Effect of nitric oxide on the transport and release of oxygen in fetal blood

It is well known that nitric oxide (NO), the most important vasodilator agent, plays an important role in lowering vascular resistance in the human umbilical-placental circulation and that its deficiency is related to the pathogenesis of pre-eclamptic disorder. Besides it has recently been demonstrated that human hemoglobin (HbA) is able to transport nitric oxide, as S-nitrosohemoglobin (SNO-Hb), from the arterial to the venous blood. In the present study we examine the functional properties of the adult and fetal nitrosated hemoglobins to see if the double transport of oxygen and NO may influence the fetal oxygenation and the relation between maternal and fetal blood. Our results show that S-nitrosation significantly increases the oxygen affinity of the adult Hb (HbA) with respect to native protein (no-nitrosated) while the functional properties of HbF are less influenced. The oxygen affinity modification, found for SNO-HbA, was ascribed to the nitrosation of cysteine beta 93: really, the same residue is also present in the gamma chains of fetal hemoglobin, while the increase of affinity is less evidenced; hence, it is probable that the 39 aminoacidic substitutions between beta and gamma chains allay the effects of S-nitrosation. As regards the physiological modulators (protons, chloride ions, 2,3-diphosphoglyceric acid, and temperature), they influence the oxygen affinity of the two hemoglobins S-nitrosated, in equal mode with respect to the native forms determining the same variation on the oxygen affinity. Hence, our results evidence the fact that the NO release by SNO-HbA "in vivo" would be limited to regions of extremely low oxygen tension (such as hypoxic regions), while in fetus, SNO-HbF would unload nitric oxide and oxygen at pressure values close to normal.     

Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability

Using the pUT-miniTn5 vector system developed by the laboratory of K.N. Timmis, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosomes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemoglobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-type P. aeruginosa and degraded benzoic acid faster than the latter strain at both normal and low aeration. In contrast, the vgb-bearing B. cepacia strain had a growth advantage over the wild-type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no difference in DNT degradation was seen between the two strains at either normal or low aeration. The results demonstrate the practicality of enhancing bioremediation with vgb stably integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial effects to be seen. Journal of Industrial Microbiology & Biotechnology (2001) 27, 27–33. Received 20 October 2000/ Accepted in revised form 04 May 2001     

Ethylene signal perception and transduction: multiple paradigms?

Current progress on the mechanisms of ethylene signal perception and transduction are reviewed with an emphasis on reconciling data from molecular genetics and from biochemical approaches. It is proposed that there exist two or more interacting transduction pathways.     

Effects of zinc supplementation to ration on some hematological parameters in broiler chicks

The aim of the study was to examine the effects of zinc supplementation on some hematological parameters. Sixty newborn male broiler chicks were utilized in the study. Zinc (Zn) was added into drinking water at levels of 0, 125, 500, and 1000 mg/kg. In the study, there was no significant difference between control and Zn-supplemented groups in erythrocyte count, hemoglobin amount, hematocrit levels, total leukocyte count, and differential leukocyte % levels, but the α-naphthyl acetate esterase ANAE(+) lymphocyte rate significantly (p<0.05) increased in the 125-ppm Zn-supplemented group compared with the control group. In conclusion, the data obtained may be beneficial in demonstrating the effects of zinc on, at least, these parameters.     

Crystal structures of unligated and CN-ligated Glycera dibranchiata monomer ferric hemoglobin components III and IV

Erythrocytes of the marine annelid, Glycera dibranchiata, contain a mixture of monomeric and polymeric hemoglobins. There are three major monomer hemoglobin components, II, III, IV (also called GMH2, 3, and 4), that have been highly purified and well characterized. We have now crystallized GMH3 and GMH4 and determined their structures to 1.4-1.8 A resolution. The structures were determined for these two monomer hemoglobins in the oxidized (Fe3+, ferric, or met-) forms in both the unligated and cyanide-ligated states. This work differs from two published, refined structures of a Glycera dibranchiata monomer hemoglobin, which has a sequence that is substantially different from any bona fide major monomer hemoglobins (GMH2, 3, or 4). The high-resolution crystal structures (presented here) and the previous NMR structure of CO-ligated GMH4, provide a basis for interpreting structure/function details of the monomer hemoglobins. These details include: (1) the strong correlation between temperature factor and NMR dynamics for respective protein forms; (2) the unique nature of the HisE7Leu primary sequence substitutions in GMH3 and GMH4 and their impact on cyanide ion binding kinetics; (3) the LeuB10Phe difference between GMH3 and GMH4 and its impact on ligand binding; and (4) elucidation of changes in the structural details of the distal and proximal heme pockets upon cyanide binding.     

Protective role of vitamin E on mefenamic acid-induced alterations in erythrocytes

Erythrocyte osmotic fragility (O.F.), acetylcholinesterase (AChE) activity,and the level of malonyl dialdehyde (MDA) of control, mefenamic acid treated, and mefenamic acid with vitamin E treated rats were investigated. Administration of mefenamic acid to albino rats brought about a significant increase in the osmotic fragility of red cells and a significant (p<0.01) decrease in the activity of AChE. We have also observed increased red cell level of MDA and decreased cholesterol (Chl), hemoglobin (Hb), and reduced glutathione (GSH) content. Supplementation of vitamin E to the mefenamic acid treated rats restored the O.F., AChE activity, level of MDA, and Chl, Hb, and GSH content almost to normal. These observations suggest that mefenamic acid causes functional impairment of red cell membrane, while vitamin E shows its protective role in maintaining normal red cell functions.     

Distal heme pocket regulation of ligand binding and stability in soybean leghemoglobin

Leghemoglobins facilitate diffusion of oxygen through root tissue to a bacterial terminal oxidase in much the same way that myoglobin transports oxygen from blood to muscle cell mitochondria. Leghemoglobin serves an additional role as an oxygen scavenger to prevent inhibition of nitrogen fixation. For this purpose, the oxygen affinity of soybean leghemoglobin is 20-fold greater than myoglobin, resulting from an 8-fold faster association rate constant combined with a 3-fold slower dissociation rate constant. Although the biochemical mechanism used by myoglobin to bind oxygen has been described in elegant detail, an explanation for the difference in affinity between these two structurally similar proteins is not obvious. The present work demonstrates that, despite their similar structures, leghemoglobin uses methods different from myoglobin to regulate ligand affinity. Oxygen and carbon monoxide binding to a comprehensive set of leghemoglobin distal heme pocket mutant proteins in comparison to their myoglobin counterparts has revealed some of these mechanisms. The "distal histidine" provides a crucial hydrogen bond to stabilize oxygen in myoglobin but has little effect on bound oxygen in leghemoglobin and is retained mainly for reasons of protein stability and prevention of heme loss. Furthermore, soybean leghemoglobin uses an unusual combination of HisE7 and TyrB10 to sustain a weak stabilizing interaction with bound oxygen. Thus, the leghemoglobin distal heme pocket provides a much lower barrier to oxygen association than occurs in myoglobin and oxygen dissociation is regulated from the proximal heme pocket.     

Novel approaches for regulating gas supply to plant systems <Emphasis Type="Italic">in vitro</Emphasis>: Application and benefits of artificial gas carriers

Summary The composition of the gaseous environment within tissue culture vessels is a critical factor in determining in vitro plant growth and morphogenic responsiveness. Consequently, the provision of an adequate and sustainable oxygen supply for cultured plant cells (including isolated protoplasts), tissues and organs is a crucial prerequisite for optimization and regulation of such cultural responses. During the past decade, research has focused on improving growth and development using artificial gas carriers based on inert perfluorocarbon (PFC) liquids and hemoglobin (Hb) solution. Supplementation of culture media with such artificial oxygen carriers has demonstrated beneficial effects of increased and sustainable cellular mitotic division and subsequent biomass production in a diverse range of plant species, during both short- and longer-term culture. Studies have targeted systems where oxygen availability is actually or potentially a major growth-limiting factor. Undoubtedly, gas carrier-facilitated improvements in regulating the supply of respiratory gases to cultured cells, tissues and organs will have increasingly important biotechnological and practical implications in the context of plant micropropagation, somatic hybridization, transgenic plant production, and secondary product biosynthesis.     

A solid phase adsorption method for preparation of bovine serum albumin-bovine hemoglobin conjugate

A solid phase adsorption method was proposed to prepare well-defined bovine serum albumin–bovine hemoglobin (Hb) conjugate. After adsorption by the solid phase, Q Sepharose Fast Flow media, bovine serum albumin (BSA) molecules were allowed to react with glutaraldehyde. The spacing out of BSA molecules on the solid phase was assumed to limit polymerization of BSA molecules, except some molecules bound closely on the solid phase resulting in minor dimer formation. Following the elution procedure, the activated monomeric BSA was separated from the dimers by gel filtration chromatography on Superdex 200 and then reacted with bovine Hb at 4 °C and pH 9.5. The 1:1 (BSA:Hb) conjugate was obtained with the yield of 64%. The P₅₀ values of the conjugates, prepared under anaerobic and aerobic conditions, were 19.1 and 14.2 mmHg, respectively. The dependence of the P₅₀ on chloride ions for the conjugate was slightly diminished, presumably due to covalent attachment of BSA to bovine Hb.