Biomedical subjects

D K Stone

Publications and source records attributed to D K Stone.

46 records · Page 3Linked to original sources

PubMed1986

Lipid requirements for reconstitution of the proton-translocating complex of clathrin-coated vesicles.

We have recently reported the reconstitution of the clathrin-coated vesicle proton-translocating complex with liposomes prepared from ethanol-extracted crude bovine brain lipids. Reconstitution of proton pumping by the isolated proton ATPase has now been achieved with liposomes prepared from pure lipids. Optimal proton pumping, as assessed by ATP-generated acridine orange quenching and 32Pi-ATP exchange, is achieved with liposomes prepared from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol at a weight ratio of 40:26.5:7.5:26, and a lipid-to-protein weight ratio of 200:1. Under such conditions, the extent of decrease of ATP-generated acridine orange quenching is 150-fold greater than that of native clathrin-coated vesicles. Cholesterol is required to stabilize the proteoliposome. Phosphatidylserine, which is most effective in activating ATP-hydrolytic activity of the solubilized enzyme, is not obligatory for reconstitution of proton pumping.

Animals↗

PubMed1986

High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry.

Calelectrins are a family of antigenically related Ca2+-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca2+. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.

Animals↗

PubMed1984

Activation and partial purification of the ATPase of clathrin-coated vesicles and reconstitution of the proton pump.

A N-ethylmaleimide-sensitive ATPase was extracted and partially purified from clathrin-coated vesicles of bovine brain. During purification the enzyme lost activity which was restored by a purified phospholipid fraction from brain. Phosphatidylserine, but no other commercial phospholipids tested, replaced the brain lipid fraction as activator. Particles depleted of the ATPase exhibited no H+ pump activity when reconstituted with brain phospholipids by the cholate dilution procedure. H+ pump activity was restored by incubating the reconstituted vesicles with the partially purified ATPase.

Animals↗

PubMed1984

Inhibition of clathrin-coated vesicle acidification by duramycin.

Clathrin-coated vesicles contain a proton translocating ATPase which operates in parallel with a chloride transporter (Xie, X.-S., Stone, D.K., and Racker, E. (1983) J. Biol. Chem. 258, 14834-14838). The polypeptide antibiotic, duramycin, has a dual inhibitory effect on clathrin-coated vesicle acidification. Low amounts of duramycin (5 micrograms/100 micrograms of protein) inhibit by 50% the proton translocation facilitated by chloride translocation. Under these conditions duramycin inhibits also 36Cl uptake when driven by either the electrogenic proton pump or by inward directed K+ movement in the presence of valinomycin. Higher amounts of duramycin (20 micrograms/100 micrograms of protein) are needed to inhibit by 50% the proton pump itself, as evidenced by reduced proton translocation facilitated by an outward potassium movement in the presence of valinomycin. In addition, the amount of duramycin needed to inhibit the proton pump corresponded well with the amount needed to inhibit the ouabain-insensitive, N-ethylmaleimide-sensitive ATPase activity of clathrin-coated vesicles.

Animals↗

PubMed1984

A Chinese hamster ovary cell mutant with a heat-sensitive, conditional-lethal defect in vacuolar function.

We describe a mutant derived from Chinese hamster ovary cells that is offt-sensitive for viability and for resistance to certain protein toxins. This mutant, termed G.7.1, grows normally at 34 degrees C but does not grow in Dulbecco's modified Eagle's medium at 39.5 degrees C. However, when this medium is supplemented with FeSO4, the mutant cells will grow at the elevated temperature. At 39.5 degrees C, G.7.1 cells acquire resistance to diphtheria toxin, modeccin, and Pseudomonas aeruginosa exotoxin A, all of which are protein toxins that require endocytosis and exposure to a low pH within vesicles before they can invade the cytosol and kill cells. The properties of mutant G.7.1 could result from a heat-sensitive lesion that impairs vacuolar acidification. We assayed the ATP-stimulated generation of pH gradients across the membrane of vesicles in cell-free preparations from mutant and parental cells by the partitioning of acridine orange into acidic compartments and found that the acidification response of the mutant cells was heat-labile. Altogether the evidence suggests that G.7.1 cells contain a heat-sensitive lesion that impairs vacuolar acidification and that they fail to grow in normal medium at 39.5 degrees C because they cannot extract Fe+3 from transferrin, a process that normally requires exposing transferrin to a low pH within endosomal vesicles.

Animals↗

PubMed1983

Determinants of clathrin-coated vesicle acidification.

The proton-translocating ATPase of clathrin-coated vesicles of bovine brain is characterized by ATP specificity. Chloride or bromide serve as co-ions balancing electrogenic proton pumping. ATP-driven proton translocation can be observed in the absence of chloride, provided the membrane potential is collapsed by K+ moving out in the presence of valinomycin. Chloride transport can be observed independent of proton movements in the absence of ATP, provided an inside positive membrane potential is generated by K+ moving from the outside to the inside in the presence of valinomycin.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1983

An ATP-driven proton pump in clathrin-coated vesicles.

Clathrin containing coated vesicles prepared from bovine brain catalyzed ATP-driven proton translocation and a 32Pi-ATP exchange reaction. Both activities were measured in the presence of 5 micrograms of oligomycin/mg of protein which completely inhibited these reactions catalyzed by submitochondrial particles. Analyses performed during the purification procedure demonstrated that the oligomycin-resistant pump was concentrated and highly purified in the fractions containing coated vesicles. Moreover, vesicles precipitated by either monoclonal or polyclonal antibodies against clathrin contained the H+ pump activity. Dicyclohexylcarbodiimide (0.5 mM) and N-ethylmaleimide (1 mM) added to the assay mixture inhibited the pump completely, whereas neither vanadate, sodium azide, efrapeptin, or mitochondrial ATPase inhibitor had an effect.

Adenosine Triphosphatases↗

PubMed1983

Anion dependence of rabbit medullary collecting duct acidification.

Rabbit medullary collecting duct (MCD) acidification has been demonstrated to occur by means of a sodium-independent, aldosterone-stimulated mechanism. We have examined the anionic dependence of this process by means of the isolated perfused tubule technique. Total replacement of perfusate chloride with gluconate enhanced tubular bicarbonate reabsorption (JHCO3), from a basal rate of 10.7 +/- 1.0 pmol X mm-1 X min-1 to a rate of 15.01 +/- 1.0 pmol X mm-1 X min-1. Removal of bath chloride, with and without removal of perfusate chloride completely abolished acidification. Bath, but not luminal 4-acetamido-4' isothiocyano-2,2'-disulfonic stilbene provoked a marked decrease in JHCO3 from 10.1 +/- 1.2 pmol X mm-1 X min-1 to 2.3 +/- 0.3 pmol X mm-1 X min-1. Measurement of chloride reabsorptive rate (JCl) revealed colinearity between JHCO3 (9.18 +/- 0.9 pmol X mm-1 X min-1) and JCl (9.75 +/- 1.18 pmol X mm-1 X min-1). We propose a model of mammalian distal nephron acidification in which (a) cellular base exit is effected by means of a basolateral membrane Cl-base exchanger and (b) net electroneutrality of electrogenic proton secretion is maintained by the parallel movement of an anionic species, functionally chloride.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1983

Mineralocorticoid modulation of rabbit medullary collecting duct acidification. A sodium-independent effect.

Rabbit medullary collecting duct (MCD) from inner stripe of outer medulla has been identified as a major distal nephron acidification site. The isolated, perfused tubule technique was used to examine the roles of mineralocorticoid and glucocorticoid in regulation of MCD acidification. Surgical adrenalectomy reduced bicarbonate reabsorptive rate (JHCO3, pmol X mm-1 X min-1) from the normal of 9.79 +/- 1.21 to 0.67 +/- 1.1. Chronic administration of deoxycorticosterone acetate (DOCA) increased JHCO3 of MCD significantly to 18.02 +/- 1.62 whereas chronic dexamethasone administration did not affect JHCO3. The direct effects of aldosterone and dexamethasone upon MCD acidification were examined by perfusing tubules harvested from adrenalectomized rabbits in the presence of aldosterone or dexamethasone. Aldosterone, at 5 X 10(-8) M, increased JHCO3 significantly from 1.27 +/- 0.28 to 3.09 +/- 0.34. At 10(-6) M, aldosterone produced a greater increase in JHCO3 from 0.67 +/- 1.1 to 9.39 +/- 1.59. In vitro dexamethasone treatment had no effect on JHCO3. Studies examining the sodium dependence of aldosterone-stimulated acidification demonstrated that JHCO3 in tubules harvested from normal and deoxycorticosterone acetate-treated animals was unaffected by total replacement of sodium with tetramethylammonium. Likewise, luminal amiloride (5 X 10(-5) M) had no effect on JHCO3 in tubules harvested from adrenalectomized and normal animals. Moreover, the acute, in vitro stimulatory effect of aldosterone was seen to occur in the presence of luminal amiloride. These studies define a mammalian distal nephron segment that possesses major acidifying capacity, which is modulated by mineralocorticoid but independent of luminal sodium.

Absorption↗

PubMedDate not supplied

Stimulation of haematopoiesis in primates by continuous infusion of recombinant human GM-CSF.

Certain proteins are known to play an important part in the proliferation, differentiation and functional activation of haematopoietic progenitor cells in vitro. These proteins include erythropoietin and various colony-stimulating factors (CSFs), one of which is granulocyte-macrophage colony-stimulating factor (GM-CSF). Recently, both murine and human GM-CSF have been purified to homogeneity and complementary DNAs encoding them have been cloned. Although the in vitro activity of recombinant human GM-CSF has been investigated intensively, little is known about the functional activity of this protein in vivo. There is strong evidence that colony-stimulating activities produced by various human and murine tumour tissues and cell lines can stimulate granulopoiesis in mice, as can human urinary extracts. A partially purified preparation of human urinary colony-stimulating factor, however, proved only marginally effective in stimulating granulopoiesis in humans. All these studies suffer from the lack of a homogeneous preparation of colony-stimulating factor. It has recently been shown that recombinant murine multi-CSF or interleukin-3 can stimulate haematopoiesis in mice in vivo. Large-scale production of recombinant human GM-CSF now permits us to examine its effects in vivo using a primate model. We find that the continuous infusion of GM-CSF in healthy monkeys rapidly elicits a dramatic leukocytosis and a substantial reticulocytosis. A similar effect has been observed in one pancytopenic, immunodeficient rhesus macaque. These results suggest that GM-CSF could prove useful in several clinical situations.

Animals↗