Search PubMed⌕ Search

Biomedical subjects

E Gruenstein

Publications and source records attributed to E Gruenstein.

At least 37 records · Page 2Linked to original sources
PubMed1988

Lithium transport in human fibroblasts: relationship to RBC lithium transport and psychiatric diagnoses.

Cultured fibroblasts were prepared from six normal controls, five DSM-III manic patients, and six DSM-III schizophrenic patients. Lithium (Li+) uptake, 24-hour Li+ ratios, and steady-state membrane potential were measured in these cell lines. The uptake of 10 mM Li+ reached maximum at 2 hours, with an intracellular concentration of approximately 15 mM. No significant difference in uptake was found among subject groups. Twenty-four hour Li+ (ratio of intracellular/extracellular Li+) ratios were determined by incubating the cell lines for 24 hours in the presence of 2 mM Li+. No significant difference was observed among groups; nor was there any significant correlation between the fibroblast 24-hour ratios and 24-hour in vitro ratios determined in donor red cells. The relationship between membrane potential and the 24 hour Li+ ratio in fibroblasts was determined. The average potential in these cell lines was -56 mV and was not affected by Li+ treatment. No correlation between the Li+ ratio and membrane potential was found.

Adult↗
PubMed1988

Immunological identification of a high molecular weight protein as a candidate for the product of the Duchenne muscular dystrophy gene.

An oligopeptide was synthesized based on translation of the nucleotide sequence of the putative exon region of clone pERT87-25 from the gene for Duchenne muscular dystrophy. Immunization of rabbits with this oligopeptide induced the formation of antibodies directed against a protein present in human, rat, and rabbit skeletal muscle. This protein, which is missing in the skeletal muscle of two patients with Duchenne muscular dystrophy, has a molecular mass of approximately equal to 320-420 kDa and is clearly different from the putative Duchenne muscular dystrophy-related protein nebulin. The data suggest that this 320- to 420-kDa protein is produced by the Duchenne muscular dystrophy gene.

Antigen-Antibody Complex↗
PubMed1988

Pathways of Cl- transport in human fibroblasts.

Three pathways of Cl- efflux were identified in normal human fibroblasts. Twenty percent of the total Cl- efflux is via an electrically conductive pathway with an efflux constant of 0.016 min-1. This pathway is insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and bumetanide but is partially inhibited by anthracene-9-carboxylic acid. Twenty-five percent of the Cl- efflux occurs via Cl- with cation cotransport having an efflux constant of 0.020 min-1. This pathway is inhibited by bumetanide and is dependent on the simultaneous presence of Na+, K+, and Cl-. Under basal conditions, the energetics of this pathway indicate that it is operating close to equilibrium. Fifty percent of the Cl- efflux occurs via an anion exchange pathway having an efflux constant of 0.040 min-1 that is inhibited by DIDS or by removal of Cl- from the extracellular medium. Together these pathways account for 95% of the total Cl- efflux.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Regulation of conductive Cl- transport in human fibroblasts.

Under normal growth conditions, approximately 20% of the efflux of Cl- from human fibroblasts occurs via an electrically conductive pathway or Cl- channel. This basal Cl- conductance is insensitive to the Cl- -anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and to the Cl- -cation cotransport inhibitor bumetanide. Exposure of the cells to dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) for 15 min increases the electrically conductive component of Cl- efflux by approximately 20%. Unlike the basal Cl- conductance, the cAMP-activated channel is DIDS sensitive, indicating that cAMP activates a different Cl- pathway from the one responsible for the basal Cl- conductance. Elevation of intracellular Ca2+ by addition of the ionophore A23187 also stimulates Cl- efflux via a DIDS inhibitable, electrically conductive Cl- pathway. That the cAMP- and Ca2+-stimulated pathways are different is suggested by the observation that simultaneous exposure of cells to optimal levels of dibutyryl cAMP and A23187 results in an increased Cl- efflux equal to the sum of the two factors acting independently. Prostaglandin E1, a known activator of adenylate cyclase, also elevates the levels of intracellular free Ca2+ in these cells and concomitantly activates both the cAMP- and the Ca2+-stimulated Cl- channels. Although regulated, Cl- channels are known to function in the modulation of nerve and muscle excitability, their role in fibroblast function is not clear.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Identification of a defective cAMP-stimulated Cl- channel in cystic fibrosis fibroblasts.

Cl- efflux from normal human fibroblasts is stimulated by elevation of cAMP and by elevation of intracellular free Ca2+. In both cases the stimulated Cl- transport occurs via electrically conductive pathways. In six lines of normal human fibroblasts, dibutyryl cAMP increased total Cl- efflux by an average of 13%. In six lines of fibroblasts from patients with cystic fibrosis, dibutyryl cAMP was without effect. The electrically conductive component of Cl- transport was increased an average of 30% by dibutyryl cAMP in normal cells and was unaffected by dibutyryl cAMP in cystic fibrosis cells. Stimulation of the Ca2+-sensitive Cl- channel by addition of A23187 increased Cl- efflux by an average of 30% in normal and 30% in cystic fibrosis fibroblasts. The data indicate that there is a defect in a cAMP-activated Cl- channel in cystic fibrosis fibroblasts.

Bucladesine↗
PubMed1985

Reversible change in the fibroblast lysosomal enzyme dipeptidyl aminopeptidase-1 (cathepsin C) related to the commercial source of fetal bovine serum in the culture medium.

The commercial source of fetal bovine serum used to supplement the growth medium of human skin fibroblasts alters the activity of the lysosomal enzyme dipeptidyl aminopeptidase-1 (DAP-1). Cells grown with one serum were found to have a threefold higher level of DAP-1 than those grown with serum from another source (P less than 0.001). The effect on DAP-1 activity was specific inasmuch as no differences were found in the activities of a variety of other lysosomal and nonlysosomal hydrolases: DAP-II, DAP-III, DAP-IV, beta-glucosidase, beta-glucuronidase, and N-acetyl-beta-galactosaminidase. The effect is reversible and is observed over a wide range of cell population doublings. Cell growth kinetics were not significantly different with the different sera.

Adult↗
PubMed1983

Increased membrane permeability to chloride in Duchenne muscular dystrophy fibroblasts and its relationship to muscle function.

Previous studies have suggested an abnormality in Cl- metabolism in Duchenne muscular dystrophy (DMD) fibroblasts. In order to further characterize this abnormality, we have studied 36Cl- distribution and permeability in 11 DMD and 12 normal fibroblast lines. Under steady-state conditions Cl- efflux in fibroblasts is observed to be biphasic, revealing the presence of two major subcellular compartments. Each compartment contains approximately half of the cellular Cl-. The faster of the two observed efflux components is significantly higher in DMD than in control fibroblasts (P less than 0.001). To determine the results of a similar increase in Cl- permeability on skeletal muscle action potentials, we have simulated the effects of increased Cl- conductance on muscle by using a computer model. Effects on the simulated action potential include lower rates of membrane depolarization, lower overpotential, longer duration, and lower input resistance. These effects are similar to those actually observed in DMD muscle.

Action Potentials↗
PubMed1982

Analysis of neurotoxin and mitogen-stimulated sodium transport in human fibroblasts.

Human fibroblasts contain two classes of ouabain-insensitive Na+ channels. One channel is activated by alkaloid neurotoxins in combination with scorpion venom, a response which is similar to that of the voltage-sensitive Na+ ionophore of nerve. The other channel is activated by fetal calf serum, several other mitogenic agents, or low extracellular Ca2+. The nervelike Na+ channel is inhibited by tetrodotoxin but not by amiloride, while the mitogen-stimulated channel is blocked by amiloride but not by tetrodotoxin. The combined effects of neurotoxins and low Ca2+ on Na+ uptake are additive, consistent with the conclusion that these Na+ pathways are different and independent. Studies of Na+ efflux show the presence of two intracellular Na+ compartments. When Na+ flux through the (Na+ + K+)-ATPase pump is inhibited by ouabain, compartment A has a high rate constant of efflux (K1 = 0.30 mn-1) and constitutes 60% of the total cellular Na+. Compartment B, with a markedly smaller efflux constant (k2 = -0.07), contains the remaining 40% of cell Na+. Stimulation of flux through the nervelike channel has a pronounced effect on the rate constants and Na+ content of both compartments (K1 = 0.56, k2 = 0.10), but activation of the mitogenic channel has a pronounced effect only on the rate of efflux from compartment A and on the size of compartment B. A cytoplasmic-nuclear model for the Na+ compartments is presented.

Batrachotoxins↗
PubMed1982

Creatine kinase activity in normal and Duchenne muscular dystrophy fibroblasts.

Cultured human skin fibroblasts from 9 patients with Duchenne muscular dystrophy (DMD) and 8 normal age- and sex-matched controls were examined for creatine kinase (CK) activity. Both the normal and the DMD fibroblasts were found to have significant levels of CK activity (approximately 10 x 10(-3) IU per milligram of fibroblast protein). The control cells had slightly higher CK activity than the DMD lines, but this difference was not significant (0.2 less than P less than 0.1). The MM (muscle) isozyme, the BB (brain) isozyme, and the MB (hybrid) isozyme, of CK were found to be present in fibroblasts. The isozymes were separated by electrophoresis and the relative amount of each was determined for both normal and DMD cells. In normal fibroblasts, approximately 48% of the total CK activity was of the MM type, 40% was of the BB type, and 12% was of the MB type with no significant differences apparent between normal and DMD groups. The presence in human fibroblasts of significant levels of CK activity with a characteristic isozyme profile is an important consideration for studies of this "marker" enzyme in the pseudohypertrophic muscle of DMD.

Adolescent↗
PubMed1982

Decreased structure-linked latency of lysosomal dipeptidyl aminopeptidase-I activity in Duchenne muscular dystrophy fibroblasts.

Crude lysosomal pellets were prepared from skin fibroblasts grown from patients having Duchenne muscular dystrophy, and from normal controls. Disruption of the lysosomes by nonionic detergents resulted in the expression of latent activity of the enzyme dipeptidyl aminopeptidase-I(DAP-I). Duchenne lysosomes showed less structure-linked latency than those from normal controls, and sedimentation studies demonstrated that the difference was not caused by increased leakage of the enzyme from lysosomes. Permeability properties of the lysosomes for an artificial substrate revealed no difference of the apparent Km. However, in intact lysosomes the apparent K, for Cl- of this chloride-requiring enzyme was found to be lower in DMD lysosomes. The apparent increase in entry for Cl- was closely related with the decreased amount of the DAP-I latency. High concentrations of extra-lysosomal Cl- corrected the abnormality.

Cathepsins↗
PubMed1982

Identification of a biochemical difference between male and female human fibroblasts: implications for the expression of Duchenne muscular dystrophy.

The lysosomal enzyme dipeptidyl aminopeptidase-I (DAP-I) was reduced in Duchenne muscular dystrophy (DMD) fibroblasts to 30% of the level found in age- and sex-matched controls (p less than 0.005). Structure-linked latency, defined as the increase in DAP-I activity caused by disruption of the lysosomal membrane, was also reduced in Duchenne fibroblasts to 70% of normal levels (p less than 0.001). Duchenne carriers and age- and sex-matched control fibroblasts had similar DAP-I activities and latency. However, the similarity of normal and carrier female DAP-I activities was not due to elevation of the carrier female activities to levels similar to normal males, but rather to reduction of all female DAP-I activities to 30% of normal male levels (p less than 0.001). This gave them comparable activities to those of the male DMD cells. One explanation for these unexpected results would require a modification of the genetics of DMD to that of a Y-influenced X-linked model.

Cathepsins↗
PubMed1981

Structural changes in lysosomes from cultured human fibroblasts in Duchenne's muscular dystrophy.

We have previously reported a decreased activity of the lysosomal enzyme dipeptidyl aminopeptidase-I (DAP-I) in cultured fibroblasts from patients with Duchenne's muscular dystrophy (DMD). Here we report that electron microscope examination of these cells reveals the presence of abundant lamellar bodies, a morphologic abnormalities commonly associated with impaired lysosomal function. Morphometric analysis of these cytoplasmic figures in dystrophic cells shows a sevenfold increase relative to normal controls (P less than 0.01). Analysis of lysosomal density profiles by density gradient centrifugation reveals similar patterns in normal and DMD cells. Treatment of lysosomes wit the nonionic detergent Triton X-100 causes an activation of DAP-I. This activation, attributable to structure-linked latency, is markedly diminished in DMD cells which show an optimal activation of only 180% compared to 255% for control fibroblasts (P less than 0.01). These data suggest an alteration in the properties of the lysosomal membrane in DMD fibroblasts. This suggestion is also supported by studies on the release of DAP-I from lysosomes by osmotic shock which show it to be a membrane-associated enzyme with membrane-binding characteristics intermediate between those of tightly bound beta-glucosidase and those of unbound N-acetylgalactosaminidase. The latency characteristics of these other lysosomal enzymes are not altered in the DMD cells, indicating that the effect is specific for DAP-I.

Cathepsins↗
PubMed1980

Decreased lysosomal dipeptidyl aminopeptidase I activity in cultured human skin fibroblasts in Duchenne's muscular dystrophy.

Several lysosomal enzymes were assayed in cultured human skin fibroblasts from patients with Duchenne's muscular dystrophy (DMD) and age- and sex-matched control patients (N). The activity of four glycosidases, cathepsin B(1), and total autoproteolysis at pH 4.0 were unchanged between the groups, but dipeptidyl aminopeptidase I (DAP-I, or cathepsin C) in the DMD cells was found to be only 30% as active as in the control cells (P < 0.003). This difference is not the result of a redistribution or loss of enzyme during homogenization because the difference occurs in all homogenate fractions. DAP-I activity existing in N and DMD fibroblasts behaves identically with respect to activation by chloride ion, activation by the sulfhydryl reducing agent dithiothreitol, changes in hydrogen ion concentration (pH), changes in substrate concentration (i.e., apparent K(m) values), and changes in temperature (i.e., apparent activation energies). Mixtures of N and DMD cell sonicates display an additivity in DAP-I activity. These results support the conclusion that the catalytic function of the DAP-I molecule is equivalent between N and DMD fibroblasts, and that the decrease in tissue-specific DAP-I activity probably results from the fact that fewer enzyme molecules are present in the DMD cells. These results are also an indication that these nonmuscle cells are expressing some of the phenotypic aspects of the genetic defect in DMD. Cultured human skin fibroblasts may therefore be a useful cellular model in DMD research.

Cathepsins↗