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C Sousa

Publications and source records attributed to C Sousa.

At least 19 recordsLinked to original sources
PubMed2001

Rational design of a bacterial transcriptional cascade for amplifying gene expression capacity.

Cascade regulatory circuits have been described that control numerous cell processes, and may provide models for the design of artificial circuits with novel properties. Here we describe the design of a transcriptional regulatory cascade to amplify the cell response to a given signal. We used the salicylate-responsive activators of Pseudomonas putida NahR of the naphthalene degradation plasmid NAH7 and XylS2, a mutant regulator of the TOL plasmid for catabolism of m-xylene and their respective cognate promoters Psal and PM: Control of the expression of xylS2 with the nahR/Psal system permitted either their selective activation with specific effectors for each protein or the simultaneous activation of both of them with salicylate. When cells face the common effector of the two regulators, both the increase in XylS2 concentration and the stimulation of its activity act synergistically on the PM: promoter, amplifying the gene expression capacity by at least one order of magnitude with respect to the individual systems. By changing the hierarchy of regulators, we showed that the specific features of the downstream regulator were crucial for the amplification effect. Directed changes in the effector profile of the regulators allowed the extension of the amplifying system to other molecular signals.

Bacterial Proteins↗
PubMed2001

Influence of the interval between the administration of doxorubicin and paclitaxel on the pharmacokinetics of these drugs in patients with locally advanced breast cancer.

PURPOSE: The combination of bolus doxorubicin followed by a 3-h infusion of paclitaxel has high antitumor activity in patients with metastatic breast cancer, but is limited by unexpected cardiac toxicity. In contrast, the administration of the two drugs 16 h apart has similar antitumor activity but less cardiac toxicity. The purpose of this study was to compare the pharmacokinetics of these drugs when doxorubicin administration preceded paclitaxel by 30 min or by 24 h. PATIENTS AND METHODS: Women with locally advanced breast cancer were treated with doxorubicin (60 mg/m2 i.v. bolus) followed 24 h later by paclitaxel (200 mg/m2 i.v. over 3 h) for six cycles (four before and two after surgery). In one of the first two cycles doxorubicin preceded paclitaxel by 30 min instead of 24 h, with plasma sampling for pharmacokinetic analysis up to 48 h. Determination of drug levels in plasma was done by HPLC. RESULTS: A total of 28 patients were included. No clinical cardiac toxicity was observed but five patients discontinued doxorubicin-paclitaxel treatment after four cycles because of a decrease in LVEF of at least 15% from baseline or to less than 50%. While paclitaxel pharmacokinetics were not changed, there was a 30% and an 80% increase in the AUC0, 24h for doxorubicin and doxorubicinol, respectively, when the drugs were administered 30 min instead of 24 h apart. Even when paclitaxel was given 24 h after doxorubicin, there was a rebound 240% increase in the plasma concentration of doxorubicinol. CONCLUSIONS: Paclitaxel interferes with the pharmacokinetics of doxorubicin leading to higher systemic exposure to both doxorubicin and doxorubicinol, which is more evident when the plasma concentration of the anthracyclines is higher. This interference may explain the higher incidence of cardiac toxicity observed when the two drugs are administered within a short interval.

Adult↗
PubMed2001

Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex.

A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin gene enod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5' and 3' regions of enod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40 action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

Base Sequence↗
PubMed2001

Regulation of nod factor sulphation genes in Rhizobium tropici CIAT899.

Rhizobium tropici CIAT899 is a tropical symbiont able to nodulate various legumes such as Leucaena, Phaseolus, and Macroptilium. Broad host range of this species is related to its Nod factors wide spectrum. R. tropici contains Nod factors sulphation nod genes, nodHPQ genes, which control nodulation efficiency in Leucaena. To study nodHPQ regulation, we carried out different interposon insertions in its upstream region. One of these generated interruptions, nodI mutant produced nonsulphated Nod factors suggesting a possible dependence of these genes on nodI upstream region. Moreover, analysis results of lacZ transcriptional fusions with these genes in symbiotic plasmid showed dependence of these genes on NodD protein. In order to determine nodHPQ organization, we studied the effect of interposon insertion upstream of each lacZ transcriptional fusion, and the data obtained was used to indicate that nodHPQ belong to the nodABCSUIJ operon. However, comparison between nodP::lacZ beta-galactosidase activity in the symbiotic plasmid and in the pHM500 plasmid (containing nodHPQ genes) suggested constitutive expression in free living, and flavonoid inducible expression in symbiotic conditions. Constitutive nodHPQ expression may play a role in bacterial house-keeping metabolism. On the other hand, the transference of R. tropici nodHPQ genes to other rhizobia that do not present sulphated substitutions demonstrated that NodH protein sulphotransference is specific to C6 at the reducing end.

Bacterial Proteins↗
PubMed2000

Diaqua6,6'-dimethoxy-2,2'-

In the title compound, [Ni(C(19)H(20)N(2)O(4))(H(2)O)(2)], the Ni atom has a distorted octahedral coordination geometry in which the tetradentate Schiff base ligand acts as a cis-N(2)O(2) donor defining an equatorial plane, and water molecules occupy the axial positions. The two parts of the molecule are related by a mirror plane that passes through the Ni atom and is perpendicular to the equatorial plane. The angular distortions from normal octahedral geometry are in the range 1-6 degrees, and the equatorial plane, defined by the donor atoms of the Schiff base, is almost square planar. The six-membered ring comprising the Ni, the imine N and the propylene C atoms adopts a half-chair conformation. The Ni-O [2.017 (2) A] and Ni-N [2.071 (2) A] distances are within the ranges expected for high-spin octahedral nickel complexes.

Journal Article↗
PubMed1999

Alteration of enod40 expression modifies medicago truncatula root nodule development induced by sinorhizobium meliloti

Molecular mechanisms involved in the control of root nodule organogenesis in the plant host are poorly understood. One of the nodulin genes associated with the earliest phases of this developmental program is enod40. We show here that transgenic Medicago truncatula plants overexpressing enod40 exhibit accelerated nodulation induced by Sinorhizobium meliloti. This resulted from increased initiation of primordia, which was accompanied by a proliferation response of the region close to the root tip and enhanced root length. The root cortex of the enod40-transformed plants showed increased sensitivity to nodulation signals. T(1) and T(2) descendants of two transgenic lines with reduced amounts of enod40 transcripts (probably from cosuppression) formed only a few and modified nodulelike structures. Our results suggest that induction of enod40 is a limiting step in primordium formation, and its function is required for appropriate nodule development.

Journal Article↗
PubMed1998

8-Methoxypsoralen potentiates the photocytotoxic effect of Photofrin II towards EMT-6 murine tumor cells.

Pretreatment of EMT-6 murine tumor cells for 24 h with 10(-4) M 8-methoxypsoralen (8-MOP) increased the photocytotoxicity of Photofrin II (P2) after cell exposure to low doses (1-1.5 J/cm2) of UVA by two- to three-fold. 8-MOP alone had no cytotoxic action under these experimental conditions and did not significantly change the amount of P2 recovered in cells. 8-MOP enhanced the lipid peroxidation end product formation measured as thiobarbituric acid reactive substances (TBARS) during cell photosensitization by P2. The psoralen alone also slightly increased the TBARS level after UVA exposure. These results suggest that 8-MOP, albeit non-photocytotoxic by itself under our experimental conditions, could enhance the efficiency of P2 by increasing cellular lipid peroxidation following light exposure.

Animals↗
PubMed1998

Sulfation of nod factors via nodHPQ is nodD independent in Rhizobium tropici CIAT899.

A cosmid from the Rhizobium tropici CIAT899 symbiotic plasmid, containing most of the nodulation genes described in this strain, has been isolated. Although this cosmid does not carry a nodD gene, it confers ability to heterologous Rhizobium spp. to nodulate R. tropici hosts (Phaseolus vulgaris, Macroptilium atropurpureum, and Leucaena leucocephala). The observed phenotype is due to constitutive expression of the nodABCSUIJ operon, which has lost its regulatory region and is expressed from a promoter present in the cloning vector. Thin-layer chromatography (TLC) analysis of the Nod factors produced by this construction shows that it is still capable of synthesizing sulfated compounds, suggesting that the nodHPQ genes are organized as an operon that is transcribed in a nodD-independent manner and is not regulated by flavonoids.

Bacterial Proteins↗
PubMed1998

Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer membrane protein LamB.

Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB153-YMT and LamB153-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. coli cells to bind Cd2+ 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.

Adsorption↗
PubMed1998

PUVA-induced cell mortality in NCTC 2544 keratinocytes: is it related to the microenvironmental properties of the excited states of psoralens?

The phototoxic effect of psoralen (PSO), 5-methoxypsoralen (5MOP), 8-methoxypsoralen (8MOP) and 4,5',8-trimetylpsoralen (TMP) has been compared on the NCTC 2544 keratinocyte cell line in terms of cell mortality and lipid peroxidation. The order of effectiveness for cell photokilling is TMP, 5MOP >> 8MOP, PSO, whereas a little lipid peroxidation is observed for the four psoralens under study. Oxygen-independent membrane damage seem to play a key role in the lethal photodamage because the biological effectiveness of the most hydrophobic lipid-soluble psoralens, TMP and 5MOP, is about an order of magnitude higher than that of the more water-soluble 8MOP and PSO. In relation to this hypothesis, and in contrast to 8MOP, TMP is readily extracted from cells by ethyl acetate, a good membrane solvent, as shown by GC/MS analysis on cell extracts. The results are discussed in terms of the highly microenvironment-dependent photophysical properties of psoralens. By the measure of the intracellular psoralen concentration, the neutral red uptake and the lipid peroxidation products, this work provides evidence that PUVA therapy-mediated cell mortality is a lipid peroxidation-independent phenomenon.

Cell Line↗
PubMed1997

Effector specificity mutants of the transcriptional activator NahR of naphthalene degrading Pseudomonas define protein sites involved in binding of aromatic inducers.

This work reports a genetic analysis of the interactions between NahR, the LysR-type regulator of the NAH operons for biodegradation of naphthalene in Pseudomonas, and its aromatic effectors. Six mutants encoding NahR variants responsive to salicylate analogs such as benzoate, which is not an inducer for the wild type regulator, were isolated with a polymerase chain reaction-based saturation mutagenesis protocol. Most mutants displaying a specific change of effector profile bore single amino acid substitutions within a short protein segment of 60 residues located at the central portion of the NahR sequence. Some of the protein variants exhibited an increased affinity for salicylate and also for otherwise suboptimal effectors, with apparent Ks' values 5-100-fold lower than those of the wild type NahR protein. In addition, all mutants were activated by inducers bearing novel substituents at positions 1 or 2 of the aromatic ring and displayed also an enhanced tolerance to changes at positions 3 and 4. Correlation between mutations in NahR and the structures of the new effectors suggested that protein sites Met116, Arg132, Asn169, and Arg248 are involved in effector recognition and binding during the earlier steps of the process leading to transcriptional activation of cognate NAH promoters.

Bacterial Proteins↗
PubMed1996

Enhanced metalloadsorption of bacterial cells displaying poly-His peptides.

The properties of Escherichia coli cells, acquired by cell surface presentation of one or two hexahistidine (His) clusters carried by the outer membrane LamB protein, have been examined. Strains producing LamB hybrids with the His chains accumulated greater than 11-fold more Cd2+ than E. coli cells expressing the protein without the His insert. Furthermore, the hexa-His chains on the cell surface caused cells to adhere reversibly to a Ni(2+)-containing solid matrix in a metal-dependent fashion. Thus, expression of poly-His peptides enables bacteria to act as a metalloaffinity adsorbent. These results open up the possibility for biosorption of heavy ions using engineered microorganisms.

Adsorption↗
PubMed1996

Characterization of Rhizobium tropici CIAT899 nodulation factors: the role of nodH and nodPQ genes in their sulfation.

We have purified and characterized the nodulation factors produced by Rhizobium tropici CIAT899. This strain produces a large variety of nodulation factors, these being a mixture of sulfated or nonsulfated penta- or tetra-chito-oligosaccharides to which any of six different fatty acyl moieties may be attached to nitrogen of the nonreducing terminal residue. In this mixture we have also found methylated or nonmethylated lipo-chitin oligosaccharides. Here we describe a novel lipo-chitin-oligosaccharide consisting of a linear backbone of 4 N-acetylglucosamine residues and one mannose that is the reducing-terminal residue and bearing a C18:1 fatty acyl moiety on the nonreducing terminal residue. In addition, we have identified, cloned, and sequenced R. tropici nodH and nodPQ genes, generated mutations in the nodH and nodQ genes, and tested the mutant strains for nodulation in Phaseolus and Leucaena plants. Our results indicate that the sulfate group present in wild-type Nod factors plays a major role in nodulation of Leucaena plants by strain CIAT899 of R. tropici.

Amino Acid Sequence↗
PubMed1996

Microenvironment effects on the excited state properties of psoralens: a clue to their photobiological activity.

The singlet and triplet excited state parameters (phi f, tau t and phi T) of psoralen (PSO) and derivatives 4,6,4'-trimethylangelicin (TMA) and 4,5',8-trimethylpsoralen (TMP) show an extreme sensitivity to solvation in dioxane/water mixtures. These effects are attributed to the variation of the S1-->S0 internal conversion rate constant kic, which is the nonradiative deactivation path dominating their photophysical behavior. Depending on the compound, kic is very high, (approximately 1 x 10(10) s-1) in nonpolar solvents and then decreases to a low value (3 x 10(8) s-1), with increasing solvent polarity. This work shows that dioxane/water mixtures display the same solvent-induced changes in the electronic structure of psoralens during solvation as those induced by the biological microenvironment sensed by the drug's localization. This mixture matches the photophysical parameters of psoralens observed in protic and aprotic pure solvents, in micelles, in liposomes and in human serum low-density lipoproteins (LDL). They can be used to probe the solvating ability of the interaction site in macrocyclic hosts. A particular localization site, i.e. the more (TMA and TMP) or less (PSO) lipophilic sites found when in interaction with LDL, determines the amount of the triplet reactive state of psoralens and the molecular mechanism available for photoreaction: oxic (type I and type II) or anoxic (type III) pathways.

Furocoumarins↗
PubMed1996

Solvent polarity and pH effects on the spectroscopic properties of neutral red: application to lysosomal microenvironment probing in living cells.

Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRH) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of lysosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal microenvironments.

Dioxanes↗
PubMed1995

Evora district as a new focus for canine leishmaniasis in Portugal.

On the basis of information acquired from local health authorities in Evora district of Portugal on cases of visceral leishmaniasis (VL), an epidemiology survey study was conducted. To determine the prevalence of anti-Leishmania antibodies in the local human and canine populations residing in Evora town and 14 adjacent villages, blood samples collected from 885 children and 3,614 dogs were tested in a direct agglutination test (DAT). Seropositivity for Leishmania parasite obtained by DAT in both endemic populations was further confirmed by an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence test (IFAT). For identification of the responsible sandfly vector, 79 biotopes within the study areas were surveyed. In the infantile population assessed, none of the children screened showed an antibody level indicative (titer, > = 1:3200) of visceral leishmaniasis in the DAT. However, agglutinating antibody rates ranging from 0.7% to 6.9% were obtained in dogs residing in Evora and 11 adjacent villages. Concordant seropositivity of 94.04% was obtained by ELISA and IFAT in the same canine population (141) identified by DAT. Of the 159 sandflies captured, 67 were identified as Phlebotomus sergenti; 15, as P. ariasi; 58, as P. perniciosus; and 19, as Sergentomyia minuta. Unlike the results previously reported in Alto-Douro and Algarve districts of Portugal, as compared with the other three species, P. sergenti appears to be more abundant in Evora district.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗