Modulation of in vitro erythropoiesis: enhancement of erythroid colony growth by cyclic nucleotides.

Mammalian erythropoiesis, as assayed by erythroid colony formation in vitro, is enhanced by cyclic adenosine nucleotides and agents which are capable of raising intracellular cyclic AMP (cAMP) levels. With canine marrow cells as target, this enhancement was shown to be specific for cAMP and its mono- and dibutyryl derivatives. Adenosine and its derivatives, such as AMP, ADP and ATP, and other cyclic nucleotides, such as cGMP, dibutyryl-cGMP, cCMP and cIMP and sodium butyrate were inactive. The phosphodiesterase inhibitor, RO-20-1724, and the adenyl cyclase stimulator, cholera enterotoxin, both markedly increased colony numbers. Studies with tritiated thymidine showed that about 50% of the cells responding to either erythropoietin (ESF) or dibutyryl cAMP (db-cAMP) were in DNA synthesis. However, by unit gravity sedimentation velocity analysis, the peak of ESF-responsive colony forming cells sedimented more rapidly (8-7 +/- 0-2 mm/hr) than the peak of db-cAMP-responsive cells (7-5 +/- 0 mm/hr). These results demonstrate that adenyl cyclase-linked mechanisms influence in vitro erythropoietic proliferation and suggest that other hormones and simple molecules might interact with surface receptors and thus modulate the action of ESF at the cellular level.     

The transbilayer distribution of phosphatidylethanolamine in erythroid plasma membranes during erythropoiesis

Fluorescamine was used to assess the transbilayer distribution of phosphatidylethanolamine in the plasma membrane of murine erythroid progenitor cells, CFU-E (colony-forming unit erythroid), at different stages of their differentiation pathway. Intact cells were exposed to increasing concentrations of fluorescamine and the amount of labeled phosphatidylethanolamine was determined by measuring the fluorescence intensity of its fluorescamine derivative. A semilogarithmic plot of the dose-response curve revealed three different pools of phosphatidylethanolamine, representing its fractions in, respectively, the inner- and outer monolayers of the plasma membrane and subcellular membrane systems. These results show that 9-11% of the total cellular phosphatidylethanolamine is present in the outer leaflet and 9-10% of it is located in the inner leaflet of the plasma membrane in early as well as late erythroblasts. This symmetric distribution of phosphatidylethanolamine over the two halves of the bilayer in the plasma membrane of CFU-E is very similar to that observed earlier in the plasma membrane of friend erythroleukaemic cells (Rawyler, Van der Schaft, Roelofsen and Op den Kamp (1985) Biochemistry 24, 1777-1783). These observations imply that the characteristic asymmetric distribution of phosphatidylethanolamine, as is found in mature erythrocytes, is accomplished at a very late stage of erythropoiesis and possibly during enucleation of the cells or shortly thereafter.     

Developmental changes in microheterogeneity of foetal plasma glycoproteins of mice

Changes in microheterogeneity of foetal plasma glycoproteins during development of mouse embryos were investigated. Analysis of foetal plasma by polyacrylamide-gel electrophoresis indicated three major zones of proteins: (1) transferrins, (2) alpha-foetoproteins and (3) albumin. Three transferrins (Tr1, Tr2, Tr3) and five alpha-foetoproteins (Fp1, Fp2, Fp3, Fp4, Fp5) were resolved. Evidence for the presence of transferrins was the binding of (59)Fe to the three electrophoretic variants. By day 15.5 of gestation, there was a marked increase in the more-acidic components (Tr3, Fp4, Fp5) and a decrease in the less-acidic ones (Tr1, Tr2, Fp1, Fp2, Fp3). Treatment of foetal plasma with neuraminidase at this time of development converted the more acidic components into Tr1 and Tr2 and Fp1, Fp2 and Fp3. Furthermore, it was shown that early in development (day 12.5) only the less-acidic components of transferrin and alpha-foetoprotein were synthesized; at the later time in development (day 14.5) new synthesis of the acidic components of both groups occurred. That these more-acidic components of alpha-foetoprotein (Fp4, Fp5) were in fact electrophoretic variants of the less-acidic alpha-foetoproteins was shown by the immunoprecipitation of labelled Fp4 and Fp5 with anti-Fp1, anti-Fp2 and anti-Fp3. From these results it is postulated that the plasma glycoproteins that are synthesized later in development contain increased amounts of sialic acid and that the observed changes in microheterogeneity of these proteins represent regulation of glycoprotein biosynthesis at the level of carbohydrate attachment.     

Fetal erythropoiesis in steel mutant mice : I. A morphological study of erythroid cell development in fetal liver

A method of definitive identification of mutant (S1/S1^d) and wild-type (+/+) mouse embryos in segregating litters is described, based on the total number of circulating erythrocytes in a unit volume of embryonic blood and the relative proportion of nonnucleated vs. nucleated red blood cells. Evidence is presented that from days 13–17 of gestation, S1/S1^d embryos have many fewer fetal liver derived nonnucleated erythrocytes whereas the number of yolk sac-derived nucleated red blood cells is similar between S1/S1^d and +/+. Erythroid precursor cells at various stages of maturation in mutant fetal livers are studied by light and electron microscopy, and their fine structure is found to be identical to those present in normal embryos. The number of hemoglobin-containing mature erythroblasts in mutant fetal livers is far fewer than that of the normal, whereas the number of immature erythroid precursors present in a unit area of fetal liver is not significantly different between S1/S1^d and +/+. It is suggested that the mutant S1 gene product(s) interferes with or fails to support the differentiation of immature erythroid precursors into hemoglobin synthesizing cells.     

Developmental changes of sepiapterin synthase activity associated with a variegated purple gene in Drosophila melanogaster

A variegated position effect on the autonomous gene, purple, has been studied enzymologically in Drosophila melanogaster. Sepiapterin synthase, the enzyme system associated with pr⁺, was examined for activity in different developmental stages of the fly. The results indicate that T(Y:2) pr^c5, cn/pr^c4 cn flies (flies in which pr⁺ has been translocated and which exhibit variegation) have a reduced amount of enzyme activity as compared with both Oregon-R and pr¹ flies. This reduction in activity was not found in larval stages, which suggests that the inactivation process probably occurs in late larval or early pupal stages. The phenotype of the variegated adult has white eyes with red-colored spots and patches where drosopterins occur. The phenotype of the fly carrying the translocation is modified by the presence of additional Y chromosomes. This extends the observation from other systems that extra heterochromatin acts to suppress the variegated position effect. The advantages of studying the variegation by measuring enzyme activity, as well as the phenotypic expression, are several; for example, the developmental time at which variegation occurs may be estimated even though drosopterin synthesis is not occurring.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the Department of Energy under Contract No. W-7405-eng-26.     

Apolipoprotein changes associated with the plasma lipid-regulating activity of gemfibrozil in cholesterol-fed rats

Gemfibrozil (Lopid) is a new plasma lipid-regulating drug that decreases very low and low density lipoprotein (VLD/LDL) and increases high density lipoprotein (HDL) concentrations in man. The present experiments tested the effects of gemfibrozil on plasma lipoproteins and apolipoproteins in rats fed high fat/high cholesterol diets. Compared to chow-fed rats, cholesterol feeding for 2 weeks (20% olive oil/2% cholesterol) produced the expected increases in VLDL and intermediate density lipoprotein (IDL) while lowering plasma HDL. This was documented by using three methods of lipoprotein isolation: sequential ultracentrifugation, density gradient ultracentrifugation, and agarose gel filtration. Gemfibrozil gavaged at 50 mg/kg per day for 2 weeks during cholesterol feeding prevented these changes such that lipoprotein patterns were similar to those in chow-fed animals. Whole plasma apoE and apoA-I concentrations were decreased and apoB increased due to cholesterol feeding as determined by electroimmunoassay, but again gemfibrozil treatment prevented these diet-induced alterations. Gradient polyacrylamide gel electrophoresis patterns of the total d less than 1.21 g/ml lipoprotein fractions reflected the changes in apolipoprotein concentrations and further demonstrated a greater increase of apoBl compared to apoBh in cholesterol-fed rats. Gemfibrozil lowered the concentration of both apoB variants and prevented the shift of apoE from HDL to lower density lipoproteins. Changes in the distribution of apoE were confirmed using agarose gel column chromatography followed by electroimmunoassay. These methods also revealed a shift of apoA-IV from HDL to the d greater than 1.21 g/ml, lipoprotein-free fraction with gemfibrozil treatment when blood was taken from fasted or postabsorptive animals. Since it was also noted that in chow-fed rats more apoA-IV was present in the d greater than 1.21 g/ml fraction in the postabsorptive or fed state compared to fasted animals, it could be postulated that the shift of apoA-IV into this fraction in gemfibrozil-treated rats is related to an accelerated clearance of chylomicrons. It is concluded that gemfibrozil largely prevents the accumulation of abnormal lipoproteins in this model of dyslipoproteinemia, and that apoE may play a critical role in this normalization process.     

Metabolic changes associated with selenium deficiency in mice

Selenium (Se), which is a central component for the biosynthesis and functionality of selenoproteins, plays an important role in the anti-oxidative response, reproduction, thyroid hormone metabolism and the protection from infection and inflammation. However, dietary Se effects have not well been established to date and the available studies often present contradictory results. To obtain a better understanding of Se intake and its influence on the metabolism of living systems, we have utilized a metabolomics approach to gain insight into the specific metabolic alterations caused by Se deficiency in mice. Serum samples were collected from two groups of C57BL/6 mice: an experimental group which was fed a Se-deficient diet and controls consuming normal chow. The samples were analyzed by ¹H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. The resulting metabolite data were examined separately for both analytical methods and in a combined manner. By applying multivariate statistical analysis we were able to distinguish the two groups and detect a metabolite pattern associated with Se deficiency. We found that the concentrations of 15 metabolites significantly changed in serum samples collected from Se-deficient mice when compared to the controls. Many of the perturbed biological pathways pointed towards compensatory mechanisms during Se deficiency and were associated with amino acid metabolism. Our findings show that a metabolomics approach may be applied to identify the metabolic impact of Se and reveal the most impaired biological pathways as well as induced regulatory mechanisms during Se deficiency.     

Developmental changes to gut microflora metabolism in mice

W.E. BRENNAN-CRADDOCK, A.K. MALLETT, I.R. ROWLAND AND S. NEALE. 1992. Developmental changes in the activities of bacterial nitrate reductase, nitroreductase and β-glucuronidase and their response to fermentable dietary fibre, were investigated in caecal contents from suckling mice (2-week-old) and in mice aged 4–24 weeks fed either a purified fibre-free diet or that diet supplemented with 5% (w/w) pectin. There was no apparent age-related trend common to the three enzymes studied. Nitrate reductase activity in the mice fed the fibre-free diet did not markedly alter with age. Pectin administration, however, was associated with a significant increase in nitrate reductase activity, particularly in 4-week-old mice. Nitroreductase activity exhibited an overall upward trend in mice from 2 to 12 weeks and thereafter decreased. Caecal β-glucuronidase activity in mice increased sharply between 2 weeks and 4 weeks of age, thereafter not changing significantly until the 24th week. Pectin feeding had no consistent effect on activities either of nitroreductase or β-glucuronidase. The changes in enzyme activities with age were not related to the concentration of bacteria in the caecum, which was highest in the 2-week-old mice.
We conclude that the weaning is a period in which marked changes in caecal bacterial enzyme activities can occur.     

Transport changes associated with growth control and malignant transformation.

We can distinguish two classes of membrane transport changes in cultured cells: (a) growth-rate contingent changes are those which occur in coordination with the onset of density-dependent inhibition of growth; (b) transformation-specific changes are those which occur when cells become transformed, and which can be detected even when normal and transformed cells are growing at the same rate. Growth-rate contingent changes include the density-dependent changes in phosphate, nucleoside, glucose, amino acid, and potassium transport. Only one transformation-specific transport change has been found in Rous-transformed chicken embryo fibroblasts: an increased rate of hexose transport. The variation in potassium transport are associated with variations in the number of ouabain binding sites in the membrane. The molecular basis for changes in the rate of hexose transport is unknown, although gross changes in membrane bilayer composition and "fluidity" seem not to be involved. In analyzing the regulation of hexose transport activity, we find that decreased cAMP may play a role in the transformation-specific increase in hexose transport, but that fibrinolytic activity is not necessary.     

A rapid radioimmunoassay method of growth hormone in dog plasma.

A rapid and sensitive homologous radioimmunoassay (RIA) system is described for the measurement of growth hormone (GH) in dog plasma. The method requires only 40 hr and is able to detect concentrations of GH as low as 1.0 ng/ml of dog plasma. Antiserum to canine growth hormone (cGH) prepared in monkeys, exhibited a complete cross-reactivity with porcine GH, suggesting that the latter can substitute cGH in a heterologous radioimmunoassay for cGH. Studies on GH regulation performed with this RIA in the unanesthetized dog showed that this species resembles the primate more than the rat or the rabbit.     

Effect of absolute and relative changes of hematocrit on erythropoiesis in mice

The serial changes of peripheral reticulocytes and marrow erythroid progenitor cells (CFU-e) in mice were monitored under the conditions of absolute or relative changes in red cell mass to study the regulatory mechanism of erythropoiesis. A decreased number of marrow CFU-e and peripheral reticulocytes was observed in the mice with relative polycythemia induced by dehydration as well as in the mice with absolute polycythemia induced by hypertransfusion. On the other hand, a transient increase in the number of marrow CFU-e followed by a gradual increase in the number of peripheral reticulocytes was seen after a considerable amount of exsanguination. Similar stimulatory effects on marrow CFU-e were also observed either by rehydrating the dehydrated mice or by overhydrating the untreated mice to relatively decrease the level of hematocrit. The results suggested that in addition to factors relating to the balance between oxygen supply and requirement, which has been well known, erythropoiesis is greatly affected by hematocrit.