[Bacteremia due to Campylobacter jejuni: report of two cases].
Explore the source record for details and available documents.
Adult↗
Biomedical subjects
C Guerrero
Publications and source records attributed to C Guerrero.
beta-agonists regulate Na,K-ATPase via novel MAPK/ERK and rapamycin-sensitive pathways.
We studied whether the beta-adrenergic agonist, isoproterenol (ISO), regulates Na,K-ATPase in alveolar epithelial cells (AEC) via a mitogen-activated protein kinase (MAPK)/extracellular signaling related kinase (ERK) dependent pathway. ISO increased ERK activity in AEC by 10 min via a beta-adrenergic receptor, protein kinase A (PKA)-dependent mechanism. Activation of the MAPK pathway by ISO, resulted in increased Na,K-ATPase beta1 and alpha1 subunit protein abundance in whole cell lysates, which resulted in functional Na, K-ATPases at the basolateral membranes. ISO did not change the alpha1 or beta1 mRNA steady state levels, but rapamycin, the inhibitor of the mammalian target of rapamycin, also blocked the ISO-mediated increase in Na,K-ATPase total protein abundance, suggesting a posttranscriptional regulation. We conclude that ISO, regulates the Na,K-ATPase in AEC via PKA, ERK and rapamycin-sensitive mechanisms.
Adrenergic beta-Agonists↗
A tomato peroxidase involved in the synthesis of lignin and suberin.
The last step in the synthesis of lignin and suberin has been proposed to be catalyzed by peroxidases, although other proteins may also be involved. To determine which peroxidases are involved in the synthesis of lignin and suberin, five peroxidases from tomato (Lycopersicon esculentum) roots, representing the majority of the peroxidase activity in this organ, have been partially purified and characterized kinetically. The purified peroxidases with isoelectric point (pI) values of 3.6 and 9.6 showed the highest catalytic efficiency when the substrate used was syringaldazine, an analog of lignin monomer. Using a combination of transgenic expression and antibody recognition, we now show that the peroxidase pI 9.6 is probably encoded by TPX1, a tomato peroxidase gene we have previously isolated. In situ RNA hybridization revealed that TPX1 expression is restricted to cells undergoing synthesis of lignin and suberin. Salt stress has been reported to induce the synthesis of lignin and/or suberin. This stress applied to tomato caused changes in the expression pattern of TPX1 and induced the TPX1 protein. We propose that the TPX1 product is involved in the synthesis of lignin and suberin.
Kinetics↗
Ras-guanine nucleotide exchange factor sos2 is dispensable for mouse growth and development.
The mammalian sos1 and sos2 genes encode highly homologous members of the Son-of-sevenless family of guanine nucleotide exchange factors. They are ubiquitously expressed and play key roles in transmission of signals initiated by surface protein tyrosine kinases that are transduced into the cell through the action of membrane-associated Ras proteins. Recent reports showed that targeted disruption of the sos1 locus results in embryonic lethality. To gain insight into the in vivo function of sos2, we disrupted its catalytic CDC25-H domain by means of gene targeting techniques. Mating among heterozygous sos2(+/-) mice produced viable sos2(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of sos2 does not interfere with embryo viability in the uterus. Adult homozygous mutant sos2(-/-) mice reached sexual maturity at the same age as their wild-type littermates, and both male and female null mutants were fertile. Histopathological analysis showed no observable differences between mutant and wild-type mice. Our results show that unlike the case for sos1, sos2 gene function is dispensable for normal mouse development, growth, and fertility.
Animals↗
Isoform-specific insertion near the Grb2-binding domain modulates the intrinsic guanine nucleotide exchange activity of hSos1.
Two human hSos1 isoforms (Isf I and Isf II; Rojas et al., Oncogene 12, 2291-2300, 1996) defined by the presence of a distinct 15 amino acid stretch in one of them, were compared biologically and biochemically using representative NIH3T3 transfectants overexpressing either one. We showed that hSos1-Isf II is significantly more effective than hSos1-Isf I to induce proliferation or malignant transformation of rodent fibroblasts when transfected alone or in conjunction with normal H-Ras (Gly12). The hSos1-Isf II-Ras cotransfectants consistently exhibited higher saturation density, lower cell-doubling times, increased focus-forming activity and higher ability to grow on semisolid medium and at low serum concentration than their hSos1-Isf I-Ras counterparts. Furthermore, the ratio of GTP/GDP bound to cellular p21ras was consistently higher in the hSos1-Isf II-transfected clones, both under basal and stimulated conditions. However, no significant differences were detected in vivo between Isf I- and Isf II-transfected clones regarding the amount, stability and subcellular localization of Sos1-Grb2 complex, or the level of hSos1 phosphorylation upon cellular stimulation. Interestingly, direct Ras guanine nucleotide exchange activity assays in cellular lysates showed that Isf II transfectants consistently exhibited about threefold higher activity than Isf I transfectants under basal, unstimulated conditions. Microinjection into Xenopus oocytes of purified peptides corresponding to the C-terminal region of both isoforms (encompassing the 15 amino acid insertion area and the first Grb2-binding motif) showed that only the Isf II peptide, but not its corresponding Isf I peptide, was able to induce measurable rates of meiotic maturation, and synergyzed with insulin, but not progesterone, in induction of GVBD. Our results suggest that the increased biological potency displayed by hSos1-Isf II is due to higher intrinsic guanine nucleotide exchange activity conferred upon this isoform by the 15 a.a. insertion located in proximity to its Grb2 binding region.
3T3 Cells↗
Invasive disease caused by ciprofloxacin-resistant uropathogenic Escherichia coli.
To evaluate the invasiveness of ciprofloxacin-resistant Escherichia coli isolated from the urinary tract, the susceptibility to ciprofloxacin of Escherichia coli strains from patients with invasive urinary tract infection was compared with that of isolates from patients with noninvasive disease. In a 14-month period, 2054 different isolates of Escherichia coli were analyzed, of which 554 (27%) were resistant to ciprofloxacin. One hundred twelve (5.4%) strains were isolated from patients with invasive disease. Resistance was significantly less frequent in isolates from patients with invasive disease (4.5%) than in isolates from patients with noninvasive disease (28.3%) (OR, 0.12; CI 95%, 0.05-0.29; P<0.001). Most ciprofloxacin-resistant strains associated with invasive disease were isolated from bacteremic patients who had recently undergone an invasive procedure involving the urinary tract. Invasive disease is caused more frequently by ciprofloxacin-susceptible strains of Escherichia coli, suggesting that resistance to ciprofloxacin may decrease the invasiveness of uropathogenic Escherichia coli.
Adult↗
Ventilator-associated lung injury decreases lung ability to clear edema in rats.
Ventilator-associated lung injury (VALI) is caused by high tidal volume (VT) excursions producing microvascular leakage and pulmonary edema. However, the effects of VALI on lung edema clearance and alveolar epithelial cells' Na,K-ATPase function have not been elucidated. We studied lung edema clearance in the isolated-perfused rat lung model after ventilation for 25, 40, and 60 min with high VT (peak airway opening pressure [Pao] of approximately 35 cm H2O) and compared them with low VT ventilation (Pao approximately 8 cm H2O), moderate VT ventilation (Pao approximately 20 cm H2O), and nonventilated rats. Lung edema clearance in control rats was 0.50 +/- 0.02 ml/h and decreased after 40 and 60 min of high VT to 0.26 +/- 0.03 and 0.11 +/- 0.08 ml/h, respectively (p < 0.01), but did not change after low VT and moderate VT ventilation at any time point. Lung permeability to small (22Na+, [3H]mannitol) and large solutes (fluorescein isothiocyanate-tagged albumin [FITC-albumin]) increased significantly in rats ventilated for 60 min with high VT, compared with low VT, moderate VT, and control rats (p < 0.01). Paralleling the impairment in lung edema clearance we found a decrease in Na,K-ATPase activity in alveolar type II (ATII) cells isolated from rats ventilated with moderate VT and high VT for 40 min without changes in alpha1 Na,K-ATPase mRNA. We reason that VALI decreases lung ability to clear edema by inhibiting active sodium transport and Na,K-ATPase function in the alveolar epithelium.
Animals↗
Stimulation of the dopamine 1 receptor increases lung edema clearance.
We previously reported that lung edema clearance was stimulated by dopamine (DA). The purpose of this study was to determine whether the DA-mediated stimulation of edema clearance occurs via an adrenergic or dopaminergic regulation of alveolar epithelial Na, K-ATPase. When isolated perfused rat lungs were coinstilled with DA and SCH 23390 (a specific D(1) receptor antagonist), there was a dose-dependent attenuation of the stimulatory effects of DA. Coinstillation with S-sulpiride (a specific D(2) receptor antagonist) or propranolol (a beta-adrenergic antagonist) did not alter DA-stimulated clearance. Similarly, the specific dopaminergic D(1) agonist fenoldopam increased lung edema clearance, but quinpirole (a specific dopaminergic D(2) agonist) did not. (125)I-SCH 23982 binding studies suggested that D(1) receptors are expressed on alveolar type II (ATII) cells with an apparent dissociation constant (K(d)) of 4.4 nM and binding maximum (Bmax) 9.8 pmol/mg. Consistent with these results, the D(1) receptor messenger RNA (mRNA) and protein were detected in ATII cells by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. These data demonstrate a novel mechanism involving the activation of dopaminergic D(1) receptors which mediates DA-stimulated edema removal from rat lungs.
Adrenergic beta-Antagonists↗
Ventilator-associated lung injury decreases lung ability to clear edema and downregulates alveolar epithelial cell Na,K-adenosine triphosphatase function.
Explore the source record for details and available documents.
Animals↗
Dopamine regulates NA,K-adenosine triphosphatase in alveolar epithelial cells via the mitogen-activated protein kinase/extracellular-signal-regulated kinase pathway.
Explore the source record for details and available documents.
Animals↗
Transformation suppressor activity of C3G is independent of its CDC25-homology domain.
The guanine nucleotide releasing protein C3G was initially identified as a Crk SH3-binding protein and recently shown to exhibit exchange activity on Rap1 proteins. Overexpression in NIH3T3 cells of a full-length C3G cDNA isolated from human placenta markedly reduced the focus forming activity of cotransfected, malignantly activated, ras oncogenes (5-7-fold). C3G also had a reverting effect on sis-mediated transformation, decreasing the number of c-sis-induced foci by a factor of 5-10-fold. The observed inhibitory effect of C3G on focus-forming activity of Ras and Sis was always higher than that observed with Rap1A, a known target of C3G. The inhibition of focus formation observed in the presence of C3G was not due to toxic effects on cell viability, since transfected C3G cells exhibited the same survival and growth rates as untransfected NIH3T3 cells or cells transfected with plasmid vector alone. Surprisingly, as opposed to Rap1A, which has no effect on Raf-1 oncogene-mediated transformation, C3G also reduced dramatically (6-8-fold) the number of v-raf-induced foci in transfected NIH3T3 cells. The inhibitory effect on Raf-induced transformation suggests that C3G has other functional targets in addition to Rap1. A C3G mutant (C3G deltaCat) lacking the catalytic domain (CDC25-H) but retaining the rest of the N-terminal sequences, including the Crk-binding domain, exhibited similar ability than full length C3G to inhibit focus formation. In contrast, a C3G mutant (C3G Cat), containing the catalytic domain only but lacking the rest of the N-terminal sequences, did not have any inhibitory effect on transformation mediated by the oncogenes tested. The C3G-derived gene products overexpressed in our transfected cell lines localized to the cytoplasm and did not change the basal MAPK or JNK activity of those cell lines nor their ability to activate the kinases in response to agonists. Our results suggest that the N-terminal region of C3G, and not its catalytic domain, may be responsible for the inhibitory effects observed.
3T3 Cells↗
Analysis of the expression of two thiolprotease genes from daylily (Hemerocallis spp.) during flower senescence.
A cDNA clone encoding a daylily (Hemerocallis spp.) thiolprotease (SEN11), whose expression is strongly upregulated in flower tepal senescence, has been isolated. The amino acid sequence, deduced from the nucleotide sequence, showed highest similarity to plant thiolproteases of Vigna mungo, Phaseolus vulgaris and Hemerocallis (SEN102), and contains a putative ER retention signal that has been described in Vigna mungo. SEN102 and SEN11 transcripts were not detectable in flower buds at the opening stage, but two peaks of transcripts were seen after 9 h and 19 h, in both petals and sepals, when wilting symptoms were apparent. The pattern of protease activity migrating on a 26.3 kDa protein was similar to the SEN102 and SEN11 transcript profiles. These two genes were also expressed in stamens and leaves, but their transcripts were undetectable in carpels and rhizomes. The expression of SEN102 was lower in the senescent leaf than in the green leaf. The pattern of expression of these genes suggests their involvement in the protein hydrolysis occurring in tepals at the late senescence stage, whereas in leaves they could be involved in the constitutive protein turnover machinery. Exogenous gibberellic acid application to cut flowers increased transcripts of both genes.
Amino Acid Sequence↗
Time course of active and passive liquid and solute movement in the isolated perfused rat lung model.
The isolated perfused rat lung model (IL) is used to study alveolar epithelial transport properties. Most of the previous studies have been done over a short period of time and have not used the same preparation as a control and intervention group. We evaluated whether the IL preparation could be used for a prolonged period of time (5 h) and studied the rates of active Na+ transport, lung liquid clearance, and passive movement of solutes. Active Na+ transport and lung liquid clearance were stable from 1 to 5 h. The passive movement of small solutes (Na+, mannitol) did not change significantly, and albumin movement increased slightly at the fifth hour. Total RNA isolated from IL after 5 h was intact, and the Na+-K+-ATPase activity in alveolar type II cells isolated at the end of 5-h experiments was equal to Na+-K+-ATPase function from freshly isolated alveolar type II cells. Finally, we measured the stimulatory effect of the beta-adrenergic-agonist terbutaline and the inhibitory effect of the Na+-K+-ATPase-antagonist ouabain by using the same animal as a control. Accordingly, the isolated perfused lung model is functionally stable for at least 5 h, and it could be utilized to evaluate the effect of different interventions by using the same preparation.
Animals↗
Chronodynamic evaluation of the stages of osseointegration in zirconium laminar implants.
Osteogenesis occurs throughout all stages in life, due to both bone turnover and reparative processes. Thus, osseointegration (OI) can be described as the final step in a cascade of processes involved in bone healing in relation to implants. Ten groups of 5 Wistar rats each (mean = 90 g b.w.) were used. Under ether anesthesia a zirconium laminar implant was placed in the tibia following the method previously described by our laboratory (Cabrini et al Imp Dent 2:264-7, 1993). The animals were killed at Ohs, and 1, 2, 3, 6, 7, 10, 12, 14, and 60 days post-implantation. Tibiae were resected, radiographed and processed for their embedding in methyl methacrylate. Three sections, perpendicular to the major axis of the tibia, were obtained per implant and histologic and histomorphometric studies were carried out. Volume occupied by blood clot, woven bone, percentage of OI and OI bone tissue thickness, were determined. Histologic and histomorphometric studies as function of time revealed: a) at 6 days the presence of non-osseointegrated woven bone around the device is evident increasing in volume from 7 to 10 days post-implantation, and disappearing from day 12 to 14., b) at 14 days after implantation lamellar bone formation on the surface of the zirconium implants (OI) is noticeable. Additional bone growth is observed after 60 days. This study enables quantification of peri-implant reparative process response in an unloaded, necrotic trabeculae free model showing, in the different phases of the osseointegration process, the role of the blood clot and of the appearance and disappearance of woven bone and the final stages of osseointegration. Further investigation will allow comparison of results obtained under the effect of local and/or systemic factors that might affect osseointegration.
Animals↗
Isolated Sos1 PH domain exhibits germinal vesicle breakdown-inducing activity in Xenopus oocytes.
Purified, bacterially expressed PH domains of Sos1, IRS-1, betaARK, and PLCdelta1 were analyzed functionally by means of microinjection into full grown, stage VI Xenopus laevis oocytes. Whereas the PH domains from IRS-1, betaARK, or PLCdelta1 did not show any effect in the oocytes, injection of the purified Sos1 PH domain resulted in induction of significant rates of germinal vesicle breakdown and meiotic maturation. Furthermore, the Sos1 PH domain exhibited also significant synergy with insulin or coinjected normal Ras protein in induction of germinal vesicle breakdown, although it did not affect the rate of progesterone-induced maturation. These results suggest that purified, isolated PH domains retain, at least in part, their functional specificity and that Xenopus oocytes may constitute a useful biological system to analyze the functional role of the Sos1 PH domain in Ras signaling pathways.
Amino Acid Sequence↗
A 15 amino acid stretch close to the Grb2-binding domain defines two differentially expressed hSos1 isoforms with markedly different Grb2 binding affinity and biological activity.
We compared structure, expression and functional properties of two hSos1 cDNA isoforms (IsfI and Isf II) isolated, respectively, from human fetal brain and adult skeletal muscle libraries. IsfI and IsfII nucleotide sequences differ only by the presence in IsfII of an inframe 45 hp insertion located near the first proline-rich motif required for Grb2 binding. Some human tissues express only one isoform whereas others express different proportions of both in fetal and adult stages. In vitro binding assays and in vivo functional studies showed that MI exhibits significantly higher Grb2 binding affinity and biological activity than IsfI. These results suggest that functionally different hSos1 isoforms, with differential tissue expression and distribution, play important regulatory roles in the mechanisms controlling Ras activation in different tissues and/or developmental stages.
Adaptor Proteins, Signal Transducing↗
Expression of alternative forms of Ras exchange factors GRF and SOS1 in different human tissues and cell lines.
DNA probes and antibodies specific for different coding regions of human SOS1 and GRF genes were used to screen expression of these genes in a variety of adult and fetal human tissues and cell lines. Despite previous reports of the exclusive expression of hGRF RNA in brain, we also observed expression of this gene in various other tissues including lung and pancreas, as well as several tumor cell lines. At least three different hGRF mRNA transcripts were observed depending on the probe used, with the larger transcripts being detected by probes corresponding to the 5' end of the gene while smaller transcripts were detected by probes corresponding to the 3' end. Expression of hSOS1-related transcripts was more ubiquitous and homogeneous than with hGRF, with similar levels of specific transcripts being detected in most tissues and cell fines tested. Three to five different transcripts were detected in human tissues when using probes for the 5' end and middle regions of this gene, whereas only two were detected with probes corresponding to the 3' end. Screening of multiple human tumor cell lines showed ubiquitous expression of three specific transcripts, although the level and ratio of each of these transcripts varied widely among individual cell lines. Consistent with the variety of transcripts detected, several protein forms were also identified in Western immunoblots with antisera raised against specific domains of hSOS1 and human Ras-GRF gene products. Fluorescence in situ chromosomal hybridization suggested that, in both cases, the multiple forms arise from single chromosomal loci. The heterogeneity of hGRF and hSOS1 gene products detected (which appear to retain in most cases a functional catalytic domain), suggests that differentially expressed, alternatively spliced hSOS1 and hGRF forms may contribute to fine regulation of Ras activation in different tissues or at different stages of development.
3T3 Cells↗
Structural studies on the PH domains of Db1, Sos1, IRS-1, and beta ARK1 and their differential binding to G beta gamma subunits.
Pleckstrin homology (PH) domains are approximately 110 amino acid residues in length and are structurally conserved in a number of intracellular signaling proteins. A role for these domains has been postulated for beta ARK, which binds to G beta gamma subunits. We have quantified the binding of individual (His)6-tag PH domains of human Db1, human Sos1, rat IRS-1, human beta ARK, and human beta ARK with an extra 33-residue C-terminal extension (beta ARK + C) to G beta gamma subunits. Our in vitro binding studies show that all of the PH domains (apart from Sos1), bind G beta gamma subunits in a dose-dependent manner, but beta ARK + C binds 4 times as much G beta gamma at saturation as the others. The IRS-1 PH domain has a similar half-maximal concentration of G beta gamma binding (18 nM) to beta ARK + C (30 nM), suggesting that the IRS-1 PH domain has sufficient determinants for G beta gamma binding. The beta ARK PH domain alone has a half-maximal value of 45 nM but a drastically reduced extent of G beta gamma binding, suggesting that both the PH domain and the C-terminal 33 residues are necessary for maximal binding. Db1 has a half-maximum concentration of G beta gamma binding of 45 nM and a maximal extent of binding similar to that of beta ARK, but it is difficult to demonstrate saturable binding of G beta gamma to Sos1. Since it was previously predicted that the C-terminal PH domain of Pleckstrin [Tyers, M., et al. (1988) Nature 333, 470-473] contains a potential calcium binding site, we have tested the different PH domains for calcium binding. Only the PH domain of Db1 bound 45Ca2+ with a Kd of 10 microM. CD spectroscopy of the purified recombinant PH domains indicated that they are predominantly beta-sheet structures.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗