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E Santos

Publications and source records attributed to E Santos.

At least 55 records · Page 3Linked to original sources
PubMed1998

Isolation and molecular characterization of the Arabidopsis TPS1 gene, encoding trehalose-6-phosphate synthase.

An Arabidopsis thaliana cDNA clone, AtTPS1, that encodes a trehalose-6-phosphate synthase was isolated. The identity of this protein is supported by both structural and functional evidence. On one hand, the predicted sequence of the protein encoded by AtTPS1 showed a high degree of similarity with trehalose-6-phosphate synthases of different organisms. On the other hand, expression of the AtTPS1 cDNA in the yeast tps1 mutant restored its ability to synthesize trehalose and suppressed its growth defect related to the lack of trehalose-6-phosphate. Genomic organization and expression analyses suggest that AtTPS1 is a single-copy gene and is expressed constitutively at very low levels.

Amino Acid Sequence↗
PubMed1998

[Photosensitivity induced by piroxicam].

In Portugal, piroxicam is one of the most prescribed non-steroidal anti-inflammatory agents (NSAIDs) and is frequently associated to photosensitivity skin reactions, well known by all dermatologists. The authors review the physiopathology and clinical patterns of this adverse drug reaction. Importance is given to the high prevalence of thimerosal sensitisation in our country, a marker of piroxicam photosensitivity. The authors propose that piroxicam prescription should be avoided in Portugal, as this drug can be substituted by any other NSAID, without marked photosensitivity.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1997

Biological activity of p27kip1 and its amino- and carboxy-terminal domains in G2/M transition of Xenopus oocytes.

p27kip1 is a general inhibitor of Cdks that preferentially accumulates and functions during G1 phase, before the restriction point of the mammalian cell cycle. We observed that injection of purified p27kip1 into Xenopus oocytes potently inhibits the G2/M transition and activation/dephosphorylation of the maturation promoting factor (MPF, p34cdc2/cyclin B complex) kinase associated with germinal vesicle breakdown (GVBD) induced by progesterone or insulin. Addition of exogenous p27kip1 in vitro to lysates of hormonally matured oocytes blocked the enzymatic activity of the activated MPF kinase present in those extracts. Interestingly, the isolated amino-terminal region of p27kip1 (p27N), encompassing only the Cdk binding site, exhibited a similar inhibitory behavior in vitro and a weaker inhibitory effect in vivo than the complete p27kip1 protein. Surprisingly, the remaining carboxy-terminal region of p27kip1 (p27C) actually induced GVBD when injected alone into the oocytes, and also accelerated the kinetics of insulin- or progesterone-induced GVBD. Consistent with the in vivo observations, p27C formed a complex with, and activated, the MPF kinase in lysates of immature oocytes, although this activation was blocked by simultaneous addition of p27N or complete p27kip1. Active MPF was able to phosphorylate p27C only in the absence of p27N or whole p27kip1, suggesting that the inhibitory activity associated with the amino terminus is dominant over the activation produced by p27C. These results demonstrate the functional interaction of p27kip1 with cyclin B/p34cdc2 complexes during G2/M progression in oocytes, and suggest that the amino and carboxy terminal portions of this protein may play opposite regulatory roles, reminiscent of the corresponding N- and C-terminal portions of p21waf. We speculate that accumulation of a truncated, C-terminal p27 fragment may play a physiological regulatory role in progression through G2 and later stages of the cell cycle.

Animals↗
PubMed1997

Carboxyl-terminal domain of p27Kip1 activates CDC2.

A variant form of p27 was unexpectedly detected in a synchronized culture of NIH3T3 cells treated with serum. The expression levels of this form of p27 which lacked its amino (NH2)-terminal region reached maximum during G2/M phase. Since the appearance of the NH2-terminal truncated form of p27 coincided with increased expression of Cdc2, we hypothesized that p27 may play a role in regulating Cdc2 catalytic activity. To test this hypothesis, wild type p27, as well as the amino-terminal (Np27) and carboxyl-terminal (Cp27), were individually expressed, purified, and examined for their ability to regulate CDC2 kinase activity in vitro. Our data showed that both p27 and Np27 inhibited CDC2 kinase activity. However, in marked contrast, Cp27 enhanced the CDC2 kinase activity. In vitro kinase assays showed that Cp27 and p27 were phosphorylated by CDC2, whereas Np27 was not. In addition, we demonstrated that deletion of the putative CDC2 phosphorylation site in the carboxyl-terminal domain of Cp27 diminished activation of CDC2 kinase activity otherwise stimulated by Cp27. A similar deletion did not have any effect on the inhibitory function of p27. Together these results suggest that the carboxyl-terminal domain of p27 may activate CDC2 kinase activity in vivo during G2/M and that this effect may be regulated by serine/threonine phosphorylation.

3T3 Cells↗
PubMed1997

Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3-L1 adipocytic differentiation.

In 3T3-L1 fibroblasts, Ras proteins mediate both insulin-induced differentiation to adipocytes and its activation of cytosolic serine/threonine kinases, including Raf-1 kinase, mitogen-activated protein kinase (MAPK), and Rsk. Here, we report that insulin- and Ras-induced activation of MAPK is not required for the differentiation process and in fact antagonizes it. The treatment of 3T3-L1 preadipocytes with MEK-specific inhibitor PD98059 blocked insulin- and Ras-induced MAPK activation but had no effect on or slightly enhanced adipocytic differentiation. Tumor necrosis factor alpha (TNF-alpha), an inhibitor of insulin-stimulated adipogenesis, activated MAPK in 3T3-L1 cells. PD98059 treatment blocked MAPK activation by TNF-alpha and reversed the blockade of adipogenesis mediated by low (1 ng/ml) TNF-alpha concentrations. 3T3-L1 transfectants containing hyperactivated MEK1 or overexpressed MAPK displayed impaired adipocytic differentiation. PD98059 treatment also reversed the blockade of differentiation in MEK1 transfectants. These results indicate that MAPK does not promote but can contribute to inhibition of the process of adipocytic differentiation of 3T3-L1 cells.

3T3 Cells↗
PubMed1997

[Delayed reposition of gastrostomy tube using Savary's dilators].

The delay in substituting a gastrostomy tube after removal leads to the gastrocutaneous tract closure and to the impossibility of putting other nourishment tube without needing another complete endoscopic procedure. We have used the technique described by Tsang through the one which and using Savary's dilators, the stenosed gastrocutaneous tract after accidental removal of the tube, is dilated permitting to put in a simple way a new one. We accomplished the procedure in three patients that attended by removal of the probe and severe stenosis of the stoma. In all they and under endoscopic control we put a new tube in a way rapid and without complications.

Aged↗
PubMed1996

Functional interactions between isolated SH2 domains and insulin/Ras signaling pathways of Xenopus oocytes: opposite effects of the carboxy- and amino-terminal SH2 domains of p85 PI 3-kinase.

Purified amino-terminal Src homology 2 (SH2) domains of GAP, PLCgamma1 and the p85alpha subunit of PI 3-kinase, as well as the carboxy-terminal SH2 domain of the latter protein and the unique SH2 domain of Grb2, were injected into full grown, stage VI Xenopus laevis oocytes. None of the injected domains showed any effect when injected alone, nor did they affect the rate of GVBD induced by progesterone, an adenylate cyclase-dependent process. On the other hand, the unique Grb2 SH2 domain and all N-terminal SH2 domains injected inhibited to various degrees the rate of insulin-induced GVBD, a tyrosine kinase dependent pathway. Interestingly, and in contrast to the behavior shown by the N-terminal domain of the same molecule, the C-terminal SH2 domain of p85 did not inhibit, but slightly accelerated the rate of GVBD induced by insulin. Furthermore, whereas the Grb SH2 domain and all N-terminal SH2 domains tested failed to co-operate with normal Ras protein to induce GVBD, the C-terminal SH2 domain of p85alpha exhibited significant synergy when coinjected with normal Ras protein, indicating that the C- and N-terminal SH2 domains of p85alpha exert opposite (positive and negative, respectively) regulatory roles in the control of oocyte insulin/Ras signaling pathways. Our results demonstrate that the purified, isolated SH2 domains retain structural and functional specificity and that Xenopus oocytes constitute an useful biological system to analyse their functional role in tyrosine kinase signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1996

Mechanisms controlling egg size and number in the rainbow trout, Oncorhynchus mykiss.

Female rainbow trout (Oncorhynchus mykiss) produce a single batch of eggs each year; synchronous growth of oocytes, all of which are ovulated at the same time, occurs in the two ovaries. To examine the regulatory mechanisms controlling egg size and number, virgin female rainbow trout were subjected to unilateral ovariectomy (ULO) during early vitellogenesis, and oocyte recruitment and growth in the remaining ovary were monitored. The study also set out to determine whether the presence of a second population of smaller oocytes in the maturing pool (induced by ULO) affected the timing of ovulation and/or the size of the eggs ovulated. Two months after ULO, there was no difference in the gonadosomatic index between ULO fish and controls. Compensatory ovarian hypertrophy resulted from the recruitment of a second population of primary oocytes into the vitellogenic pool. This population of smaller maturing oocytes in the ULO fish displayed growth rates up to twice those of the population of larger oocytes in the same ovary and of oocytes in controls. The growth rate of the population of larger oocytes in the ULO fish was not altered by the recruitment of a second maturing population. One month after ULO, fish had a lower concentration of plasma estradiol-17 beta than did controls; subsequently the concentrations of plasma estradiol-17 beta in the ULO and control groups were similar. After ULO, plasma levels of vitellogenin in the ULO fish did not differ from those in the control group throughout the study. At or close to ovulation, the fecundity of ULO fish was 75-80% that of controls. In the control group, oocytes appeared to reach a certain critical size before they were ovulated, and fish with higher fecundity ovulated later than their less fecund counterparts. ULO did not affect the timing of ovulation, and ULO fish ovulated eggs with a considerably greater size-range than did controls.

Animals↗
PubMed1996

The insulin/Ras pathway of adipocytic differentiation of 3T3 L1 cells: dissociation between Raf-1 kinase and the MAPK/RSK cascade.

Insulin-induced differentiation of 3T3 L1 cells can be mimicked by expression of transfected ras oncogenes but is completely blocked by expression of dominant negative Ras mutants, demonstrating that Ras proteins mediate insulin signaling in these mammalian cells. In contrast, transfection of tyrosine kinase oncogenes including trk and src dose not result in adipocytic differentiation. Transfected raf-1 oncogenes induce partial adipocytic differentiation, while dominant negative raf mutants block partially the insulin-induced differentiation process. Exposure of 3T3 L1 cells to insulin results in formation of the active Ras-GTP complex without GAP tyrosine phosphorylation. Insulin treatment of untransfected 3T3 L1 cells also induced quick activation of cytosolic 42 kDa mitogen-activated protein kinase (MAPK) and a 90 kDa S6 kinase (RSK). The activation of these cytosolic serine-threonine kinases was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with inducible ras oncogenes. Furthermore, insulin-induced activation of MAPK and RSK could be blocked by expression of a transfected, inducible dominant negative Ras mutant (N17). These results indicate that Ras proteins are obligatory intermediates in the activation of cytosolic ERKs by insulin. Insulin treatment of 3T3 L1 cells or expression of transfected ras oncogenes resulted also in hyperphosphorylation of cellular Raf-1. Insulin-induced Raf hyperphosphorylation was inhibited by expression of an inducible, dominant negative Ras mutant (N17). Interestingly, however, expression of transfected raf oncogenes did not induce MAPK or RSK activation, and the insulin-induced activation of these kinases was not blocked by expression of transfected dominant negative raf mutants. These results suggest a functional dissociation between Raf-1 and MAPK/RSK activation in insulin/Ras signaling pathways leading to 3T3 L1 differentiation and are consistent with Raf-1 kinase acting in a parallel pathway to the MAPK/RSK pathway after Ras activation in these cells.

3T3 Cells↗
PubMed1995

A novel insertional mutation in the TC21 gene activates its transforming activity in a human leiomyosarcoma cell line.

TC21 is the fourth member of the ras gene family to exhibit oncogenic activation in human tumor cells. To assess the prevalence of activated TC21 oncogenes in human tumors, we have developed sensitive single-strand conformational polymorphism (SSCP) conditions and immunological reagents for the detection of both single base alterations and/or overt overexpression in a wide spectrum of human tumor cell lines and surgical samples. In an initial examination of 33 human tumor specimens, we observed a novel nine basepair three amino acids insertion at TC21 codon 24 in one human uterine leiomyosarcoma cell line, SK-UT-1. This mutant allele when transfected into NIH3T3 cells, displayed high transforming activity comparable to that of the Leu72 oncogenic mutant identified by expression cDNA cloning from a human ovarian carcinoma cell line. Comparing the level of GTP-binding by the mutant and normal TC21 products revealed that this novel lesion increases the GTP-bound form of the TC21 molecule. These findings imply that the mechanism by which mutations activate the oncogenic properties of this ras-related molecule is analogous to that of previously known ras family members.

3T3 Cells↗
PubMed1995

Vav and Ras induce fibroblast transformation by overlapping signaling pathways which require c-Myc function.

Recent evidence has suggested that the Vav oncoprotein may function as a hematopoietic-specific GTP exchange factor for the Ras superfamily of proteins. However, transformation of NIH3T3 fibroblast cells by Vav is morphologically distinct from that induced by activated Ras oncogenes, suggesting that the two oncoproteins induce separate signal transduction pathways which promote transformation. To address this issue, the effects of dominant negative mutants of H-ras and proto-Vav (proto-VavR695L, a mutation in the VavSH2 domain) were tested on Vav- and Ras-induced transformation. These mutants partially inhibited both Vav- and Ras-induced transformation, suggesting that they may induce a common downstream signaling pathway which potentiates transformation. As an independent measure of Vav function we also tested the ability of the purified protein encoded by VavSH2 to influence Germinal Vesicle Breakdown (GVBD) during Xenopus oocyte maturation. Microinjection of the VavSH2 protein alone, but not mutant VavR695L SH2 protein, was sufficient to induce GVBD and accelerated maturation induced by normal Ras, suggesting that in this system as well Vav and Ras signals overlap through a common effector. A key target of multiple signalling pathways is c-Myc. Dominant negative versions of c-Myc totally abolished morphologic transformation of NIH3T3 cells by both Vav and Ras oncogenes. These results suggest that distinct, but overlapping, signalling pathways are induced by Vav and Ras and that fibroblast cell transformation by either oncogene requires c-Myc functions.

3T3 Cells↗
PubMed1995

Human dual specificity phosphatase VHR activates maturation promotion factor and triggers meiotic maturation in Xenopus oocytes.

Bacterially expressed, dual specificity phosphatase VHR protein induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes, albeit with slower kinetics than that observed in progesterone- or insulin-induced maturation. A mutant VHR protein missing an essential cysteine residue for its in vitro phosphatase activity completely lacked activity in injected oocytes. VHR injection done in conjunction with progesterone or insulin treatment resulted in highly synergized GVBD responses showing much faster kinetics than that produced by VHR or either hormone alone. The delayed kinetics of VHR-induced GVBD and the synergistic responses obtained in the presence of hormones suggested that this protein may be promoting G2/M transition by weakly mimicking the action of cdc25, the dual specificity phosphatase that physiologically activates the maturation promotion factor. Various experimental observations are consistent with such a role for the injected VHR in oocytes: 1) as opposed to hormone-treated oocytes, histone H1 kinase activation is not preceded by MAPK activation in the process of GVBD in VHR-injected oocytes; 2) incubation of purified VHR with highly concentrated cell-free extracts of untreated oocytes resulted in activation of histone H1 kinase activity in the lysates; 3) coinjection of VHR with activated Ras proteins resulted in synergized responses, faster than those produced by either protein alone; 4) coinjection of VHR with the purified amino-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (which blocks insulin-induced GVBD) does not affect VHR-induced maturation. The biological actions of VHR in oocytes clearly distinguish it from other dual specificity phosphatases, which have shown inhibitory effects when tested in oocytes. We speculate that VHR may represent a dual specificity phosphatase responsible for activation of cdk-cyclin complex(es) at a still undetermined stage of the cell cycle.

Animals↗
PubMed1995

A divalent metal ion binding site in the kinase insert domain of the alpha-platelet-derived growth factor receptor regulates its association with SH2 domains.

To investigate the effects of metal ion binding to the alpha-PDGFR kinase insert domain, a PCR product representing amino acid residues 691-795 (104 amino acids) was bacterially expressed and purified. Secondary structure prediction and circular dichroism spectroscopy indicated this domain to be a mixed alpha + beta protein with a large coil/turn contribution. This 16 kDa, soluble, nonphosphorylated domain bound to 45Ca2+ and 65Zn2+ through a common shared site. Of the unlabeled divalent and trivalent metal ions tested, Ho3+ = Zn2+ > Ni2+ > Ca2+ = Mn2+ > Mg2+, Ba2+ in competing for 45Ca2+ binding to this domain. In the presence of Ca2+ ions, the conformation of the KI domain changed significantly, and this changed conformation was resistant to subtilisin proteolysis. However, in the presence of Zn2+ ions, the conformation of the KI domain changed only slightly. Nevertheless, Zn2+ ions were more effective in rendering the KI domain resistant to proteolysis as compared to that shown by Ca2+ ions. In vitro binding studies using purified baculovirus-expressed alpha-PDGFR showed a marked increase in binding the p85 N-SH2 domain in the presence of Ca2+ or Zn2+ ions (KD = 0.5 microM), suggesting that metal ion binding enhances association of the p85 N-SH2 domain with the receptor. To confirm this, association of the alpha-PDGFR with the p85 N-SH2 domain was tested in the presence of the KI domain. The nonphosphorylated KI domain was effective in competing with the alpha-PDGFR for the binding of the p85 N-SH2 domain. This effect was more pronounced in the presence of Ca2+ ions. Microinjection of this domain into Xenopus oocytes delayed maturation in the presence of insulin but not progesterone. This suggests that the KI domain has a correctly folded three-dimensional structure compatible with biological activity. Together these findings indicate that the recombinant alpha-PDGFR KI domain binds the p85 N-SH2 domain and this binding is modulated by the presence of a novel divalent metal ion binding site within its structure.

Amino Acid Sequence↗