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R Serrano

Publications and source records attributed to R Serrano.

At least 109 records · Page 6Linked to original sources
PubMed1991

Analysis of the regulatory domain of yeast plasma membrane H+-ATPase by directed mutagenesis and intragenic suppression.

The yeast plasma membrane H+-ATPase is activated in vivo by glucose metabolism, and previous deletion analysis has shown the C-terminus of the enzyme to be involved in this regulation. Site-directed mutagenesis demonstrates that Arg909 and Thr912 at the C-terminus are important for the increase in Vmax of the ATPase induced by glucose. Other changes in kinetic parameters induced by glucose are largely independent of these amino acids. Arg909 and Thr912 form a potential phosphorylation site for calmodulin-dependent multiprotein kinase. A double mutation of Ser911 and Thr912 to Ala results in no cell growth in glucose medium and greatly reduced activation of the ATPase by glucose. Growth and activity are restored by a third mutation (Ala547----Val) at the catalytic domain, providing genetic evidence for domain interaction.

Amino Acid Sequence↗
PubMed1991

Domains of yeast plasma membrane and ATPase-associated glycoprotein.

In yeast homogenates the plasma membrane H(+)-ATPase and a major surface glycoprotein of about 115 kDa are present in two membrane fractions with peak densities in sucrose gradients of 1.17 and 1.22. Immunogold electron microscopy of frozen yeast sections indicates that the ATPase is exclusively (greater than 95%) present at the surface membrane. Therefore the two ATPase-containing fractions appear to correspond to different domains of the plasma membrane. The 115 kDa glycoprotein is tightly associated with the ATPase during solubilization and purification of the enzyme. However, in a mutant lacking the glycoprotein the activity of the plasma membrane H(+)-ATPase is similar to wild type, suggesting that this association is fortuitous. The ATPase and the glycoprotein are difficult to separate by electrophoresis and therefore binding of concanavalin A to the ATPase cannot be unambiguously demonstrated in wild-type yeast. By utilizing the mutant without glycoprotein it was shown that the ATPase band of 105 kDa binds concanavalin A.

Cell Membrane↗
PubMed1990

Isolation of a novel tumor protein that induces resistance to natural killer cell lysis.

The human metastatic tumor cell line CAP-2, produces a soluble factor that induces resistance to NK lysis of K-562 susceptible leukemia cell line, and does not inhibit the cytotoxic capacity of effector cells. The use of sequential HPLC, hydrophobic interaction chromatography, and reverse phase chromatography, coupled with cytotoxic assays, resulted in the isolation and separation to homogeneity of a novel protein responsible for this biologic activity. Size estimation studies based on TSK HPLC columns showed that this protein has a mass of 8 to 12 kDa. The amino acid composition analysis of the CAP-2 protein calculated from HPLC chromatograms shows that this protein contains around 108 amino acids. Subsequent gas phase sequence analysis, however, was hampered because the N terminus of this protein was blocked and therefore unsuitable for sequencing by Edman degradation. The functional studies showed that the NK lysis-resistance activity of the CAP-2 protein is mediated by interaction with and nonspecific binding to NK target cells. The lymphokine-activated killer and macrophage-mediated cytotoxicity and mitogen-induced proliferation is not affected. Unexpectedly, the CAP-2 protein appears to be mitogenic to its own cell line. Thus, the induction of NK lysis-resistance and the mitogenic activity showed by CAP-2 protein could contribute to the tumor growth and metastatic establishment.

Amino Acids↗
PubMed1990

Analysis of the mechanisms involved in NK resistance induced by a new tumor factor NK-RIF.

The mechanisms involved in susceptibility or resistance of neoplasic cells to lysis by NK cells are not well known. We have recently described a 12-kDa factor (NK-RIF), produced and released by different tumor cell lines, making K562 resistant to NK lysis without affecting the cytotoxic function of NK effector cells. In this paper we further study the mechanism involved in NK resistance of K562 mediated by NK-RIF and its biological implications. The results show that NK-RIF does not affect the binding capacity of target and effector cells nor the levels of HLA class I antigen expression on the target cells, as a proof that resistance to NK-mediated lysis is not always associated with a defect in target effector binding or with an increased MHC class I antigen expression. However NK-RIF-treated K562 loses its capacity to induce NK cell activation and the subsequent capacity to release NKCF and makes K562 resistant to lysis by NKCF. Therefore our results show that induction of resistance to NK cytotoxicity can be the result of the modulation of target structures responsible for inducing effector cell activation without affecting target/effector binding molecules. This indicates that the structures involved in adherence and activation of NK cells have a different nature and that molecules other than HLA participate in NK resistance.

Biological Factors↗
PubMed1990

Catalytic and regulatory sites of yeast plasma membrane H(+)-ATPase studied by directed mutagenesis.

More than 35 site-directed mutants of the plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae have been constructed and expressed to investigate the function of N- and C-termini and of conserved amino acids. Conserved motif TGES seems to form part of both the catalytic machinery for the hydrolysis of the phosphorylated intermediate and the vanadate binding site. In addition, it is involved in the coupling of ATP hydrolysis to H+ transport. The phosphorylated intermediate is also essential for this coupling, but not for ATP hydrolysis. The aspartate residues of conserved motifs DPPR, TGD and TGDGVND (the last one) seem to form part of the ATP binding site. The positive charge of the conserved motif KGAP is important for the kinase or phosphorylating activity. A conserved proline and a conserved aspartate predicted to have a transmembrane location are essential for activity. The N-terminus contains a conserved acidic region which may be involved in assembly into the plasma membrane. All the hydrophobic stretches at the C-terminus are also required for assembly. The last 11 amino acids constitute a non-essential inhibitory domain involved in regulation of the enzyme by glucose metabolism.

Amino Acid Sequence↗
PubMed1990

Natural killer susceptibility is independent of HLA class I antigen expression on cell lines obtained from human solid tumors.

The susceptibility to natural killer (NK) cell-mediated cytotoxicity of 20 cell lines obtained from human solid tumors and their class I histocompatibility antigen (HLA) levels were studied in an attempt to determine whether major histocompatibility complex (MHC) products expressed on cells derived from human solid tumors influence NK susceptibility. The effect of interferon-gamma (IFN-gamma) treatment on these elements was also analyzed. The MHC class I (HLA-ABC, HLA-A and HLA-B) antigen levels and degree of NK lysis were very heterogeneous and no correlation was found on comparison. After treatment with IFN-gamma a marked decrease in NK susceptibility was observed in all the cell lines, including the control line K-562. However, the level of HLA class I expression was not modified in any of the lines with the exception of the K-562, which increased. In some cell lines the expression of HLA class I-like antigens. CDla, b and c, was also measured before and after IFN treatment; however, no correlation was found between CD1 levels and NK susceptibility. Consequently, from our results it is possible to conclude that HLA class I antigens do not play a decisive role in NK susceptibility of cell lines derived from human solid tumors and to suggest that molecules which are not HLA class I antigens but IFN-gamma inducible may confer NK resistance to these lines.

Antigens, CD1↗
PubMed1990

Tumorigenic 3T3 cells maintain an alkaline intracellular pH under physiological conditions.

One of the earliest events in the response of mammalian cells to mitogens is activation of Na+/H+ exchange, which increases intracellular pH (pHin) in the absence of HCO3- or at external pH values below 7.2. The proliferative response can be blocked by preventing the pHin increase; yet, the proliferative response cannot be stimulated by artificially raising pHin with weak bases or high medium pH. These observations support the hypothesis that optimal pHin is a necessary, but not sufficient, component of the proliferative-response sequence. This hypothesis has recently been challenged by the observation that transfection of NIH 3T3 cells with yeast H(+)-ATPase renders them tumorigenic. Although previous measurements indicated that these transfected cells maintain a higher pHin in the absence of HCO3-, whether H(+)-ATPase transfection raised the pHin under physiologically relevant conditions was not known. The current report shows that these transfected cells do maintain a higher pHin than control cells in the presence of HCO3-, supporting the possibility that elevated pHin is a proliferative trigger in situ. We also show that these cells are serum-independent for growth and that they glycolyze much more rapidly than phenotypically normal cells.

Animals↗
PubMed1990

Immunocytolocalization of Plasma Membrane H-ATPase.

The localization of plasma membrane H(+)-ATPase has been studied at the optical microscope level utilizing frozen and paraffin sections of Avena sativa and Pisum sativum, specific anti-ATPase polyclonal antibody, and second antibody coupled to alkaline phosphatase. In leaves and stems the ATPase is concentrated at the phloem, supporting the notion that it generates the driving force for phloem loading. In roots the ATPase is concentrated at both the periphery (rootcap and epidermis) and at the central cylinder, including endodermis and vascular cells. This supports a ;two-pump' mechanism for ion absorption, involving active uptake at the epidermis, symplast transport across the cortex, and active efflux at the xylem. The low ATPase content of root meristem and elongation zone may explain the observed transorgan H(+) currents, which leave nongrowing parts and enter growing tips.

Journal Article↗
PubMed1990

Transformation and pH homeostasis of fibroblasts expressing yeast H(+)-ATPase containing site-directed mutations.

Mouse fibroblasts expressing a yeast proton-pumping ATPase show tumorigenic transformation (R. Perona, and R. Serrano, Nature (London) 334:438-440, 1988). By expressing site-directed mutations of the yeast ATPase with different levels of activity, a close correlation has been found between enzyme activity, tumorigenic transformation, and intracellular pH measured by weak-acid distribution. Fibroblasts expressing the yeast proton-pumping ATPase showed increased capability to grow at acidic pH and to resist lethal acidification mediated by reversal of the Na(+)-H+ antiporter. Measurements with microelectrodes in individual cells demonstrated electrical hyperpolarization and confirmed the increased pH of cells expressing yeast ATPase. These results indicate that the yeast enzyme expressed in mouse fibroblasts has electrogenic proton-pumping activity and that this activity deregulates fibroblast growth. This suggests a connection between the biophysical phenomena of proton transport, intracellular pH, and membrane potential and the biochemical regulatory circuits based on protein kinases and transcription factors.

Animals↗
PubMed1990

Primary cerebral abscess due to nocardia presenting as 'ghost tumor'. Clinical and pathological study.

We report a case of a primary cerebral abscess due to Nocardia asteroides in a nonimmunocompromised patient with a particular clinical course. The first symptom (right subacute brachial palsy) and the lesion in a computed tomographic (CT) scan (left parietofrontal edema suggestive of brain tumor) disappeared after corticosteroid treatment and the patient was discharged with total recovery. After 2 months she complained of headache and visual disturbance. A new CT scan showed an annular lesion in the left occipital lobe. A cerebral biopsy was diagnosed of nocardia infection. The patient died 2 weeks after this biopsy. A postmortem study showed an occipital brain abscess but not structural abnormalities were seen in the left parietofrontal area. We believe that the first episode could be a local inflammatory response to cerebral implantation of nocardia which disappeared clinically in the CT scan and in the postmortem study after corticosteroid treatment. Then the nocardia could have displaced by the hematological route to the second and definitive cerebral lesion.

Aged↗
PubMed1990

[The superior vena cava syndrome secondary to a benign intrathoracic goiter: a case report and review of the literature].

The clinical case of a female patient suffering a benign retrosternal goitre which first appeared as a superior vena cava syndrome, is presented. After confirming the diagnosis with an intrathoracic CT scan, a subtotal bilateral thyroidectomy was performed resulting in a complete remission of the clinical picture. A literature review is performed commenting on some clinical, diagnostic and therapeutical aspects.

Aged↗
PubMed1989

Growth control strength and active site of yeast plasma membrane ATPase studied by site-directed mutagenesis.

Several amino acids which are conserved in cation-pumping ATPases with phosphorylated intermediate have been mutagenized in the yeast plasma membrane H+-ATPase. The mutant genes have been selectively expressed in a yeast strain where the wild-type ATPase is only expressed in galactose medium. A series of mutants with decreasing levels of activity demonstrates that the ATPase is rate-limiting for growth and that decreased ATPase activity correlates with decreased intracellular pH. Enzymatic and transport studies of mutant ATPases indicate that (a) Lys474 is the target for the inhibitor fluorescein 5'-isothiocyanate and this residue can be replaced by either arginine or histidine with partial retention of activity; (b) the sensitivity to inhibition by vanadate is affected by the mutations Thr231----Gly, Cys376----Leu, Lys379----Gln and Asp634----Asn; (c) the mutation Ser234----Ala causes uncoupling between ATP hydrolysis and proton transport and reduces the ATP content of the cells; (d) the mutation Asp730----Asn, which affects a polar residue conserved in hydrophobic stretches of H+-ATPases, abolishes ATPase activity and proton transport but not the formation of a phosphorylated intermediate.

Adenosine Triphosphate↗