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P Navas

Publications and source records attributed to P Navas.

At least 73 records · Page 4Linked to original sources
PubMed1992

Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus.

Trans cisternal elements of the Golgi apparatus from rat liver, identified by thiamin pyrophosphatase cytochemistry, were isolated by preparative free-flow electrophoresis and were found to undergo acidification as measured by a spectral shift in the absorbance of acridine orange. Acidification was supported not only by adenosine triphosphate (ATP) but nearly to the same degree by inorganic pyrophosphate (PPi). The proton gradients generated by either ATP or PPi were collapsed by addition of a neutral H+/K+ exchanger, nigericin, or the protonophore, carbonyl cyanide m-chlorophenylhydrazone, both at 1.5 microM. Both ATP hydrolysis and ATP-driven proton translocation as well as pyrophosphate hydrolysis and pyrophosphate-driven acidification were stimulated by chloride ions. However, ATP-dependent activities were optimum at pH 6.6, whereas pyrophosphate-dependent activities were optimum at pH 7.6. The Mg2+ optima also were different, being 0.5 mM with ATP and 5 mM with pyrophosphate. With both ATPase and especially pyrophosphatase activity, both by cytochemistry and analysis of free-flow electrophoresis fractions, hydrolysis was more evenly distributed across the Golgi apparatus stack than was either ATP- or PPi-induced inward transport of protons. Proton transport colocalized more closely with thiamin pyrophosphatase activity than did either pyrophosphatase or ATPase activity. ATP- and pyrophosphatase-dependent acidification were maximal in different electrophoretic fractions consistent with the operation of two distinct proton translocation activities, one driven by ATP and one driven by pyrophosphate.

Adenosine Triphosphatases↗
PubMed1991

Ascorbate is regenerated by HL-60 cells through the transplasmalemma redox system.

Ascorbate was maintained in the media during a long-term culture by HL-60 cells. The chemical oxidation of ascorbate was reversed in vitro by living HL-60 cells and was related to the amount of cells added. The increase of NADH concentration by lactate addition to cells was accompanied by an increase of both ascorbate regeneration and ferricyanide reduction. Further, plasma membrane enriched fractions from HL-60 cells revealed enhancement of both ascorbate regeneration and ferricyanide reduction in the presence of NADH when previously treated with detergent. The blockage of cell surface carbohydrates by wheat germ agglutinin (WGA) and Concanavalina ensiformis (Con A) lectins significantly inhibited the regeneration of ascorbate caused by the cells. These results support the idea that ascorbate is externally regenerated by the NADH-ascorbate free radical reductase as a part of the transplasma membrane redox system.

Ascorbic Acid↗
PubMed1990

Ascorbate free radical stimulates the growth of a human promyelocytic leukemia cell line.

Ascorbate free radical stimulates the growth of human promyelocytic leukemia cells (HL-60) in the presence of a limited amount of serum (1%) when added to the cells under conditions where it is impermeable. Maximum growth stimulation occurs at concentrations from 5 x 10(-9) to 2 x 10(-8) M. Ascorbate mimicks the stimulation effect of its free radical but stimulates at higher concentrations. Autoxidation of ascorbate by oxygen produces its free radical, which apparently causes growth stimulation. Ascorbate could be regenerated by intact cells in vitro, since prevention of autoxidation of ascorbate in the presence of cells is observed. Neither dehydroascorbate nor isoascorbate increases HL-60 cell growth. Short term incubation of cells in the presence of ascorbate free radical induced intracellular NADH oxidation. We propose that the stimulation of growth of HL-60 cells shown here could be caused by activation of the transplasma membrane electron transport system by the ascorbate free radical.

Ascorbic Acid↗
PubMed1990

Hypertrophy of renal mitochondria.

Compensatory renal hypertrophy leads to an increase in the size and metabolic capacity of renal tubular cells. Increased transport and metabolic activities must be sustained by an augmented rate of energy production, which is largely dependent on mitochondrial processes. Although previous studies have suggested that mitochondria proliferate in the hypertrophying cell, the data to support this have not been convincing. This study was designed to determine whether the mitochondria of the hypertrophied renal proximal tubular cell undergo hypertrophy or proliferation. Flow cytometric analysis of proximal tubular cells obtained from the kidneys of uninephrectomized rabbits revealed an increase in cell size and RNA content compared with control cells but showed no change in DNA content and nuclear size and no evidence of entry into the S/G2/M phases of the cell cycle. Histomorphometric analysis of cortical proximal tubules revealed that although cytoplasmic volume increased, mitochondrial density remained constant, indicating that mitochondrial volume increases in proportion to the increase in cell volume. By day 14, mitochondrial volume had increased 66% above control values. Electron microscopic examination of isolated S2 proximal tubules from 5/6 nephrectomized rabbits with maximal hypertrophy revealed mitochondrial cristae which appeared to be more densely packed than that in normal cells. The size of the functional mitochondrial pool per cell was determined by rhodamine-123 fluorescence. This increased within 24 h of uninephrectomy, peaked at approximately 80% above control levels at 5 days, and remained elevated throughout the 16 days of observation. The initial increase (days 1 and 2) occurred before a measurable increase in mitochondrial volume occurred and presumably reflects an increase in mitochondrial membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Isolation of purified plasma membranes from cultured cells and hepatomas by two-phase partition and preparative free-flow electrophoresis.

This report describes and documents the isolation of plasma membranes from hepatomas and tissue culture cells by aqueous two-phase partition. The method used previously for normal liver was effective, rapid, and reproducible. Preparations from both cells and hepatomas were more than 90% plasma membrane-derived based on electron microscope morphometry and assays of marker enzyme activities. Relative enrichments over starting homogenates were near theoretical as determined by electron microscope morphometry of starting cells and tissues. Recoveries were about 10% or greater. Briefly, the membranes to be separated were mixed with a combination of two different polymers that themselves separated into two phases. For tissue culture cells and hepatomas, a mixture of 6.6% (w/w) dextran and 6.6% (w/w) polyethylene glycol containing 0.25 M sucrose and 5 mM potassium phosphate (pH 7.2) was used. A potassium-stimulated, ouabain-inhibited, p-nitrophenylphosphatase was employed as a plasma membrane marker to monitor yield and recovery. In combination with free-flow electrophoresis, the preparations of plasma membranes from cultured cells were resolved further into fractions enriched in vesicles of right side-out or inside-out orientations. Unlike centrifugation methods, the same type of two-phase separations provided useful plasma membrane fractions when applied to different types of cultured cells as well as to solid tumors and normal tissues. The lower phase membranes, after two-phase partition, provided a plasma membrane-depleted source of membranes other than plasma membrane for use as reference fractions. The procedures should find wide application to problems of cancer research where facile and decisive separations of surface and internal membranes may be required.

Animals↗
PubMed1989

Differential morphometric values induced in Golgi apparatus of higher plant cells by aldehyde and permanganate fixation.

In order to determine the best conditions to carry out quantitative ultrastructural studies in plant specimens, five different fixation techniques, including some of the most reported electron microscopy fixatives (glutaraldehyde-paraformaldehyde, osmium tetroxide, potassium permanganate), were assayed in onion root meristems to check their ability to induce morphometric changes in Golgi apparatus ultrastructure. Although the parameters evaluated showed in all cases the same tendency, values obtained after permanganate fixation were always higher than those found after aldehyde techniques (especially aldehyde-osmium). Aldehyde followed by osmium fixation appears as the most indicated fixation method when accurate quantitative ultrastructural studies are to be developed.

Aldehydes↗
PubMed1989

Polarization of plasma membrane glycoconjugates in amphibian epidermis during metamorphosis.

Wheat germ agglutinin (WGA) binding sites have been examined in tadpole epidermal cells at the level of both light and electron microscopy using the WGA-ovomucoid-gold technique. In premetamorphic tadpoles the reaction was observed on the plasma membranes of epithelial cells showing a gradient from inner to outer membranes. These glycoconjugates were polarized during development, and at the end of metamorphic climax they were only located in plasma membranes of stratum corneum. The existence of an apical cell surface coat is needed to facilitate the absorption of water through the adult epidermis. The possible implications of this polarization process are discussed.

Amphibians↗
PubMed1988

Cell surface glycoconjugates control the activity of the NADH-ascorbate free radical reductase of rat liver plasma membrane.

Plasma membrane isolated by two-phase partition from rat liver showed rates of ascorbate free radical reduction by NADH of 4-5 nmoles of oxidized NADH/min/mg protein. This activity was inhibited 80% by ConA and up to 97% by WGA and LFA lectins. NADH-ascorbate free radical reductase was also inhibited in rat liver plasma membranes preincubated with neuraminidase or trypsin, but no additional inhibition was observed in the presence of LFA after enzyme digestion. It appears that the integrity of glucan moieities of the cell surface glycoconjugates are necessary for the optimal function of this activity that could be considered as part of the transplasma membrane electron transport system.

Animals↗
PubMed1988

Transformation of Tetrahymena thermophila by electroporation and parameters effecting cell survival.

We have successfully transformed Tetrahymena thermophila by electroporation, a process of electrically introducing DNA. The DNA used for transformation contains a mutant ribosomal RNA gene (rDNA) that confers resistance to paromomycin on the transformed cells. This mutant rDNA replicates more rapidly than the endogenous rDNA of the transformed cells so that the mutant rDNA becomes predominant within several generations. This mutant rDNA also carries a restriction polymorphism that readily distinguishes it from the endogenous rDNA of the transformed cells. Substantial nuclease activity is released from the cells during electroporation and must be neutralized in order for transformation to be effective. Cell survival is inversely proportional to the electrical energy dissipated (joules) in the medium. Electroporation is a convenient and effective means of introducing transforming DNA into T. thermophila.

Animals↗
PubMed1988

Thyroxin specifically stimulates anuran larvae epidermal sodium pump during metamorphosis.

1. Sodium pump, measured as K+-dependent, ouabain sensitive pNPPase, but not the non-specific, ouabain insensitive pNPPase, was stimulated by thyroxin in epidermis of tadpoles of Rana perezi. 2. Epidermal K+-pNPPase of thyroxin treated tadpoles was only stimulated in those stages already showing activity and reached levels similar to adult frogs in tadpoles at metamorphic climax.

Animals↗
PubMed1988

Detection of glycosaminoglycans in the Golgi complex of chondrocytes.

Elongation and sulfation of glycosaminoglycans are pivotal roles of the Golgi complex during the biosynthesis of proteoglycan monomers. In the present work the spatial relationship between these processes has been investigated by using a combination of immunocytochemical and cytochemical techniques. Chondroitin sulfate and keratan sulfate glycosaminoglycans were immunocytochemically localized in 1 to 2 transmost cisternae, also in a system of narrow tubules at the trans face of the Golgi complex of chick epiphyseal chondrocytes. At these same locations sulfate groups were revealed with the high iron diamine (HID) method, proteoglycan monomers being visualized with ruthenium red. Several treatments were assayed in order to reversibly block the secretory pathway. Chondrocytes incubated at a low temperature, 15 degrees C, before fixation, showed both glycosaminoglycans in the middle cisternae of the Golgi stack as well as the above mentioned locations. After low temperature treatment both HID and ruthenium red stained the middle, but not the cis cisternae. Incubation of the cells for 30 min with either diethylcarbamazine or monensin before fixation permitted detection of glycosaminoglycans and proteoglycan monomers in the middle cisternae, whereas HID staining of the Golgi complex, but not that of secretory vesicles, was abolished. The results show that elongation of both chondroitin sulfate and keratan sulfate glycosaminoglycans takes place in the same Golgi compartments. These include the middle cisternae and probably also the trans cisternae and tubules. Also suggested is that sulfation of one or both types of glycosaminoglycans begins in the middle cisternae.

Animals↗
PubMed1987

NADH diferric transferrin reductase in liver plasma membrane.

Evidence is presented that rat liver plasma membranes contain a distinct NADH diferric transferrin reductase. Three different assay procedures for demonstration of the activity are described. The enzyme activity is highest in isolated plasma membrane, and activity in other internal membranes is one-eighth or less than in plasma membrane. The activity is inhibited by apotransferrin and antitransferrin antibodies. Trypsin treatment of the membranes leads to rapid loss of the transferrin reductase activity as compared with NADH ferricyanide reductase activity. Erythrocyte plasma membranes, which lack transferrin receptors, show no diferric transferrin reductase activity, although NADH ferricyanide reductase is present. The transferrin reductase is inhibited by agents that inhibit diferric transferrin reduction by intact cells and is activated by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate) detergent. Inhibitors of mitochondrial electron transport have no effect on the activity. We propose that the NADH diferric transferrin reductase in plasma membranes measures the activity of the enzyme that causes the reduction of diferric transferrin by intact cells. This transmembrane electron transport system requires the transferrin receptor for diferric transferrin reduction. Because the transmembrane electron transport has been shown to stimulate cell growth, the reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth, in addition to its role in iron transport.

Animals↗
PubMed1987

Lectin inhibition and kinetics of microsomal K+-dependent p-nitrophenyl phosphatase of frog epidermis.

The specific activity of K+-dependent p-NPPase (paranitrophenylphosphatase) from frog (Rana ridibunda) epidermis microsomal preparation was determined. The activity was proportional to time of incubation and protein concentrations under our assays conditions. Optimal phosphatase activity was at pH from 8 to 9 and over 35 degrees C. 10(-3) M ouabain inhibited 100% of the activity and the Ki was estimated about 5 X 10(-5) M. The Km for p-NPP was 3.8 mM and 2.1 for K+. The lectins GSI and GSII produced 80-90% of non-competitive inhibition of the activity. 50% of inhibition by GSI was obtained at 2 micrograms/ml. The Km for p-NPP did not change but the Vmax of activity was clearly reduced for both GSI and GSII lectins.

4-Nitrophenylphosphatase↗
PubMed1987

Lectin binding patterns in amphibian epidermis.

A battery of 6 different horseradish peroxidase conjugated lectins has been employed for structural localization of glycoconjugates in amphibian epidermis. Lens culinaris (LCA) lectin stained the basal membrane and gave no significant reaction on the epidermal layers. Canavlia ensiformis (Con A) and Griffonia simplicifolia II (GS II) lectins bound the keratinocyte cytoplasm and the basal membrane as well. Ulex europaeus I (UEA I) lectin had only reactivity with flask cells. Griffonia simplifolia I (GS I) and Glycine max (SBA) lectins preferentially bound the cell membranes of keratinocytes, being the intensity of the staining gradually increasing from the stratum spinosum to the stratum granulosum. These results show that UEA I, GS I, and SBA are good markers to distinguish different cell types and the degree of keratinocytes differentiation.

Animals↗
PubMed1987

The role of ascorbate in biomembrane energetics.

The mechanism(s) whereby membrane translocations are energized are poorly understood. Our work has focused on transmembrane microsomal and plasma membrane redox constituents as a means to energize membranes via alternative mechanisms complementary to ATP-driven processes. One such component is NADH-ascorbate free radical (mono- or semidehydroascorbate) oxidoreductase. This activity is associated with the trans or exit face of the Golgi apparatus, transport vesicles that move between the Golgi apparatus and the plasma membrane, and with the plasma membrane itself. Various lines of evidence, mostly indirect, link this activity to membrane translocations. Included is an apparent activation of the reductase in membranes when coated with clathrin, a single large polypeptide chain involved in exocytosis and in receptor-mediated and absorptive endocytosis. The results are consistent with a role of the ascorbate free radical as an acceptor for electron transport-mediated transfer of electrons from NADH perhaps to oxygen by coated membranes as a part of a mechanism to drive membrane translocations via generation of a proton gradient or of a membrane potential. Additionally, plasma membrane redox may be important in the regulation of cell growth, but a strict dependence on ascorbate free radical for the latter seems less likely than with internal endomembranes, where redox function may strictly depend upon the restricted pool of regeneratable acceptor that the ascorbate free radical provides.

Animals↗
PubMed1987

Isolation of plasma membrane from amphibian epidermis: evidence for a basal-to-apical charge and activity gradient.

Aqueous two-phase partition and preparative free-flow electrophoresis were used in series to isolate the plasma membranes of amphibian epidermis. Fractions obtained by two-phase partition were 40-fold enriched in a K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase relative to the total homogenate and based on morphology were representative isolates of all epidermal cells together. Small mucosal granules and mucin aggregates were the primary contaminants. Based on activities of marker enzymes, contents of mitochondria, Golgi apparatus and endoplasmic reticulum were low (0.15 that of total homogenate) or absent. When plasma membranes isolated by aqueous two-phase partition were subjected to preparative free-flow electrophoresis, they were distributed toward the anode in a series of fractions of increasing net negative charge, sialic acid content and specific activity of the K+-stimulated, ouabain-inhibited, p-nitrophenylphosphatase reminiscent of the activity gradient from base to apex for frog epidermis observed from cytochemical investigations. The most electronegative fractions nearest the anode and to the left of the main protein peak were enriched in both sulfate groups and thick membranes of the stratum corneum. A fraction migrating less toward the anode and to the right of the main protein peak contained hemidesmosomes together with the lowest enrichments of sialic acid, sulfate and the phosphatase. The results suggest that the plasma membranes isolated from mixed cell populations, such as those encountered in epidermal homogenates, may be resolved by free-flow electrophoresis according to cell type of origin following activity gradients present in the original tissue. Additionally, the findings provide independent biochemical confirmation of a base-to-apex gradient of transport (ATPase) activity associated with the plasma membranes of cells of the different strata of the amphibian epidermis.

4-Nitrophenylphosphatase↗