Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O 2 supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe 2؉ ) form, indicating a His-Fe 2؉ -His binding scheme. O 2 or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe 3؉ ) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k on ) and a slow dissociation rate (k off ) for both O 2 and CO, indicating a high intrinsic affinity for these ligands. However, because the ratelimiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O 2 and CO binding is suggested to be slow in vivo. Because of this competition, the observed O 2 affinity of recombinant human neuroglobin is average (1 torr at 37°C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37°C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E o ‫؍‬ ؊129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfidedependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.In addition to the well known hemoglobins (Hbs) 1 and myoglobins (Mbs), a third type of globin has recently been described in vertebrates that is predominantly expressed in the brain and other nerve tissues (1). These neuroglobins (NGBs) consist of single chains with 151 amino acids (M r ϭ ϳ17,000) that share only little sequence similarity with the vertebrate globins (Mb Ͻ 21%; Hb Ͻ 25%). Nevertheless, all key determinants of genuine globins are conserved (2). Although NGB was initially discovered in mouse and man, recent data show its presence in many different mammalian species as well as in fish, suggesting the universal occurrence of NGB in vertebrate brains. Nerve-specific globins have been sporadically observed in mollusc, annelid, arthropod, and nemertean species (3-5). These invertebrate nerve globins reach high local concentrations up to the millimolar range, which may be sufficient to facilitate O 2 diffusion or store O 2 that supports cell function during temporary hypoxia (5). The latter assumption is supported by the observation that the nervous function in the mollusc Tellina alternata under anoxic conditions depends on the oxygenation of a nerve globin (6, 7). However, the estimated amount of NGB in the vertebrate brain under nonpathological conditions is only in the micromolar range and thus is much lower than that of a typical invertebrate nerve globin (1). The physiological role of such lowly expressed globins is not well understood. Wittenberg (8) proposed that cytoplasmic globins at low concentrat...

We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.

Neuroglobin and cytoglobin reversibly bind oxygen in competition with the distal histidine, and the observed oxygen affinity therefore depends on the properties of both ligands. In the absence of an external ligand, the iron atom of these globins is hexacoordinated. There are three cysteine residues in human neuroglobin; those at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. Both cysteine residues in cytoglobin, although localized in other positions than in human neuroglobin, may form a disulfide bond as well. The existence and position of these disulfide bonds was demonstrated by mass spectrometry and thiol accessibility studies. Mutation of the cysteines involved, or the use of reducing agents to break the S-S bond, led to a decrease in the observed oxygen affinity of human neuroglobin by an order of magnitude. The critical parameter is the histidine dissociation rate, which changes by about a factor of 10. The same effect is observed with human cytoglobin, although to a much lesser extent (less than a factor of 2). These results suggest a novel mechanism for the regulation of oxygen binding; contact with an appropriate electron donor would provoke the release of oxygen. Hence the oxygen affinity would be directly linked to the redox state of the cell.

In vitro RBC production from stem cells could represent an alternative to classic transfusion products. Until now the clinical feasibility of this concept has not been demonstrated. We addressed the question of the capacity of cultured RBCs (cRBCs) to survive in humans. By using a culture protocol permitting erythroid differentiation from peripheral CD34 ؉ HSC, we generated a homogeneous population of cRBC functional in terms of their deformability, enzyme content, capacity of their hemoglobin to fix/release oxygen, and expression of blood group antigens. We then demonstrated in the nonobese diabetes/severe combined immunodeficiency mouse that cRBC encountered in vivo the conditions necessary for their complete maturation. These data provided the rationale for injecting into one human a homogeneous sample of 10 10 cRBCs generated under good manufacturing practice conditions and labeled with 51