To study the existence of the erythropoietin receptor (Epo-R) mRNA in brain capillary endothelial cells, the reverse transcription (RT) PCR was performed using total RNAs from rat brain capillary endothelial cells (RBECs) and MBEC4, which is one of the established mouse brain capillary endothelial cell lines. Southern analysis of the RT-PCR products indicated that both RBECs and MBEC4 expressed an authentic form of Epo-R mRNA as a minor form and an intron-5-inserted form of Epo-R mRNA, thus a soluble form of Epo-R mRNA, as a major form. Furthermore, the effect of recombinant human erythropoietin (rHuEpo) on the DNA synthesis in RBECs was analyzed. rHuEpo showed a dose-dependent mitogenic action on RBECs as a competence factor. Radioiodinated rHuEpo was bound specifically to RBECs with time, cell number and dose dependencies. Binding studies with "'I-rHuEpo showed that RBECs had a single class of receptors with low-affinity (K,, = 860 pM) and that the number of siteskell (10300) was abundant. These results suggest that brain capillary endothelial cells express not only an authentic form of Epo-R but also a soluble form of Epo-R and that erythropoietin acts directly on brain capillary endotheha1 cells as a competence factor.Keywords: erythropoietin ; recombinant human erythropoietin ; erythropoietin receptor; brain capillary endothelial cells.Hypoxemia resulting from lung and heart diseases, anemia and high-altitude residence induces a series of modifications in the mammals. As an adaptation mechanism to hypoxia, mammals increase the number of the capillaries/tissue mass [I] and the erythrocytes in the blood (21 to maintain an adequate 0, delivery.Erythropoietin (Epo) is a serum glycoprotein hormone required for survival, proliferation, and differentiation of committed erythroid progenitor cells and its production is accelerated in the kidney by hypoxia [3-51. Recently, i t has been reported that Epo may act on such non-erythroid cells as endothelial cells [6-91, smooth Chemistry, Osaka Prefecture University, Sakai, Osaka, Japan 593 [9]. Although these results suggest that Epo may function as an angiogenic factor for adapting to hypoxia, after embryogenesis, angiogenesis proceeds by the growth of new capillary vessels from an established microvasculature following stimulation by various physiological or pathological processes [18, 191. Furthermore, the mean microvessel density increases in brain [20] and no new capillaries develop in muscle [21] under hypoxic conditions such as exposure to high altitudes, implying that capillary endothelial cells from various tissues each has specific characteristics.HUVECs express Epo-R mRNA [7]. In the present study, we use rat brain capillary endothelial cells (RBECs) and MBEC4, which is one of the established mouse brain capillary endothelial cell lines [22], to analyze the existence of Epo-R mRNA in brain capillary endothelial cells, which have been thought to be associated with angiogenesis under hypoxic conditions, and provide evidence that both RBECs and MBEC4 express ...

Aerobically grown Euglena gracilis, a bleached mutant, shows a prompt synthesis of wax esters with the concomitant fall of the paramylon (a β‐1,3‐glucan) content upon exposure to anaerobiosis. Bringing the anaerobic cells back to aerobiosis causes the reverse conversions. The anaerobic wax ester formation is accompanied by a net synthesis of ATP. The transition between the fermentation and respiration occurs at 10−5–10−7 M of the O2 concentration.

A malonyl-CoA-independent fatty acid synthetic system, different from the systems in other subcellular fractions, occurred in mitochondria of Euglena gracilis. The system had ability to synthesize fatty acids directly from acetylCoA as both primer and C , donor using NADH as an electron donor. Fatty acids were synthesized by reversal of fi-oxidation with the exception that enoyl-CoA reductase functioned instead of acyl-CoA dehydrogenase in degradation system. A fairly high activity of enoyl-CoA reductase was found on various enoyl-CoA substrates (C, -C,,) with NADH or NADPH. Three species of enoyl-CoA reductase, distinct from each other by their chain-length specificity, were found in Euglena mitochondria, and one of them was highly specific for crotonyl-CoA. It is also discussed that the mitochondrial fatty-acid synthetic system contributes to wax ester fermentation, the anaerobic energy-generating system found in the organism.We have reported in the preceding paper [I] that when aerobically grown Euglena gracilis is placcd under anaerobic conditions, paramylon, the reserve poly5accharide, is promptly converted into wax esters with concomitant generation of ATP, and this phenomenon has been named as the wax ester fermentation. In Euglena occurrence of four systems of fatty acid synthesis has been reported [2], namely, one involving multifunctional fatty acid synthetase in cytosol [3 -71, two acyl-carrier-protein-dependent systems in chloroplasts (one being a chain-elongation system) [4-6 , 8 -101 and one involving a fatty acid synthetase in microsomes [II, 121. In the wax ester fermentation, thesc known systems of fatty acid synthesis cannot explain the net gain of ATP, since consumption of ATP by formation of malonyl-CoA and transfer of acetyl unit from mitochondria to other subcellular sites exceeds generation of ATP in glycolysis. Existence of an unknown system of fatty acid synthesis which explain the net gain of ATP in wax ester fermentation is accordingly suggested.In the present paper we report that a malonyl-CoA independent fatty acid-synthetic system occurs in mitochondria of E. gracilis and that the system has ability to synthesize fatty acids directly from acetyl-CoA as both primer and C, donor unlike other malonyl-CoA independent systems of fatty acid synthesis in mammalian mitochondria [I 3 -161, chloroplasts of spinach [17] and Mycohacterium smegmutis [18, 191. It is also discussed that the mitochondrial system of fatty acid synthesis contributes to the wax ester fermentation.

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