Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity.

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an approximately 3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with > 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

The structure of Ascaris hemoglobin domain I at 2.2 A resolution: molecular features of oxygen avidity.

The perienteric hemoglobin of the parasitic nematode Ascaris has an exceptionally high affinity for oxygen. It is an octameric protein containing two similar heme-binding domains per subunit, but recombinant constructs expressing a single, monomeric heme-binding domain (domain 1; D1) retain full oxygen avidity. We have solved the crystal structure of D1 at 2.2 A resolution. Analysis of the structure reveals a characteristic globin fold and illuminates molecular features involved in oxygen avidity of Ascaris perienteric hemoglobin. A strong hydrogen bond between tyrosine at position 10 in the B helix (tyrosine-B10) and the distal oxygen of the ligand, combined with a weak hydrogen bond between glutamine-E7 and the proximal oxygen, grips the ligand in the binding pocket. A third hydrogen bond between these two amino acids appears to stabilize the structure. The B helix of D1 is displaced laterally by 2.5 A when compared with sperm whale myoglobin. This shifts the tyrosine-B10 hydroxyl far enough from liganded oxygen to form a strong hydrogen bond without steric hindrance. Changes in the F helix compared with myoglobin contribute to a tilted heme that may also be important for oxygen affinity.

The hemoglobin function of blood stored at 4⁰ C., (progress report),

Presented at the First International Conference on Red Cell Metabolism and Function, Ann Arbor, Michigan, 1-3 October 1969. Work Unit No. 164, Combat Surgery Task No. 00, Combat Surgery DA Project No. 3A062110A821.

Hookworm aspartic protease, Na-APR-2, cleaves human hemoglobin and serum proteins in a host-specific fashion

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term nemepsins for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease.

Efficacy of twice-weekly multiple micronutrient supplementation for improving the hemoglobin and micronutrient status of anemic adolescent schoolgirls in Bangladesh

Background: Although iron deficiency is a major cause of anemia, other micronutrient deficiencies may also play a role. Objective: We examined whether multiple micronutrient supplementation is more efficacious than is supplementation with iron and folic acid alone for improving the hemoglobin and iron status of anemic adolescent girls in Bangladesh. Design: Anemic (hemoglobin < 12.0 g/dL) girls (n = 197) aged 14-18 y from rural schools in Dhaka District were entered into a randomized double-blind trial and received twice-weekly supplements of iron and folic acid (IFA group) or multiple micronutrients (15 micronutrients, including iron and folic acid; MMN group) for 12 wk. Results: At recruitment, the characteristics of the girls in the 2 groups were not significantly different, except for family size and body mass index. At the end of the study, although both groups benefited significantly from supplementation, mean changes in hemoglobin and serum ferritin concentrations were not significantly different between groups. Compared with the IFA group, girls in the MMN group had significantly greater increases in mean serum vitamin A, plasma vitamin C, red blood cell folic acid, and riboflavin concentrations (assessed as erythrocyte glutathione reductase activation coefficient). After 12 wk of supplementation, only the prevalence of vitamins A and C and riboflavin deficiencies decreased more significantly in the MMN group than in the IFA group. Conclusions: Twice-weekly MMN supplementation for 12 wk significantly improved the status of the micronutrients assessed but was not more efficacious than was supplementation with iron and folic acid alone in improving the hematologic status of anemic adolescent girls. More frequent doses may be needed to achieve full benefit.

The association between glycated haemoglobin levels and P100 visual evoked potentials in diabetes mellitus

Diabetes mellitus (DM) is a metabolic disorder which is characterised by hyperglycaemia resulting from defects in insulin secretion, insulin action or both. The long-term specific effects of DM include the development of retinopathy, nephropathy and neuropathy. Cardiac disease, peripheral arterial and cerebrovascular disease are also known to be linked with DM. Type 1 diabetes mellitus (T1DM) accounts for approximately 10% of all individuals with DM, and insulin therapy is the only available treatment. Type 2 diabetes mellitus (T2DM) accounts for 90% of all individuals with DM. Diet, exercise, oral hypoglycaemic agents and occasionally exogenous insulin are used to manage T2DM. The diagnosis of DM is made where the glycated haemoglobin (HbA1c) percentage is greater than 6.5%. Pattern-reversal visual evoked potential (PVEP) testing is an objective means of evaluating impulse conduction along the central nervous pathways. Increased peak time of the visual P100 waveform is an expression of structural damage at the level of myelinated optic nerve fibres. This was an observational cross sectional study. The participants were grouped into two phases. Phase 1, the control group, consisted of 30 healthy non-diabetic participants. Phase 2 comprised of 104 diabetic participants of whom 52 had an HbA1c greater than 10% (poorly controlled DM) and 52 whose HbA1c was 10% and less (moderately controlled DM). The aim of this study was to firstly observe the possible association between glycated haemoglobin levels and P100 peak time of pattern-reversal visual evoked potentials (PVEPs) in DM. Secondly, to assess whether the central nervous system (CNS) and in particular visual function is affected by type and/or duration of DM. The cut-off values to define P100 peak time delay was calculated as the mean P100 peak time plus 2.5 X standard deviations as measured for the non-diabetic control group, and were 110.64 ms for the right eye. The proportion of delayed P100 peak time amounted to 38.5% for both diabetic groups, thus the poorly controlled group (HbA1c > 10%) did not pose an increased risk for delayed P100 peak time, relative to the moderately controlled group (HbA1c ≤ 10%). The P100 PVEP results for this study, do however, reflect significant delay (p < 0.001) of the DM group as compared to the non-diabetic group; thus, subclincal neuropathy of the CNS occurs in 38.5% of cases. The duration of DM and type of DM had no influence on the P100 peak time measurements.

Use of Hemoglobin A1C to Detect Haitian-Americans with Undiagnosed Type 2 Diabetes

OBJECTIVE: To evaluate the validity of hemoglobin A1C (A1C) as a diagnostic tool for type 2 diabetes and to determine the most appropriate A1C cutoff point for diagnosis in a sample of Haitian-Americans. SUBJECTS AND METHODS: Subjects (n = 128) were recruited from Miami-Dade and Broward counties, FL. Receiver operating characteristics (ROC) analysis was run in order to measure sensitivity and specificity of A1C for detecting diabetes at different cutoff points. RESULTS: The area under the ROC curve was 0.86 using fasting plasma glucose ≥ 7.0 mmol/L as the gold standard. An A1C cutoff point of 6.26% had sensitivity of 80% and specificity of 74%, whereas an A1C cutoff point of 6.50% (recommended by the American Diabetes Association – ADA) had sensitivity of 73% and specificity of 89%. CONCLUSIONS: A1C is a reliable alternative to fasting plasma glucose in detecting diabetes in this sample of Haitian-Americans. A cutoff point of 6.26% was the optimum value to detect type 2 diabetes.

Factors affecting BMI and hemoglobin levels of mothers and infants in Kuwait

If you are the rightful copyright holder of this dissertation or thesis and wish to have it removed from the Open Access Collection, please submit a request to dcc@fiu.edu and include clear identification of the work, preferably with URL.

Selective disappearance of individuals with high levels of glycated haemoglobin in a free-living bird

Peer reviewed

Impact of Hemoglobin Levels and Anemia on Mortality in Acute Stroke : Analysis of UK Regional Registry Data, Systematic Review, and Meta‐Analysis

Funding Sources The NNUH Stroke and TIA Register is maintained by the NNUH NHS Foundation Trust Stroke Services and data management for this study is supported by the NNUH Research and Development Department through Research Capability Funds.

Antioxidant status in J774A.1 macrophage cell line during chronic exposure to glycated serum

Advanced glycation end-products (AGEs) are linked to aging and correlated diseases. The aim of present study was to evaluate oxidative stress related parameters in J774A.1 murine macrophage cells during chronic exposure to a subtoxic concentration of AGE (5% ribose-glycated serum (GS)) and subsequently for 48 h to a higher dose (10% GS). No effects on cell viability were evident in either experimental condition. During chronic treatment, glycative markers (free and bound pentosidine) increased significantly in intra- and extracellular environments, but the production and release of thiobarbituric acid reactive substances (TBARs), as an index of lipid peroxidation, underwent a time-dependent decrease. Exposure to 10% GS evidenced that glycative markers rose further, while TBARs elicited a cellular defence against oxidative stress. Nonadapted cultures showed an accumulation of AGEs, a marked oxidative stress, and a loss of viability. During 10% GS exposure, reduced glutathione levels in adapted cultures remained constant, as did the oxidized glutathione to reduced glutathione ratio, while nonadapted cells showed a markedly increased redox ratio. A constant increase of heat shock protein 70 (HSP70) mRNA was observed in all experimental conditions. On the contrary, HSP70 expression became undetectable for a longer exposure time; this could be due to the direct involvement of HSP70 in the refolding of damaged proteins. Our findings suggest an adaptive response of macrophages to subtoxic doses of AGE, which could constitute an important factor in the spread of damage to other cellular types during aging.Key words: in vitro cytotoxicity, AGE, pentosidine, glycoxidation, oxidative stress, TBARs.

Hemoglobin conformational properties and ionic composition of red blood cells in vascular diseases patients

This is an abstract of a presented talk at the European Biotechnology Conference held in Latvia during 05–07 May 2016

On the temperature stability of extracellular hemoglobin of Glossoscolex paulistus, at different oxidation states: SAXS and DLS studies

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). DLS melting curves were measured for met-HbGp at different concentrations. SAXS temperature studies were performed for oxy-, cyanomet- and met-HbGp forms, at several pH values. At pH 5.0 and 6.0, the scattering curves are identical from 20 to 60 degrees C, and R-g is 108 angstrom, independent of the oxidation form. At pH 7.0, protein denaturation and aggregation occurs above 55 degrees C and 60 degrees C, for oxy and met-HbGp, respectively. Cyanomet-HbGp, at pH 7.0, is stable up to 60 degrees C. At alkaline pH (8.0-9.0) and higher temperature, an irreversible dissociation process is observed, with a decrease of R-g, D-max and I(0). Analysis by p(r), obtained from GNOM, and OLIGOMER, was used to fit the SAXS experimental scattering curves by a combination of theoretical curves obtained for HbLt fragments from the crystal structure. Our results show clearly the increasing contribution of smaller molecular weight fragments, as a function of increasing pH and temperature, as well as, the order of thermal stabilities: cyanomet-> oxy- > met-HbGp. (C) 2012 Elsevier B.V. All rights reserved.

Ceramic membranes for separation of proteins and DNA by in situ growth of alumina nanofibres with a nanomesh of metal oxide nanofibres

Ceramic membranes were fabricated by in situ synthesis of alumina nanofibres in the pores of an alumina support as a separation layer, and exhibited a high permeation selectivity for bovine serum albumin relative to bovine hemoglobin (over 60 times) and can effectively retain DNA molecules at high fluxes.