Interleukin-6 induces the synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity (TIMP-1/EPA).

This study examines the role of interleukin-6 (IL-6) in connective tissue metabolism. Effects of different preparations of IL-6 on production of collagenase and tissue inhibitor of metalloproteinases-1/erythroid potentiating activity production are studied in human fibroblasts, synoviocytes, and articular chondrocytes. In contrast to interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha), IL-6 does not stimulate the production of collagenase, nor does it modulate the stimulatory effects of IL-1 beta and TNF alpha on the production of this proteinase. Furthermore, IL-6 has no detectable effect on prostaglandin E2 production, an additional proinflammatory response induced by IL-1 beta and TNF alpha. IL-6, however, is identified as a potent inducer of de novo synthesis of tissue inhibitor of metalloproteinases-1/erythroid potentiating activity in all types of connective tissue cells examined. These results define new biological activities of IL-6 and provide further insight into the regulation of connective tissues by cytokines.     

Macromolecular synthesis in organ cultures of neonatal rat lung

Organ cultures of newborn rat lungs synthesize and accumulate DNA, RNA, collagen and noncollagenous proteins almost at a linear rate for at least 5 days. During this period the synthesis of collagen consistently exceeds the synthesis of noncollagenous proteins in a pattern similar to neonatal lung growth in vivo. Although some morphological characteristics of lung architecture are distorted after culture, fundamental structural similarities to lungs growing in intact animals are retained. When these cultures are maintained in atmospheres rich in oxygen, increased collagen synthesis is observed, a response similar to that of lungs in intact animals exposed to high oxygen concentrations in vivo. Our studies suggest that lung organ cultures may be a suitable system for investigating the biochemical aspects of lung tissue-environmental interaction.     

Macromolecular synthesis in organ cultures of neonatal rat lung

Organ cultures of newborn rat lungs synthesize and accumulate DNA, RNA, collagen and noncollagenous proteins almost at a linear rate for at least 5 days. During this period the synthesis of collagen consistently exceeds the synthesis of noncollagenous proteins in a pattern similar to neonatal lung growth in vivo. Although some morphological characteristics of lung architecture are distorted after culture, fundamental structural similarities to lungs growing in intact animals are retained. When these cultures are maintained in atmospheres rich in oxygen, increased collagen synthesis is observed, a response similar to that of lungs in intact animals exposed to high oxygen concentrations in vivo. Our studies suggest that lung organ cultures may be a suitable system for investigating the biochemical aspects of lung tissue-environmental interaction. These studies were supported in parts by NIH Grant HL-19668, a contract (68-03-2005) from the U.S. Environmental Protection Agency, and grants from the California Lung Association.     

Hemoglobin switching in sheep and goats. VI. Commitment of erythroid colony-forming cells to the synthesis of betaC globin

Bone marrow from mature goats and sheep was cultured in plasma clots, and three erythropoietin (ESF)-dependent responses-growth (colony formation), differentiation (globin production), and initiation of hemoglobin C (alpha2beta2C) synthesis--were quantitated. ESF concentrations below 0.01 U/ml supported colony growth and adult hemoglobin production in cultures of goat marrow, while maximal hemoglobin C synthesis (70%), as measured between 72 and 96 h in culture, required a 100-fold higher ESF concentration. Sheep marrow was cultured in a medium enriched to enhance growth and to permit complete maturation of colonies. These colonies active in hemoglobin synthesis between 24 and 96 h produced mainly adult hemoglobin, and only between 96 and 120 h did sheep colonies develop which produced mainly hemoglobin C (up to 70%). A similar heterogeneity may exist among goat colonies. Thus, when goat bone marrow was fractionated by unit gravity sedimentation, more hemoglobin C synthesis was observed in colonies derived from cells of intermediate sedimentation velocity than in colonies derived from the most rapidly sedimenting cells. Brief exposure of sheep (in vivo) and goat (in vitro) bone marrow to a high ESF concentration committed precursor cells to the generation of colonies which, even at low ESF concentration, produced hemoglobin C. Committment to hemoglobin phenotype appears to be an early and probably irreversible event in the development of an erythroid cell.     

Hemoglobin switching in sheep and goats. V. Effect of erythropoietin concentration on in vitro erythroid colony growth and globin synthesis

Erythroid colonies were generated in response to erythropoietin in plasma clot cultures of sheep and goat bone marrow cells. At low concentration erythropoietin only hemoglobin A (betaA globin) was synthesized in goat cultures, but at high concentrations 50% of the hemoglobin synthesized was hemoglobin C (betaC globin). This effect of erythropoietin on the expression of a specific beta globin gene was manifested only after 72 h in vitro and followed the development of erythroid colonies. Sheep colonies behaved differently from those of goat in that little or no betaC globin synthesis occurred even at high erythropoietin concentration. To investigate this difference, sheep marrow cells were fractionated by unit gravity sedimentation. The erythroid colony-forming cells sedimented more rapidly (3.5-6mm/h) than the hemoglobinized eththroid precursors (1-3.5 mm/h), suggesting that the colonies were formed from an early erythroid precursor, However, the colonies formed from the sheep marrow fractions synthesized only betaA globin even at concentrations of erythropoietin sufficient to stimulate betaC globin synthesis in goat colonies. Morphologically, the goat colonies were larger and more mature than those of the sheep. By 96 h in vitro three-fourths of the goat colonies contained enucleated red cells compared to only 3% of the sheep colonies. Thus, erythropoietin had an equivalent effect in stimulating erythroid colony growth from the marrow of both species although there were both biochemical and morphological differences between the colonies. Hemoglobin switching appeared to require exposure of an early precursor to high erythropoietin concentration, but the results with sheep marrow suggested that the rate of colony growth and cellular maturation might also be important.     

Determination of antidiabetic activity in Allium cepa (onion) tissue cultures.

Seedling, seedling parts and callus cultures of onion were tested for their antidiabetic activity by feeding the tissue-extracts to diabetic rats. The results indicated much higher antidiabetic activity in callus cultures as compared to natural bulbs of onion. These results may be of pharmaceutical significance since the callus can be used as an alternative source for the isolation of antidiabetic compounds.     

Increase in erythroid colony formation in rabbits following the administration of testosterone.

Changes in the numbers of erythroid colonies formed in cultures from rabbits pretreated with either testosterone or busulfan plus testosterone were studied using a methyl cellulose gel system. The mean number of erythroid colonies in bone marrows from rabbits treated with testosterone in vivo was significantly higher than that of controls. However, this increase in erythroid colonies in cultures seen following testosterone treatment was completely blocked by the concurrent administration of busulfan as seen in the rabbits treated with busulfan orally and followed by testosterone injections. Busulfan has been postulated block the formation of new erythropoietin responsive cells (ERC) from hematopoietic stem cells (CFU). Thus, these findings suggest that testosterone may act directly on CFU to enhance their differentiation into the ERC compartment causing an increase in nucleated erythroid cells.     

Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.     

Increase of degree of spermidine stimulation of polypeptide synthesis in the presence of phosphate

The addition of phosphate caused an increase in the degree of spermidine stimulation of polypeptide synthesis in an Escherichia coli and a wheat germ cell-free system. Optimal stimulation of polypeptide synthesis was observed at 20 mm phosphate for both systems, but concentrations of phosphate up to 40 mm had no additional effect. The increase of degree of spermidine stimulation in the presence of phosphate in an E. coli cell-free system occurred at the level of aminoacyl-tRNA binding to ribosomes and not at the level of peptide bond formation, translocation, or aminoacyl-tRNA formation. From the results of studies on RNase A sensitivity of ribosomal subunits and the effect of antibiotics known to act on the 30 S ribosomal subunits, it is suggested that the nature of the 30 S ribosomal subunits is changed by phosphate so that the degree of spermidine stimulation of polypeptide synthesis is increased.     

Hemoglobin of tree sparrows (Passer montanus, Passeriformes): Sequence of the major (Hb A) and minor (Hb D) components

Blood of the adult Tree Sparrow (Passer montanus) contains two hemoglobin components, Hb A (ca. 85%), Hb D (ca. 15%). They differ in their alpha-chains (alpha A, alpha D), the beta-chains are identical. The complete primary structures of alpha A-, alpha D- and beta-chains are presented. Comparison with the Greylag Goose (Anser anser) hemoglobin (Hb A) showed that the alpha A-chains differ by 22 amino-acid exchanges, the beta-chains by 16. Comparison with the minor component of the Pheasant (Phasianus colchicus colchicus) hemoglobin (Hb D) showed that the alpha D-chains differ by 34 amino-acid exchanges. Proline is found incorporated in an internal position of an alpha-helix (pos. 124, H7). In comparison to that of the Starling (Sturnus vulgaris) the ratio of amino-acid exchanges for beta: alpha A: alpha D chains is 1 : 7 : 4; in comparison to other birds this ratio is found to be 1 : 2 (1.4-2.2):3 (2.2-4).     

Regulation of product synthesis in cell cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle Catharanthus roseus (L) G. Don were maintained on Gamborg's B5 medium and their growth monitored by measuring cellular fresh and dry weight, cell number and mitotic activity. Samples of cells of different ages and physiological states were subcultured onto an alkaloid production medium and their rates of growth and alkaloid accumulation measured over a period of 30–45 days. In two experiments the rate of biomass accumulation was directly related to the rate of cellular serpentine accumulation. Possible mechanisms underlying this phenomenon are discussed in relation to the properties of cells comprising the inocula.     

The effect of progesterone on hemoglobin synthesis in suspension cultures of fetal erythroid cells from calf liver

When fetal calf liver erythroid cells were incubated in the presence of small amounts of progesterone (10(-7)-10(-8) M), the hemoglobin synthesis in these cells was significantly increased. The increase in the amount of radioactivity in de novo synthesized hemoglobins could be demonstrated when techniques such as isoelectric focusing, chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100 were used to isolate the hemoglobin fraction. Using the latter technique, it was shown that the synthesis of cytoplasmic non-hemoglobin proteins in erythroid-cell lysates was also stimulated by progesterone. The presence of hepatocytes in culture nullified the hormone action. It was necessary that progesterone was present during the first hours of culture. Delayed addition of the steroid to the cells had no effect on hemoglobin synthesis. Erythropoietin was necessary to obtain stimulation by progesterone. These results suggest that the target cell of the hormone is an erythropoietin-sensitive cell. High concentrations of progesterone (10(-4) M) strongly inhibited hemoglobin synthesis in fetal calf erythroid cells. Culture of cells under this condition, however, gives rise to a cell population that preferentially synthesizes adult hemoglobin. Our results suggest that in the erythropoietic calf liver, high concentrations of progesterone may preferentially stimulate adult hemoglobin synthesis, or that those cells which have a high capacity to synthesize adult hemoglobins are less sensitive to toxic concentrations of the hormone. The effects of stimulation of hemoglobin synthesis in fetal calf erythroid cells occur at hormone concentrations that suggest a possible physiological role of progesterone in fetal, and eventually also in maternal, erythropoiesis.     

Phenolic synthesis and phenylalanine ammonia-lyase activity in suspension cultures of Acer pseudoplatanus L.

Summary Phenolic metabolism is influenced by the levels of sucrose, nitrogen and 2,4 dichlorophenoxyacetic acid (2,4-D) in the growth medium. Chromatographic evidence suggests that the principle products are polymers of leucocyanin, (-) epicatechin and (+) catechin, constituting condensed tannins. Comparison of ethanolic cell extracts with extracts from plant organs shows that although these compounds are present in parts of the plant they are not the major phenolics.Cells maintained in a modified Heller's medium containing 9.0×10^–7 M 2,4-D produce increased levels of tannins from mid passage (day 12) onwards. The presence of 2,4-D at 9.0×10^–6 M supresses this response and increased initial sucrose levels cause the amount of tannins to be greater. At the period when tannin levels increase the standard medium is exhaused of its nitrogen sources, urea and nitrate. Increased initial nitrogen levels delay the beginning of increased tannin production and the addition of urea or 2,4-D to cultures already containing high levels of tannins causes the tannin content per gram fresh weight and per culture to decline. These results indicate an antagonism between tannin synthesis and nitrogen metabolism. The activity of phenylalanine ammonia-lyase EC 4.1.1.5. (PAL) estimated by a spectrophotometric method in acetone powders derived from Acer cells increases three to four fold at the onset of increased tannin synthesis and then declines sharply. The phase of high PAL activity correlates with the exhausion of the medium nitrogen sources.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid One of the authors (R.J.W.) was supported by a Science Research Council Studentship during the course of this work.     

Glycosylated Haemoglobin (Hb A1) in Normal Rhesus Monkeys (Macaca mulatta)

Glycosylated haemoglobins were measured in 23 healthy juvenile rhesus monkeys by the use of commercially available minicolumn chromatography (Quick Sep., Isolab Inc., Ohio, USA) to establish the normal range. Values obtained (mean ± 1 standard deviation (SD) 1.57 ± 0.68%) were significantly lower than that of 17 adult healthy human volunteers by the use of the same method of estimation (mean ± 1 SD of 5.34 ± 0.78%).     

Increase in nitrate reductase activity in the presence of sucrose in bean leaf segments

The supply of sucrose to leaf segments from light-grown bean seedlings caused a substantial increase in substrate inducibility of in vivo and in vitro nitrate reductase activity but only a small increase in total protein. Cycloheximide and chloramphenicol inhibited the increase in enzyme activity by nitrate and sucrose. The in vivo decline in enzyme activity in nitrate-induced leaf segments in light and dark was protected by sucrose and nitrate. The supply of NADH also protected the decline in enzyme activity, but only in the light. In vitro stability of the extracted enzyme was, however, unaffected by sucrose. The size of the metabolic nitrate pool was also enhanced by sucrose. The experiments demonstrate that sucrose has a stimulatory effect on activity or in vivo stability ' of nitrate reductase in bean leaf segments, which is perhaps mediated through increased NADH level and/or mobilization of nitrate to the metabolic pool.