diseases such as beta thalassemia, sickle cell anemia, and Fanconi anemia can be PCR-screened from either a micro-manipulated blastomere, or a pre-fertilization ovarian polar body. Derivation of human embryonic stem cell (hESC) lines from diseased IVF blastocysts has recently been reported, and these PGD-hESC are untested yet potentially valuable tools for investigating cellular and molecular events of human embryogenesis in diseased states. For example, a great deal of interest has recently been generated in treating hematologic diseases with genetically-corrected hematopoietic stem cells (HSC) derived from patient-specific pluripotent stem cells. Generation of hematopoietic progenitors from PGD-hESC affected with genetic syndromes may thus provide novel opportunities for testing cell-based and gene therapeutic strategies. An important candidate for such cell-based therapy includes sickle cell disease (SSD) hemoglobinopathy, a classic inherited single gene disorder resulting from the substitution of glutamic acid by valine at position 6 of the beta chain of hemoglobin. Human pluripotent stem cells derived from PGD-selected blastocysts or induced pluripotency (iPS; e.g., using patient's somatic cells), will serve as critical reagents for testing such therapeutic strategies. In these studies, we report the characterization and erythropoietic differentiation of a novel PGD-hESC line affected with SSD hemoglobinopathy. The sickle point mutation was confirmed in this PGD-hESC line with direct genomic sequencing of the beta globin locus. This hESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SSD-hESC can be efficiently differentiated to the hematopoietic lineage under serum-free conditions, and gave rise to robust primitive and definitive erythropoieses. The expression of embryonic, fetal and adult globin genes in SSD PGD-hESC derived erythroid cells was confirmed by qRT-PCR, intracytoplasmic FACS, and in situ immunostaining of PGD-hESC teratoma sections. Moreover, we defined culture conditions for massive, long-term liquid culture expansion of sickle affected erythroid progenitors that remained in an undifferentiated erythroblastic phenotype for at least two months. These sickle erythroblasts were continuously maintained as a primary cell line that could be frozen and thawed without loss of viability. In vitro-expanded sickle erythroblasts expressed CD71+CD36+ and CD71+CD235a+ phenotypes, and underwent developmentally appropriate embryonic, fetal, and adult hemoglobin switching over a period of several months. Moreover, hESC-derived erythroblasts were readily infected with Plasmodium falciparum malaria parasites, thus demonstrating their potential utility in studying the effects of this important pathogen in normal and diseased erythropoiesis. These data demonstrate the utility of using patient-specific, hemoglobinopathic hESC for generating significant numbers of erythroid progenitors for molecular, developmental, gene therapeutic, pharmacologic, and microbiological studies. We are currently conducting a comparative erythropoietic differentiation study using SSD PGD-hESC vs. SSD iPS that were generated from somatic fibroblasts using defined pluripotency factors.

Tyrosine Residues as Redox Cofactors in Human Hemoglobin: IMPLICATIONS FOR ENGINEERING NONTOXIC BLOOD SUBSTITUTES

Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and protein-based free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves "through-protein" electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the alpha and beta chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the alpha chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the beta chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.