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J J Gargus

Publications and source records attributed to J J Gargus.

36 records · Page 2Linked to original sources
PubMed1992

Blockers of platelet-derived growth factor-activated nonselective cation channel inhibit cell proliferation.

In serum-deprived G(o)-arrested cells, the addition of serum or growth factors initiates a cascade of events that culminates in DNA synthesis and mitosis. Recently, we showed that in mouse L-M(TK-) fibroblasts a 28-pS nonselective cation channel (NS channel) becomes quiescent at G(o) arrest and rapidly active within seconds of platelet-derived growth factor (PDGF) or serum addition, placing this response very early in the postreceptor signaling cascade. However, lack of specific channel blockers hindered determination of whether channel activation was necessary for mitogenesis. Derivatives of N-phenylanthranilic acid (DCA) have been reported to block a pancreatic nonselective channel. Therefore, using single-channel analysis, we examined the effect of these agents on the L-M(TK-) NS channel. Flufenamic acid and mefenamic acid rapidly produced reversible channel block with an inhibitory constant (Ki) approximately 10 microM. Furthermore, the component of the macroscopic K+ efflux shown to be mediated by the NS channel was blocked with a similar Ki value. DCA effects on cell proliferation were tested by measuring cloning efficiency and growth rate. Both were inhibited over the range of concentration that affected channel activity, and a 50% inhibitory dose of 50-100 microM was determined. This observation further substantiates the hypothesis that NS channel activation forms a necessary component in the transduction of the mitogenic signal from the PDGF receptor.

Animals↗
PubMed1992

Urinary concentrating ability in patients with Jk(a-b-) blood type who lack carrier-mediated urea transport.

Water homeostasis is regulated in large part by the proper operation of the urinary concentrating mechanism. In the renal inner medulla, urea recycling from the inner medullary collecting duct to the inner medullary interstitium is thought to be essential for the production of a concentrated urine; however, it has not been possible to test this hypothesis in humans. Recently, a unique combination of genetic abnormalities has been described: absence of Kidd blood group antigens and absence of carrier-mediated urea transport in erythrocytes. Because animal studies indicate a similarity between urea transport in red blood cells and the nephron, it was postulated that patients without the Kidd antigen might lack facilitated urea transport in their kidneys. Hence, their ability to concentrate urine maximally was measured. Current models of nephron function would predict that in the complete absence of urea transport, the maximal concentrating ability would be around 800 to 900 mosM/kg H2O. Two homozygous patients had a moderate decrease in maximal concentrating ability (UosM,max = 819 mosM/kg H2O); a heterozygote also had some limitation. These studies raise the possibility that the erythrocyte urea transporter and the kidney urea transporter are encoded by a single gene (detected by the mutational loss of the Kidd antigen) and that a lack of facilitated urea transport impairs urea recycling in the kidney and, hence, maximal urinary concentrating ability.

Adolescent↗
PubMed1991

Urea transport deficiency in Jk(a-b-) erythrocytes.

The activity of the urea transporter was determined in human erythrocytes of the Kidd blood type Jk(a-b-) by measuring unidirectional urea and thiourea fluxes in tracer flux experiments and urea net fluxes in light-scattering experiments. When compared with control cells, Jk(a-b-) cells exhibited diminished urea and thiourea fluxes and lacked the kinetic characteristics of mediated transport, suggesting that in these cells urea and thiourea moved only by simple diffusion through the lipid bilayer. Control experiments showed that anion, water, and ethylene glycol permeabilities were the same in Jk(a-b-) and control cells. These experiments thus demonstrate that Jk(a-b-) cells lack mediated urea transport and strongly support the notion that urea transport function is separate from the transport for anions, water, and ethylene glycol, probably because different proteins are responsible for these transport functions.

Biological Transport↗
PubMed1991

Ethylmalonic/adipic aciduria: effects of oral medium-chain triglycerides, carnitine, and glycine on urinary excretion of organic acids, acylcarnitines, and acylglycines.

A 9-y-old girl with ethylmalonic/adipic aciduria was hospitalized to determine the possible therapeutic efficacy of oral carnitine and glycine supplementation. To provoke a mild metabolic stress, her diet was supplemented with 440 mg/kg/d of medium-chain triglycerides. She was treated successively with carnitine (100 mg/kg/d) for 5 d, neither carnitine nor glycine for 2 d, and then glycine (250 mg/kg/d) for 6 d. Consecutive 12-h urine collections were obtained throughout the entire period. The urinary excretion of eight organic acids, four acylglycines, and four acylcarnitines, which accumulate as a result of a metabolic block of five mitochondrial acyl-CoA dehydrogenases, were quantitatively determined by capillary gas chromatography, stable isotope dilution gas chromatography/mass spectrometry, and radioisotopic exchange HPLC, respectively. The excretion of each group of metabolites was calculated as the mean percentage of total output (mumol/24 h) during the four phases of the protocol (organic acids/acylglycines/acylcarnitines = 100.0%): 1) regular diet (3 d); 88.1/10.8/1.1; 2) medium-chain triglyceride supplementation (4); 82.5/15.6/1.9; 3) medium-chain triglycerides plus carnitine (5); 79.2/8.2/12.6; and 4) medium-chain triglycerides plus glycine (6); 81.0/18.7/0.3. Comparison between total and individual excretion of acylglycines and acylcarnitines indicates that oral glycine supplementation enhanced the conjugation and excretion of fatty acyl-CoA intermediates as efficiently as carnitine. We propose that oral glycine supplementation should be considered in the treatment of other inborn errors of metabolism associated with abnormal urinary excretion of acylglycines.

Acylation↗
PubMed1989

Activation of single-channel currents in mouse fibroblasts by platelet-derived growth factor.

A nonselective cation channel that we characterized in the mouse L-cell membrane becomes quiescent with serum deprivation (arrested cell growth) and rapidly active upon readdition of serum or, specifically, platelet-derived growth factor (PDGF). Using the patch-clamp technique, we find that the predominant channel in the LMTK- cell line is a bursting nonselective cation channel (the NS channel). In cell-attached and inside-out patches, the channel has a conductance of 28 pS; equal selectivity for Na+, K+, and Cs+; and no anion or divalent cation permeability. The channel open probability is voltage insensitive and in inside-out patches does not correlate with intracellular calcium (0.5 nM to 50 microM). When cultures are rendered quiescent by incubation in serum-free medium, channel open probability is virtually 0 as compared to 0.26 (+/- 0.17) in exponentially growing cultures. If mitogenesis is initiated by readdition of serum to quiescent cells while maintaining cell-attached recording, there is a rapid (15-30 s) activation of the channel (n = 12). The open probability of the patch increases (greater than 0.75) for 2-3 min and then decreases. We have attempted applications of several growth factors (fibroblast-derived growth factor, epidermal growth factor, insulin, bombesin, alpha-thrombin, and vasopressin, individually or in combination) but find that only PDGF (5-100 ng/ml; n = 9) produces channel activation. This activation should provide a Na+ entry pathway parallel to that of the Na/H exchanger.

Animals↗
PubMed1988

Gene transfer of a putative mammalian K+ channel gene from genomic and cosmid DNA.

We have successfully transfected mouse genomic DNA containing a mutant putative K+ channel gene into murine LMTK- cells. Incorporation of the mutant gene allowed primary transformants to express the mutant cell phenotype of growth at a subthreshold K+ concentration and, furthermore, caused these primary transformants to display the same characteristics of growth and unidirectional K+ flux as the mutant cell line, LTK-1, from which the genomic DNA was derived. A cosmid genomic library was formed from LTK-1 mutant DNA and shown by transfection experiments to contain at least one unique cosmid clone containing a functional copy of the mutant putative K+ channel gene.

Acyclovir↗
PubMed1988

Physiological processes revealed through an analysis of inborn errors.

To develop the significance of human inborn errors of membrane function in terms of their potential for the elucidation of physiological processes, an overview of the genetics of inherited metabolic disorders is first presented, concisely covering classical Mendelian inheritance and progressing to molecular applications currently being used in the isolation of a number of disease-causing genes. Second, approaches to the isolation of several genes implicated in inherited disorders of membrane function are discussed. Examples are drawn from studies of the Shaker/IA K+ channel mutants of Drosophila and human inborn errors in the low-density lipoprotein receptor and Kidd antigen/urea transporter. These examples illustrate the techniques of "reverse" genetics, restriction fragment-length polymorphism analysis, chromosomal walking, and cDNA library screening with oligonucleotides and antibodies. Finally, the need to develop a physiological functional analysis of the protein structures derived from isolated human disease-causing genes is presented as a necessary direction for future research.

Animals↗
PubMed1987

Molecular biology in physiology.

The aim of this symposium on molecular biology in physiology was to introduce molecular biology to physiologists who had relatively little exposure to the new developments in this field, so that they can become conversant on this topic and contribute to the advancement of physiology by incorporating molecular biological approaches as a part of their research arsenal. After the discussion of the basic concepts, terminology, and methodology used in molecular biology, it was shown how these basic principles have been applied to the study of the genes encoding two membrane proteins that have important transport functions (band 3 and ATPase). The second half of the symposium consisted of papers on the state-of-the-art developments in the application of molecular biology to the studies of the atrial natriuretic factor and renin genes, adenylate cyclase-coupled adrenergic receptors, acetylcholine receptors and sodium channel, and long-term and short-term memories. The ultimate goal is that these examples will provide an impetus for the opening of new frontiers of research in physiology by taking advantage of the tools developed from recent advances in molecular biology.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1987

Mutant isolation and gene transfer as tools in study of transport proteins.

Somatic cell and molecular genetic concepts, techniques, and approaches relevant to the analysis of a complex mechanism such as a mammalian ion transport system are reviewed and illustrated with examples taken from studies of K+ transport mutants. The reasons for undertaking a study of mutants and mutations are stated and followed by directly applicable approaches to transport mutant isolation. Examples of the types of information that can be extracted from a characterization of such mutants is then presented. The parasexual system of somatic cell hybridization is reviewed, again, followed by applicable approaches and examples of how data from such studies can be used. Finally, the technique of DNA-mediated gene transfer is presented as a method that allows the actual isolation of a transporter gene which has been characterized as above.

Carrier Proteins↗
PubMed1987

Selectable mutations altering two mechanisms of mammalian K+ transport are dominant.

Somatic cell mutants with altered K+ transport have previously been isolated from mutagenzied LMTK- cells for their ability to grow at subthreshold low-potassium concentrations (0.2 mM). These mutants fall into two classes: one class, LTK-5, possesses a functionally altered furosemide-sensitive Na+-K+-Cl- cotransport system and the other, LTK-1, an altered K+-conducting channel. Somatic cell hybrids have been formed between each of these cell lines and a wild-type L-cell line, making use of complementing selectable marker mutations carried by these parents, to establish the dominance of the K+ transport mutations. Hybrids were isolated and studied in two ways: clonal hybrid cell lines were selected in a manner unbiased toward their K+ transport phenotype, which was later assayed; and the number of independent hybrids arising in this single-selective condition was compared with the number arising in a condition which is double selective for the mutant phenotype as well. By both assays, hybrids formed with LTK-1 or LTK-5 as a parent uniformly exhibited the mutant phenotype by growth and cloning, whereas control hybrids with LMTK- as parent never did. This demonstrates both transport mutations to be dominant and thus potentially isolatable.

Animals↗
PubMed1986

Chromosome deletion 1q42-43.

We report on a newborn male and a female of 3 years 9 months with de novo 1q42 or 43-qter deletions. These cases are compared with ten other reported cases.

Child, Preschool↗
PubMed1984

Brief clinical report: lethal multiple pterygium syndrome in an 18-week fetus with hydrops.

We present the case of an 18-week abortus with lethal multiple pterygium syndrome and hydrops. Radiographic and anatomic study showed none of the bony abnormalities reported in live-born children with multiple pterygium syndrome. The pathogenesis of the hydrops was not apparent. The findings of cleft palate, pulmonary hypoplasia, muscular atrophy, gracile thoracic bones, and fetal death are typical of the lethal variant.

Abnormalities, Multiple↗
PubMed1984

Rapid changes in bidirectional K+ fluxes preceding DMSO-induced granulocytic differentiation of HL-60 human leukemic cells.

When grown in medium containing 5 mM potassium and 140 mM sodium, HL-60, a human promyelocytic cell line, maintained a steady-state intracellular K+ concentration of 145 mmol/L cells and a steady-state intracellular Na+ concentration of 30 mmol/L cells. Nearly 90% of the unidirectional 42K+ influx could be inhibited by the cardiac glycoside ouabain with a Ki of 5 X 10(-8) M. This ouabain-sensitive component of influx rose as a saturating function of the extracellular K+ concentration with a K1/2 of 0.85 mM. The component of 42K+ influx resistant to ouabain inhibition was a linear function of the extracellular K+ concentration and was insensitive to inhibition by the diuretic furosemide. Unidirectional K+ efflux followed first order kinetics with a half-time of 55 min. Addition of 1.5% dimethyl sulfoxide (DMSO) to a culture of HL-60 cells allowed two population doublings followed by the cessation of growth without an impairment of cell viability. Beginning 2 to 3 days after DMSO addition, the cells underwent a dramatic reduction in volume (from 925 microns 3 to 500 microns 3) and began to take on the morphological features of mature granulocytes. Throughout this process of differentiation there was no change in the intracellular sodium or potassium concentration. However, immediately following the addition of DMSO to a culture of cells, there began an immediate, coordinated reduction in bidirectional K+ flux. The initial rate of the ouabain-sensitive component of K+ influx fell with a half-time of 11 h to a final rate, at 6 days induction, equal to one ninth that of the uninduced control, and over the same period, the rate constant for K+ efflux fell with a half-time of 14 h to a final value one fourth that of the uninduced control. The rapidity with which these flux changes occur raises the possibility that they play some role in the control of subsequent events in the process of differentiation.

Cell Differentiation↗
PubMed1978

Genetic alterations in potassium transport in L cells.

Starting with mutagenized cultures of the mouse fibroblastic cell line LM(TK-), we have selected mutant clones by their ability to grow at 0.2 micrometer K+, a concentration unable to support the growth of the parent cell. The mutants fall into two classes on the basis of their potassium transport properties. Both classes maintain a high intracellular K+ concentration when growing in low-potassium medium, and both are unaltered in the ouabain-sensitive Na/K pump. One class shows an increased activity of a ouabain-resistant, furosemide-sensitive K+ transport system; the other class shows a decreased activity of a specific component of K+ efflux.

Biological Transport↗
PubMed1974

Chemomechanical coupling without ATP: the source of energy for motility and chemotaxis in bacteria.

The source of energy for bacterial motility is the intermediate in oxidative phosphorylation, not ATP directly. For chemotaxis, however, there is an additional requirement, presumably ATP. These conclusions are based on the following findings. (i) Unlike their parents, mutants of Escherichia coli and Salmonella typhimurium that are blocked in the conversion of ATP to the intermediate of oxidative phosphorylation failed to swim anaerobically, even when they produced ATP. When respiration was restored to the mutants, motility was simultaneously restored. (ii) Carbonylcyanide m-chlorophenylhydrazone, which uncouples oxidative phosphorylation, completely inhibited motility even though ATP remained present. (iii) Arsenate did not inhibit motility in the presence of an oxidizable substrate, though it did reduce ATP levels to less than 0.3% (iv) Arsenate completely inhibited chemotaxis under conditions where motility was normal.

Adenosine Triphosphate↗
PubMedDate not supplied

Phenotype and genotype variation in primary carnitine deficiency.

PURPOSE: Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the carnitine transporter gene SLC22A5. The objective of this study was to extend mutational analysis to four additional families with this disorder and determine whether recurrent mutations could be found. METHODS: The SLC22A5 gene encoding the OCTN2 carnitine transporter was sequenced, and the missense mutations identified were expressed in Chinese hamster ovary (CHO) cells. RESULTS: DNA sequencing revealed four novel mutations (Y4X; dup 254-264, 133X; R19P; R399Q). Alleles introducing premature STOP codons reduced the levels of OCTN2 mRNA. Carnitine transport in CHO cells expressing the R19P and R399Q mutations was reduced to < 5% of normal. The 133X mutation was found in two unrelated European families. Two patients within the same family, both homozygous for the same mutation (R399Q) had completely different clinical presentation. CONCLUSIONS: Heterogeneous mutations in the SLC22A5 gene cause primary carnitine deficiency. Different presentations are observed even in children with identical mutations.

Animals↗