Recombination of embryonic haemoglobin chains during the development of the chick.

Embryonic differentiation is at present interpreted as the expression of variable gene activity. It is commonly thought that derepression of operator gene groups is the main cause of progress during development. However it is equally possible that gene repression plays a role in the appearance of new phenotypic characteristics. This paper illustrates such a possibility. It is known that in chickens embryonic haemoglobins exist which are replaced by other haemoglobins at about the sixth day of incubation. Analyses of globin chain composition of these haemoglobins by chromatography and urea/starch gel electrophoresis as well as TLC-fingerprinting and amino acid analyses of the individual globin chains showed that the haemoglobin switch was not associated with appearance of new globin chains but rather with disappearance of a number of embryonic chains. Moreover the relative proportion of the various chains changed at that time. From these findings we conclude that new haemoglobins arise from a recombination ('hybridization in vivo') of those globin chains which remain after the repression of a gene coding for embryonic chains.     

Cell technologies and the development of skin substitutes

Large skin lesions might be of different cause and sometime resistant to the conventional and surgical treatment. For many years, skin grafting used to serve one of the most important methods to treat large and deep skin lesions. However, a limited availability of the substrate for transplantation restricts wider application of the approach. Utilization of wound covers and skin replacements of various types (including synthetic, biological and biosynthetic ones) provides an alternative. Skin substitutes--biosynthetic covers of the complex structure--constitute the most important niche in a wide spectrum of wound covers developed and actively utilized by today. Cell substrates used in the former include dermal fibroblasts, neonatal foreskin fibroblasts and keratinocytes. In the current review, key properties of the modern skin substitutes, structure features of the most widespread types. and principle of the work with the particular cell types are analyzed. Comparative analysis of the efficiency of wound covers is provided.     

Derivatization of haemoglobin with periodate-generated reticulation agents: evaluation of oxidative reactivity for potential blood substitutes

Periodate modification of the sugar moiety in sugars, including adenosine triphosphate (ATP), has previously been employed in order to prepare dialdehyde-type reagents, which were then utilized in crosslinking reactions on haemoglobin, yielding polymerized material with useful dioxygen-binding properties and hence proposed as possible artificial oxygen carriers ('blood substitutes'). Here, the periodate protocol is shown to be applicable to a wider range of oxygen-containing compounds, illustrated by starch and polyethylene glycol. Derivatization protocols are described for haemoglobin with such periodate-treated crosslinking agents, and the dioxygen-binding properties and redox reactivities are investigated for the derivatized haemoglobins, with emphasis on pro-oxidative properties. There is a general tendency of the derivatization to result in higher autooxidation rates. The peroxide reactivity of the met (ferric) form is also affected by derivatization, as witnessed, among others, by varying yields of ferryl [Fe (IV)-oxo] and free radical generated. In cell, culture tests (human umbilical vein epithelial cells, HUVEC), the derivatization protocols show no toxic effect.     

Recombinant E. coli expressing Vitreoscilla haemoglobin prefers aerobic metabolism under microaerobic conditions: A proteome-level study

Vitreoscilla haemoglobin (VHb) expression in heterologous host was shown to enhance growth and oxygen utilization capabilities under oxygen-limited conditions. The exact mechanism by which VHb enhances the oxygen utilization under oxygen-limiting conditions is still unknown. In order to understand the role of VHb in promoting oxygen utilization, changes in the total protein profile of E. coli expressing the vgb gene under its native promoter was analysed. Two-dimensional difference gel electrophoresis (2D DIGE) was employed to quantify the differentially expressed proteins under oxygen-limiting conditions. Overexpression of proteins involved in aerobic metabolic pathways and suppression of proteins involved in non-oxidative metabolic pathways shown in this study indicates that the cells expressing VHb prefer aerobic metabolic pathways even under oxygen limitation. Under these conditions, the expression levels of proteins involved in central metabolic pathways, cellular adaptation and cell division were also found to be altered. These results imply that Vitreoscilla haemoglobin expression alters aerobic metabolism specifically, in addition to altering proteins involved in other pathways, the significance of which is not clear as of now.     

Recombinant DNA approaches for the development of metabolic systems used in in vitro toxicology.

In the past few years there has been considerable progress in the development of mammalian cell systems for use in genetic toxicology by the stable transfer of genes/cDNAs coding for drug metabolizing enzymes directly into the target cell. Alternative approaches have also been developed in which mammalian cells are transiently transfected with cDNAs coding for drug-metabolizing enzymes and S9 preparations expressing a single metabolizing enzyme isolated and used for metabolic activation. Progress in these areas is reviewed here and the relative merits of the different approaches are discussed. Work to date has focused primarily on the cytochrome P450 family of enzymes, although other enzyme systems involved in xenobiotic metabolism have been used. The central theme of this review is the transfer of genetic information to improve the metabolic capability of cell systems used in genetic toxicology. However, a basic philosophy of the review is that genetic manipulation of cultured mammalian cells has the potential for developing systems to be used to better understand chemically induced toxicological effects.     

Ion transport by lasalocid A across red-blood-cell membranes. A multinuclear NMR study

Na+ and K+ fluxes mediated by lasalocid A across erythrocyte membranes have been determined from 23Na-NMR peak areas and chemical shifts, respectively. In similar experiments, Cl- transport has been monitored by NMR signal intensities. Taking into account the external pH variations, the results are readily explainable in terms of charge-balance conservation. The effect of disodium 4,4'-diisothiocyanostilbene-2,2'-disulfonate, an anion-exchange inhibitor, has also been studied.     

Organic phosphates increase the solubility of avian haemoglobin D and embryonic chicken haemoglobin.

The isolated minor haemoglobin fractions (haemoglobin D) of ostrich, chicken and duck haemoglobin, which constitute about 30% of total intracellular haemoglobin, form crystalline aggregates upon deoxygenation at physiological temperature, ionic strength and pH and at haemoglobin concentrations even well below those present in the red cell. The aggregation is reversed by oxygenation, and can be inhibited by addition of organic phosphates or the corresponding major haemoglobin fraction in a stoichiometric ratio of 1:1. Embryonic haemoglobin from chicken has similar characteristics with respect to its solubility. The results indicate close functional homology of alpha D and embryonic pi-chains as well as a novel role for organic phosphates in the regulation of haemoglobin function.     

Current status of erythrocyte substitutes.

During the last two decades the search for alternatives to whole blood transfusions has led to promising developments in the field of erythrocyte substitutes. Hemoglobin solutions free of fragments of erythrocyte stroma and fluorocarbon emulsions are not blood-type-specific and appear likely to satisfy some proportion of our blood requirements. Both must be modified before becoming clinically useful. The oxygen affinity of the hemoglobin solution must be reduced and its intravascular persistence improved. Fluorocarbons cannot yet contribute significantly to the oxygen supply unless the patient breathes hyperbaric oxygen. Recent advances are leading to solutions for these problems.     

Oxidative degradation of haemoglobin by nitrosobenzene in the erythrocyte.

Substitutions on the benzene ring of nitrosobenzene did not have the same effect on oxidative haemolysis as substitutions on phenylhydrazine. We previously found that the haemolytic effect of arylhydrazines paralleled their oxidative conversion into ligands of ferrihaemoglobin. In contrast, although most substituted nitrosobenzenes that are ligands of ferrohaemoglobin caused haemolysis and most that are not ligands failed to cause nitrosoarenes appeared to be related more closely to the ease of their reduction to arylhydroxylamines than to their properties as ligands. We propose a mechanism of oxidative degradation whereby the cyclic formation of phenylhydroxylamine from nitrosobenzene within an erythrocyte leads to the accumulation of H2O2, which then reacts with ferrohaemoglobin to initiate the oxidative cleavage of haem. The posulated active intermediate in this reaction is the same as that previously proposed in the oxidative degradation of haemoglobin by phenylhydrzine and in the coupled oxidation of ascorbic acid and haemoglobin.     

Fluorocarbons as blood substitutes. Toxicity in the rat]

Liquid fluorocarbons, having high solubility for gases (O2, CO2...) have been used as artificial blood substitutes in animals with variable results. The great diversity of these products and the lack of reproductiveness in their composition have not permitted, up to now, a standardization of their utilisation norms. Our work was to study the toxicity, in the rat, of a new kind of fluorocarbon emulsion (E-66, Ugine-Kuhlmann, France) used as an artificial blood substitute during exchange-perfusion. The short survival of the rats is in opposition to the good in vitro results obtained in other experiments (high solubilities of oxygen and dioxide carbon). The toxicity of this fluorinated emulsion is demonstrated by histologic lesions in lungs, liver and kidneys and the great amount of fluor stored in these organs. The mechanism of this toxicity is still to be demonstrated. Hepatic lesions (hyperhemia with dilatation of central veins and sinusoids) and pulmonary lesions (vascular congestion, alveolar oedema) prove a circulatory disturbance leading to right-sided cardiac failure. However, cellular degenerescence lesions, observed in hepatocytes and renal tubular cells, do not permit to exclude formally a cellular toxicity of the E-66 emulsion.