Biomedical subjects

M L Rodrigues

Publications and source records attributed to M L Rodrigues.

At least 19 recordsLinked to original sources

PubMed2006

Crystallization and preliminary structure determination of the membrane-bound complex cytochrome c nitrite reductase from Desulfovibrio vulgaris Hildenborough.

The cytochrome c nitrite reductase (cNiR) isolated from Desulfovibrio vulgaris Hildenborough is a membrane-bound complex formed of NrfA and NrfH subunits. The catalytic subunit NrfA is a soluble pentahaem cytochrome c that forms a physiological dimer of about 120 kDa. The electron-donor subunit NrfH is a membrane-anchored tetrahaem cytochrome c of about 18 kDa molecular weight and belongs to the NapC/NirT family of quinol dehydrogenases, for which no structures are known. Crystals of the native cNiR membrane complex, solubilized with dodecylmaltoside detergent (DDM), were obtained using PEG 4K as precipitant. Anomalous diffraction data were measured at the Swiss Light Source to 2.3 A resolution. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 79.5, b = 256.7, c = 578.2 A. Molecular-replacement and MAD methods were combined to solve the structure. The data presented reveal that D. vulgaris cNiR contains one NrfH subunit per NrfA dimer.

Cell Membrane↗

PubMed2006

Comparative study of ortho- and meta-nitrated inhibitors of catechol-O-methyltransferase: interactions with the active site and regioselectivity of O-methylation.

In this work, we present a comparative case study of "ortho-" and "meta-nitrated" catecholic inhibitors of catechol-O-methyltransferase (COMT), with regard to their interaction with the catalytic site of the enzyme and the in vitro regioselective formation of their mono-O-methyl ether metabolites. In particular, the effects of altering the attachment position of the inhibitors' side-chain substituent, within the classic nitrocatechol pharmacophore, were investigated. For this purpose, we compared two simple regioisomeric nitrocatechol-type inhibitors of COMT, BIA 3-228 and BIA 8-176, which contain the benzoyl substituent attached at the meta and ortho positions, respectively, relative to the nitro group. The two compounds were slowly O-methylated by COMT in vitro, but the particular substitution pattern of each compound was shown to have a profound impact on the regioselectivity of their O-methylation. To provide a plausible interpretation of these results, a comprehensive analysis of the protein-inhibitor interactions and of the relative chemical susceptibility to O-methylation of the catechol hydroxyl groups was performed by means of docking simulations and ab initio molecular orbital calculations. The major structural and chemical factors that determine the enzyme regioselectivity of O-methylation were identified, and the X-ray structure of the complex of COMT with S-adenosyl-l-methionine and BIA 8-176 is herein disclosed. This is the first reported structure of the soluble form of COMT complexed with a nitrocatecholic inhibitor having a bulky substituent group in adjacent position (ortho) to the nitro group. Structural and dynamic aspects of this complex are analyzed and discussed, in the context of the present study.

Animals↗

PubMed2006

Metabolic responses to water deficit in two Eucalyptus globulus clones with contrasting drought sensitivity.

We compared the metabolic responses of leaves and roots of two Eucalyptus globulus Labill. clones differing in drought sensitivity to a slowly imposed water deficit. Responses measured included changes in concentrations of soluble and insoluble sugars, proline, total protein and several antioxidant enzymes. In addition to the general decrease in growth caused by water deficit, we observed a decrease in osmotic potential when drought stress became severe. In both clones, the decrease was greater in roots than in leaves, consistent with the observed increases in concentrations of soluble sugars and proline in these organs. In roots of both clones, glutathione reductase activity increased significantly in response to water deficit, suggesting that this enzyme plays a protective role in roots during drought stress by catalyzing the catabolism of reactive oxygen species. Clone CN5 has stress avoidance mechanisms that account for its lower sensitivity to drought compared with Clone ST51.

Adaptation, Physiological↗

PubMed2005

Antimicrobial activity of Croton cajucara Benth linalool-rich essential oil on artificial biofilms and planktonic microorganisms.

We have previously demonstrated that a linalool-rich essential oil from Croton cajucara Benth presents leishmanicidal activity. In the present study, we demonstrate that this essential oil inhibits the growth of reference samples of Candida albicans, Lactobacillus casei, Staphylococcus aureus, Streptococcus sobrinus, Porphyromonas gingivalis and Streptococcus mutans cell suspensions, all of them associated with oral cavity disease. The purified linalool fraction was only inhibitory for C. albicans. Microbes of saliva specimens from human individuals with fixed orthodontic appliances, as well as the reference strains, were used to construct an artificial biofilm which was exposed to linalool or to the essential oil. As in microbial suspensions, the essential oil was toxic for all the microorganisms, while the purified linalool fraction mainly inhibited the growth of C. albicans. The compounds of the essential oil were separated by thin layer chromatography and exposed to the above-cited microorganisms. In this analysis, the proliferation of the bacterial cells was inhibited by still uncharacterized molecules, and linalool was confirmed as the antifungal component of the essential oil. The effects of linalool on the cell biology of C. albicans were evaluated by electron microscopy, which showed that linalool induced a reduction in cell size and abnormal germination. Neither the crude essential oil nor the purified linalool fraction is toxic to mammalian cells, which suggests that the essential oil or its purified components may be useful to control the microbial population in patients with fixed orthodontic appliances.

Acyclic Monoterpenes↗

PubMed2004

Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex.

Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson's disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 A resolution on a synchrotron-radiation source and belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 A, beta = 91.14 degrees.

Animals↗

PubMed2002

How plants cope with water stress in the field. Photosynthesis and growth.

Plants are often subjected to periods of soil and atmospheric water deficit during their life cycle. The frequency of such phenomena is likely to increase in the future even outside today's arid/semi-arid regions. Plant responses to water scarcity are complex, involving deleterious and/or adaptive changes, and under field conditions these responses can be synergistically or antagonistically modified by the superimposition of other stresses. This complexity is illustrated using examples of woody and herbaceous species mostly from Mediterranean-type ecosystems, with strategies ranging from drought-avoidance, as in winter/spring annuals or in deep-rooted perennials, to the stress resistance of sclerophylls. Differences among species that can be traced to different capacities for water acquisition, rather than to differences in metabolism at a given water status, are described. Changes in the root : shoot ratio or the temporary accumulation of reserves in the stem are accompanied by alterations in nitrogen and carbon metabolism, the fine regulation of which is still largely unknown. At the leaf level, the dissipation of excitation energy through processes other than photosynthetic C-metabolism is an important defence mechanism under conditions of water stress and is accompanied by down-regulation of photochemistry and, in the longer term, of carbon metabolism.

Acclimatization↗

PubMed2001

Crystallization and preliminary crystallographic characterization of catechol-O-methyltransferase in complex with its cosubstrate and an inhibitor.

Catechol-O-methyltransferase (COMT) is involved in the metabolism of catecholamines, catechol steroids and xenobiotic catechols. A precise knowledge of the enzyme-inhibitor structural interactions could help in the design of better inhibitors. Soluble rat COMT was expressed in Escherichia coli and the recombinant protein was crystallized with a new tight-binding inhibitor, BIA 3-335 [1-(3,4-dihydroxy-5-nitrophenyl)-3-(n-3'-trifluoromethylphenyl)piperazine-1-propanone dihydrochloride]. The crystals were obtained by the sitting-drop vapour-diffusion method using PEG 6K as a precipitant. These crystals diffracted to better than 1.9 A and belong to the trigonal space group P3(2)21. The unit-cell parameters for the crystal measured at room temperature were a = b = 51.5, c = 168.3 A; each shrank by about 1 A on freezing.

Animals↗

PubMed2001

Association of the maternal experience and changes in adiposity measured by BMI, waist:hip ratio and percentage body fat in urban Brazilian women.

The aim of the present study was to determine the association between the maternal experience and changes in adiposity measured by BMI, percentage body fat (PBF) and waist:hip ratio (WHR). In a cross-sectional study, 203 women were recruited at health care and educational facilities in Brasilia, Brazil. These women were divided into three groups: fifty-three nulliparous (no full-term pregnancy); sixty-three primiparous (one full-term pregnancy); eighty-seven multiparous (two or more full-term pregnancies). Socio-economic, behavioural, reproductive and dietary data were collected. All the women were measured for weight, height, skinfold thicknesses and waist and hip circumferences. Analysis of covariance was used to measure the differences among the three groups in relation to BMI, PBF, and WHR, controlling for the following covariates: age; socio-economic status; use of oral contraceptives; smoking; energy intake level; cooking oil intake; physical activity level; lactation score; parity. The three groups of women differed significantly for BMI (P = 0.04), PBF (P = 0.0008), and WHR (P = 0.0001). Multiparous women presented higher BMI (P = 0.01) and PBF (P = 0.03) compared with primi- and nulliparous groups. PBF was also associated with age and high socio-economic status. Primi- and multiparous women showed a higher WHR than nulliparous women (P < 0.0001). Age and smoking habit were also factors associated with higher WHR.

Adipose Tissue↗

PubMed2001

Direct current decreases cell viability but not P-glycoprotein expression and function in human multidrug resistant leukemic cells.

Inhibition of tumor growth induced by treatment with direct current (DC) has been reported in several systems. In the current work, the cellular effects generated by the DC treatment of the human leukemic K562 cell line and its vincristine-resistant derivative K562-Lucena 1 were analyzed by trypan blue staining and transmission electron microscopy. DC stimulation induced cell lysis, alterations in shape, membrane extraction or discontinuity, and intense vacuolization of some cells. In addition, treatment of K562 and K562-Lucena 1 cells caused a marked decrease in viability. Since multidrug resistance is a major factor contributing with failure of chemotherapy in many tumors, the expression and function of P-glycoprotein (P-gp) in K562-Lucena 1 cells were also studied. The expression of mdr1, the gene encoding P-gp, was analyzed by reverse transcription polymerase chain reaction, which showed that this gene was equally expressed in either treated or untreated cells. These results were confirmed by flow cytometry with a monoclonal anti P-gp antibody and the Rhodamine 123 extrusion method, which revealed that P-gp surface expression and function were unaltered after DC treatment. Our results suggest that DC treatment does not affect P-gp in human leukemic cells, but affects their viability by mechanisms that would involve clear cellular effects, but also additional targets, whose relevance in dc treated tumoral cells is currently discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Cellular damage and altered carbohydrate expression in P815 tumor cells induced by direct electric current: an in vitro analysis.

Treatment with direct electric current (DC) can inhibit tumor growth in several systems. To evaluate the cellular reactions generated by this treatment, we stimulated mouse mastocytoma P815 cells with DC and examined their viability and ultrastructural characteristics, as well as the effect of DC on surface carbohydrate expression. DC treatment affected cell viability and caused marked alterations in vital structures of P815 cells. Alterations varied depending on the duration of stimulation and polarity of electrode. Anodic and cathodic treatments caused decrease in cell viability, although the latter was more effective in generating cell lysis. DC stimulation also induced changes such as membrane damage, alterations in cell shape and chromatin organization, mitochondrial swelling and condensation, cytoplasmic swelling, and matrix rarefaction. Stimulation of P815 cells without contact with electrodes produced no alterations, suggesting that this contact might be essential for the occurrence of the cellular modifications. DC treatment also altered the membrane distribution of anionic sites of P815 cells, as well as the surface carbohydrate exposition, involving a diminished binding of Concanavalin A to the cell surface after cathodic stimulation, and an increased binding of sialic acid- and fucose-specific lectins after anodic treatment. In this work we describe important cellular targets for the action of DC, which may contribute to the understanding of the mechanisms by which DC supresses several kinds of tumors.

Animals↗

PubMed2000

Crystallization and preliminary x-ray diffraction analysis of beta-cinnamomin, an elicitin secreted by the phytopathogenic fungus Phytophthora cinnamomin.

Cinnamomin (CIN) belongs to a family of 10 kDa proteins designated as elicitins. Some of these proteins induce a hypersensitive response in diverse plant species, leading to resistance against fungal and bacterial plant pathogens. CIN was crystallized by the vapour-diffusion method using either ammonium sulfate or polyethyleneglycol (PEG) as precipitants in solutions buffered at around pH 7. These crystals are isomorphous and belong to the triclinic space group, with unit-cell parameters a = 31.69, b = 36. 99, c = 44.09 A, alpha = 76.86, beta = 84.41, gamma = 80.26 degrees. A frozen crystal diffracted X-rays beyond 1.45 A resolution on a synchrotron-radiation source.

Algal Proteins↗

PubMed2000

Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth.

A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a beta-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the beta-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal beta-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro.

Antibodies, Fungal↗

PubMed1999

Pathogenicity of Cryptococcus neoformans: virulence factors and immunological mechanisms.

Cryptococcus neoformans is the causative agent of cryptococcosis and cryptococcal meningitis, which are serious pathological conditions affecting up to 10% of patients with AIDS. Mechanisms of pathogenicity of C. neoformans and the host defenses against this fungus are reviewed, incorporating recent data and perspectives.

AIDS-Related Opportunistic Infections↗

PubMed1999

Elastase substrate specificity tailored through substrate-assisted catalysis and phage display.

The catalytic histidine of human neutrophil elastase was replaced with alanine (H57A) to determine if a substrate histidine could substitute for the missing catalytic group-'substrate-assisted catalysis'. H57A and wild-type elastase were recovered directly from Pichia pastoris following expression from a synthetic gene lacking the elastase pro sequence, thereby obviating the need for zymogen activation. Potential histidine-containing substrates for H57A elastase were identified from a phage library of randomized sequences. One such sequence, REHVVY, was cleaved by H57A elastase with a catalytic efficiency, k(cat)/K(M), of 2800 s(-1) M(-1), that is within 160-fold of wild-type elastase. In contrast, wild-type but not H57A elastase cleaved the related non-histidine containing sequence, REAVVY. Ten different histidine-containing linkers were cleaved by H57A elastase. In addition to the requirement for a P2 histidine, significant preferences were observed at other subsites including valine or threonine at P1, and methionine or arginine at P4. A designed sequence, MEHVVY, containing the preferred residues identified at each subsite proved to be a more favorable substrate than any of the phage-derived sequences. Extension of substrate-assisted catalysis to elastase suggests that this engineering strategy may be widely applicable to other serine proteases thereby creating a family of highly specific histidine-dependant proteases.

Amino Acid Sequence↗

PubMed1998

Transferrin production by the ciliary body of rabbits: a biochemical and immunocytochemical study.

PURPOSE: We have previously reported that transferrin is one of several glycoproteins synthesized within the eye and secreted into the vitreous. The present investigation was designed to determine the role of the ciliary body in the production of this vitreous transferrin. METHODS: Isolated ciliary body-iris were incubated with 3H-fucose, 3H-tyrosine or 35S-methionine and afterwards the culture media were processed for affinity chromatography using columns of Sepharose conjugated with antibody to rabbit plasma transferrin. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using total RNA extracted from fresh ciliary body-iris and primers constructed on the basis of the known sequence of transferrin mRNA from rabbit liver. The fragment obtained was employed as a probe in northern-blots of total RNA of ciliary body-iris. Furthermore, paraffin sections of eyes were treated for immunocytochemical visualization of transferrin. RESULTS: A labeled polypeptide, specifically eluted from the antitransferrin columns, was detected in the incubation medium, transferrin mRNA was found in extracts of whole ciliary body-iris, and transferrin antigenicity was identified in the ciliary and iridial epithelial cells by immunocytochemistry. CONCLUSIONS: These results demonstrate the ciliary epithelium as one of the sources of the vitreous transferrin.

Animals↗

PubMed1997

Identification of N-acetylneuraminic acid and its 9-O-acetylated derivative on the cell surface of Cryptococcus neoformans: influence on fungal phagocytosis.

Sialic acids from sialoglycoconjugates present at the cell surface of Cryptococcus neoformans yeast forms were analyzed by high-performance thin-layer chromatography, binding of influenza A and C virus strains, enzymatic treatment, and flow cytofluorimetry with fluorescein isothiocyanate-labeled lectins. C. neoformans yeast forms grown in a chemically defined medium contain N-acetylneuraminic acid and its 9-O-acetylated derivative. A density of 3 x 10(6) residues of sialic acid per cell was found in C. neoformans. Sialic acids in cryptococcal cells are glycosidically linked to galactopyranosyl units as inferred from the increased reactivity of neuraminidase-treated yeasts with peanut agglutinin. N-Acetylneuraminic acids are alpha-2,6 and alpha-2,3 linked, as indicated by using virus strains M1/5 and M1/5 HS8, respectively, as agglutination probes. The alpha-2,6 linkage markedly predominated. These findings were essentially confirmed by the interaction of cryptococcal cells with the lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin. We also investigated whether the sialyl residues present in C. neoformans are involved in the fungal interaction with a cationic solid-phase substrate and with mouse resident macrophages. Adhesion of yeast cells to poly-L-lysine was mediated, in part, by sialic acid residues, since the number of adherent cells was markedly reduced after treatment with bacterial neuraminidase. The enzymatic removal of sialic acids also made C. neoformans yeast cells more susceptible to endocytosis by macrophages. The results show that sialic acids are components of the cryptococcal cell surface that contribute to its negative charge and protect yeast forms against phagocytosis.

Acetylation↗

PubMed1997

Correlation between adrenal central vein parasitism and heart fibrosis in chronic chagasic myocarditis.

The correlation between Trypanosoma cruzi parasitism of the adrenal central vein (ACV) wall and fibrous connective tissue neoformation in the left ventricular myocardium (LVM) of patients with chronic Chagas' disease who were autopsied was evaluated using the following procedures: 1) a comparison of the incidence of fibrosis in the LVM among 18 chagasic patients with ACV parasitism and 18 individuals without phleboparasitism; 2) a determination of fibrosis intensity in the LVM in 12 cases with ACV parasites and in 12 cases without phleboparasitism, matched with respect to age, sex, race, and anatomoclinical form of the disease (indeterminant, cardiac, and digestive forms); and 3) in the cases with ACV parasitism, a calculation of Pearson's correlation coefficient between T. cruzi nests in the vessel and the intensity of fibrous connective tissue neoformation in the LVM. Among chagasic individuals with adrenal phleboparasitism, there was an increased incidence and intensity of fibrous connective tissue neoformation in the LVM, both highly significant, compared with patients without adrenal phleboparasitism. Furthermore, there was a positive, though nonsignificant, correlation (r = +0.19) between the density of nests in the ACV and the intensity of myocardial fibrosis. These results are consistent with previous data showing a higher intensity of the leukocyte exudate in the LVM and increased heart weight in individuals with T. cruzi nests in the ACV, suggesting a role of parasitism at that site in terms of the development of chronic chagasic cardiopathy.

Adrenal Glands↗

PubMed1995

Development of a humanized disulfide-stabilized anti-p185HER2 Fv-beta-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug.

The humanized anti-p185HER2 antibody, humAb4D5-8, has completed Phase II clinical trials for p185HER2-overexpressing breast cancer. Here, this antibody is used as a building block to engineer a disulfide-linked Fv (dsFv) beta-lactamase fusion protein for use in antibody-dependent enzyme-mediated prodrug therapy using cephalosporin-based prodrugs. Three Fv variants were designed with an interchain disulfide bond buried at the VL/VH interface and secreted from Escherichia coli. One variant, dsFv3 (VL L46C VH D101C0, has similar affinity for antigen (Kd = 0.7 nM) as the wild-type Fv and was used to construct a fusion protein in which beta-lactamase, RTEM-1, is joined to the carboxy terminus of VH. The dsFv3-beta-lactamase fusion protein secreted from E. coli efficiently activates a cephalothin doxorubicin prodrug (PRODOX, kcat/km = 1.5 x 10(5) s-1 M-1). PRODOX is approximately 20-fold less toxic than free doxorubicin against breast tumor cell lines SK-BR-3 and MCF7, which express p185HER2 at elevated and normal levels, respectively. Prebinding the dsFv3-beta-lactamase fusion protein specifically enhances the toxicity level of PRODOX to that of doxorubicin against SK-BR-3 but not MCF7 cells. The fusion protein retains both antigen-binding plus kinetic activity in murine serum and is cleared rapidly as judged by pharmacokinetic analysis in nude mice (initial and terminal half-lives of 0.23 and 1.27 h, respectively). Development and characterization of the dsFv3-beta-lactamase fusion protein is an important step toward targeted prodrug therapy of p185HER2-overexpressing tumors.

Animals↗