Tissue producing the serum factor stimulating the release of cathepsin D from lysosomes in vitro

Pancreatectomy as well as thyroparathyroidectomy resulted in the quick disappearance of a serum factor (stimulating cathepsin D release from lysosomes in vitro) from the rat or mouse blood. Extirpation of other organs such as duodenum, stomach, spleen, kidney, submaxillary gland, testis, adrenal gland or hypophysis, showed no effect on the serum factor level. Glucagon (but not insulin or thyroxine) given to the pancreatectomized animals restored the serum factor level in a dose-dependent manner. The serum factor-like activity was detected only in the parathyroids (but not thyroid), and the release of activity from parathyroid-slices was stimulated by glucagon, suggesting that the parathyroid may produce and/or secrete the serum factor under the influence of glucagon.     

Pyruvate flux into resealed ghosts from human erythrocytes

The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ranged from 15.0 mM at $\text{μ = 0.05}$ to 3.7 mM at $\text{μ = 0.01}$, while the $\text{V}$ went from $\text{0.611 · 10}{}^{-15}$ to $\text{0.137 · 10}{}^{-15}\text{mol}\text{·}\text{min}{}^{-1}\text{·}\text{ghost}{}^{-1}$. Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, α-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4′-isothiocyanate-stilbene-2,2′-disulfonic acid and o-phenanthroline/CuSO₄ mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.     

Pyruvate flux into resealed ghosts from human erythrocytes.

The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The Km ranged from 15.0 mM at mu = 0.05 to 3.7 mM at mu = 0.01, while the V went from 0.611 - 10(15) to 0.137 - 10(-15) mol - min-1 - ghost-1. Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, alpha-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4'-isothiocyanate-stilbene-2,2'-disulfonic acid and o-phenanthroline/CuSO4 mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.     

Amino acid transport by resealed ghosts from pigeon erythrocytes.

Resealed ghosts from pigeon erythrocytes were shown to haemolyse during incubation in isotonic media with pH values greater than about 7 and high concentrations of Na+ inside the ghosts seemed to enhance this effect. At lower pH values the ghosts were stable but still highly permeable to Na+ and K+, and moderately permeable to sucrose. Under the latter conditions the ghosts transported amino acids in a way qualitatively but not quantitatively similar to intact erythrocytes. The Na+-dependent transport of serine and alanine by the ghosts consisted essentially of an exchange of extracellular for intracellular amino acids, with no significant net flux. In contrast, net fluxes of glycine in the direction of the Na+-concentration gradient across the ghost membrane were demonstrated. However, under one condition a small net influx of glycine occurred against the prevailing Na+-concentration gradient. Unlike Na+-dependent glycine uptake, the uptake of six other amino acids by intact pigeon erythrocytes was not influenced by the nature of the anion present. The significance of these findings in relation to previous work on the Na+-gradient hypothesis of membrane transport is discussed.     

Isolation of an erythropoietic stimulating factor from the serum of anemic chicks

A factor stimulating fowl erythropoiesis in vitro has been isolated from the blood of anemic chick by gel filtration and ion exchange chromatography. This purification procedure yields a preparation whose specific activity is enriched 44 times and which has been relieved of toxic factors present in the crude anemic serum. In vitro erythropoiesis requires addition into the culture medium of erythropoietic stimulating factor and of another factor present in the serum of anemic as well as non-anemic chick. This last factor is lost during purification of the erythropoietic stimulating factor and is not present in fetal calf serum.     

A heparin-binding erythroid cell stimulating factor from fetal bovine serum has the N-terminal sequence of insulin-like growth factor II

An 8 kd heparin-binding peptide which stimulates thymidine incorporation in cultures of fetal calf liver erythroid cells was isolated from fetal bovine serum by affinity chromatography on Heparin-Sepharose, ion exchange chromatography, gel filtration and reversed-phase HPLC. The N-terminal sequence of the isolated peptide was identical to the N-terminal sequence of bovine erythrotropin or insulin-like growth factor II (IGF II). The potential heparin-binding site of IGF II is probably situated in the arginine-rich C-peptide region. The affinities of human recombinant IGF I and II were compared with those of apolipoprotein H (a plasma heparin-binding protein) and bovine insulin in a heparin-affinity column. The retention times were in the order: Apolipoprotein H greater than hrIGF II greater than hrIGF I greater than insulin (no retention). This unusual property of IGF II suggests that it may be captured in the extracellular matrix in a similar way to fibroblast growth factor, interleukin 3 or granulocyte/macrophage colony-stimulating factor.     

Thiamin transport by human erythrocytes and ghosts

Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [³H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na⁺ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K _m =0.12, 0.11 and 0.10 m andJ _max=0.01, 0.02 and 0.03 pmol·l^–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K _m =0.16 and 0.51 m andJ _max=0.01 and 0.04 pmol·l^–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN^– showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.     

Thyroid stimulating hormone upregulates secretion of cathepsin B from thyroid epithelial cells

Constant levels of thyroid hormones in the blood are principal requirements for normal vertebrate development. Their release depends on the regulated proteolysis of thyroglobulin which is extracellularly stored in the follicle lumen under resting conditions. Thyroglobulin is proteolytically degraded to a major part in lysosomes, but in part also extracellularly leading to the release of thyroxine. Extracellularly occurring lysosomal enzymes are most probably involved in the proteolytic release of thyroxine. In this study we have analyzed the secretion of cathepsin B by thyroid follicle cells (primary cells as well as FRTL-5 cells) and its regulation by thyroid stimulating hormone, which stimulated the secretory release of the proenzyme as well as of mature cathepsin B. Within one to two hours of stimulation with thyroid stimulating hormone, the cathepsin B activity associated with the plasma membrane increased significantly. This increase correlated closely with the localization of lysosomes in close proximity to the plasma membrane of cultured thyrocytes as well as with the thyroxine liberating activity of thyrocyte secretion media. These observations indicate that thyroid stimulating hormone induces the secretion of cathepsin B, which contributes to the extracellular release of thyroxine by thyrocytes.     

A serum factor capable of stimulating hyaluronic acid synthesis in cultured rat fibroblasts.

Calf serum as well as rat and mouse sera has a factor that stimulates hyaluronic acid synthesis in cultured rat fibroblasts. Such a factor was partially purified from calf serum and characterized. It has a molecular weight of approximately 150,000. The activity of the factor is lost by treatment with pronase and by periodate oxidation. It is suggested, therefore, that the factor is a glycoprotein. Its susceptibility to alpha-mannosidase and affinity for Con A-Sepharose may suggest that the factor contains a mannose residue(s) which is essential for the activity to induce hyaluronic acid synthesis.     

Fractionation of Plasmodium chabaudi-infected erythrocytes into parasites and ghosts

We have developed a new procedure for subfractionation of Plasmodium chabaudi-infected erythrocytes into parasites and ghosts. Trophozoite-infected erythrocytes enriched over a percoll-step (:1.10 g/cm3) are subjected to a glycerol-enhanced osmotic shock. This induces the release of parasites and the emergence of erythrocyte ghosts, which can be separated on a continuous percoll gradient (:1.02----1.10 g/cm3). The parasites are intact in terms of ultrastructure and incorporation of 14C-isoleucine. The erythrocyte ghosts are purified over a two-step percoll gradient (:1.01-1.02 g/cm3). Our method recovers about 40%-50% of the initial ghosts and 70%-95% of the initially freed parasites.     

Diffusional water permeability of human erythrocytes and their ghosts

The diffusional water permeability of human red cells and ghosts was determined by measuring the rate of tracer efflux by means of an improved version of the continuous flow tube method, having a time resolution of 2-3 ms. At 25 degrees C, the permeability was 2.4 x 10(3) and 2.9 x 10(3) cm s-1 for red cells and ghosts, respectively. Permeability was affected by neither a change in pH from 5.5 to 9.5, nor by osmolality up to 3.3 osmol. Manganous ions at an extracellular concentration of 19 mM did not change diffusional water permeability, as recently suggested by NMR measurements. A "ground" permeability of 1 x 10(3) cm s-1 was obtained by inhibition with 1 mM of either p- chloromercuribenzoate (PCMB) or p-chloromercuribenzene sulfonate (PCMBS). Inhibition increased temperature dependence of water permeability for red cells and ghosts from 21 to 30 kJ mol-1 to 60 kJ mol-1. Although diffusional water permeability is about one order of magnitude lower than osmotic permeability, inhibition with PCMB and PCMBS, temperature dependence both before and after inhibition, and independence of osmolality showed that diffusional water permeability has qualitative features similar to those reported for osmotic permeability, which indicates that the same properties of the membrane determine both types of transport. It is suggested that the PCMB(S)- sensitive permeability above the ground permeability takes place through the intermediate phase between integral membrane proteins and their surrounding lipids.     

A low-cost automated device incorporating a hollow fiber filtration cartridge for large-scale production of ghosts from human erythrocytes

Readily available elements were used to build an automatic apparatus dedicated to the preparation of erythrocyte ghosts. The apparatus is designed around a low-cost re-usable hollow-fiber filtration cartridge (marketed for therapeutic plasmapheresis). The apparatus is controlled by a simple programmer (based on a diode matrix and low cost timers and liquid level sensors): once the apparatus is loaded with whole red blood cells, washing of cells, as well as hemolysis and washing of ghosts, is performed by the machine in about 4.5 h without any operator intervention. Automatic filter cleaning takes a further 110 min.     

Characteristics of a sporulation stimulating factor from Clostridium perfringens type A

A product in the culture supernatant fluid of Clostridium perfringens NCTC 8239 stimulated the sporulation of a test strain, NCTC 8679, of the same organism. The responsible factor, termed sporulation factor (SF), was present in seven cultures of Cl. perfringens grown in either a defined or complex medium. The SF reversed glucose-mediated catabolite repression of sporulation by this organism. Preliminary characterization of the SF demonstrated a resistance to elevated temperatures and proteases and a molecular weight of less than 500 Da. The known association of Cl. perfringens enterotoxin with sporulation highlights the importance of interactions between strains of this organism as may occur in the human intestine during foodborne illness.