Biomedical subjects

Laura Ferreira

Publications and source records attributed to Laura Ferreira.

8 recordsLinked to original sources

PubMed2006

Urinary proteomics.

Protein measurement in urine has been used for many years for the diagnosis and monitoring of renal disease. The pattern of urinary protein excretion can be used to identify the cause of the disease and to classify proteinuria. In recent years, proteomics has proven to be a powerful tool in investigation and clinical medicine. Proteomics employs a protein separation method and the identification of proteins using mass spectrometry. One of the objectives of clinical proteomics is the identification of biological markers of disease. To accomplish this, it is necessary to have a normal proteome of the medium in question, which in our case is urine. Comparison of the normal urinary proteome with the urinary proteome from patients with a defined disease can detect proteins expressed differentially from one another. The aim of this review is to present the situation of urinary proteomics, putting special emphasis on its application in the diagnosis of glomerular diseases, renal allograft rejection, urological cancers and urolithiasis.

Biomarkers↗

PubMed2005

N-terminal cleavage of proTGFalpha occurs at the cell surface by a TACE-independent activity.

ProTGFalpha (transforming growth factor alpha precursor) maturation and conversion into soluble TGFalpha is a complex process that involves three proteolytic steps. One, that occurs co-translationally, eliminates the signal sequence. Another, occurring at the juxta-membrane domain, solubilizes TGFalpha. A third cleavage removes the N-terminal extension of proTGFalpha. This latter step has been poorly studied, mainly because of the rapid kinetics of this cleavage. In the present study, we have designed a strategy to analyse several aspects regarding this N-terminal cleavage. In vivo treatment with the hydroxamate-based metalloprotease inhibitors BB3103 or TAPI-2 (tumour necrosis factor-alpha protease inhibitor 2) reversibly induced accumulation of forms of proTGFalpha that included the N-terminal extension. N-terminal shedding was rapid, and occurred at the cell surface. However, the machinery responsible for the N-terminal cleavage was inactive in other cellular sites, such as the endoplasmic reticulum. Experiments of proTGFalpha expression and maturation in cells deficient in TACE (tumour-necrosis-factor-alpha-converting enzyme) activity indicated that this protease was dispensable for N-terminal processing of proTGFalpha in vivo, but was required for regulated cleavage at the C-terminus. These findings indicate that TACE is not involved in N-terminal processing of proTGFalpha, and suggest differences in the machineries that control the cleavage at both ends of TGFalpha within its precursor.

ADAM Proteins↗

PubMed2005

Mapping of the RNA promoter of Newcastle disease virus.

The RNA promoters of the genome and antigenome of Newcastle disease virus (NDV) were studied by mutational analysis of their 3' terminal ends. Similarly to other paramyxoviruses, NDV RNA replication follows the rule of six, and the genomic and antigenomic promoters require two discontinuous regions: conserved region I (first 18 nucleotides) and conserved region II (nucleotides 73-90). Proper spacing between those regions and the phase of six in region II is critical for efficient RNA promoter activity. As expected, the gene start signal at the 3' end of the NDV genome was required for mRNA transcription, but not for RNA replication. Surprisingly, mutation of the polyadenylation signal in the 5' end did not affect gene expression transcription. Although the conserved region I of NDV (avulavirus) promoter appears to be more similar to that of Sendai virus (SeV) (respirovirus), conserved region II is analogous to that of rubulaviruses.

Cell Line↗

PubMed2004

Gangliosides and N-glycoproteins function as Newcastle disease virus receptors.

The interaction of enveloped viruses with cell surface receptors is the first step in the viral cycle and an important determinant of viral host range. Although it is established that the paramyxovirus Newcastle Disease Virus binds to sialic acid-containing glycoconjugates the exact nature of the receptors has not yet been determined. Accordingly, here we attempted to characterize the cellular receptors for Newcastle disease virus. Treatment of cells with tunicamycin, an inhibitor of protein N-glycosylation, blocked fusion and infectivity, while the inhibitor of O-glycosylation benzyl-N-acetyl-alpha-D-galactosamide had no effect. Additionally, the inhibitor of glycolipid biosynthesis 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol blocked viral fusion and infectivity. These results suggest that N-linked glycoproteins and glycolipids would be involved in viral entry but not O-linked glycoproteins. The ganglioside content of COS-7 cells was analyzed showing that GD1a was the major ganglioside component; the presence of GM1, GM2 and GM3 was also established. In a thin-layer chromatographic binding assay, we analyzed the binding of the virus to different gangliosides, detecting the interaction with monosialogangliosides such as GM3, GM2 and GM1; disialogangliosides such as GD1a and GD1b, and trisialogangliosides such as GT1b. Unlike with other viruses, our results seem to point to the absence of a specific pattern of gangliosides that interact with Newcastle disease virus. In conclusion, our results suggest that Newcastle disease virus requires different sialic acid-containing compounds, gangliosides and glycoproteins for entry into the target cell. We propose that gangliosides would act as primary receptors while N-linked glycoproteins would function as the second receptor critical for viral entry.

Animals↗

PubMed2004

Nitric oxide protects against pancreatic subcellular damage in acute pancreatitis.

OBJECTIVES: Oxidative stress involvement in damage to the pancreas in acute pancreatitis (AP) is well documented. However, little is known about oxidative damage occurring in the different subcellular fractions of pancreatic cells. The aim of this study was to ascertain the main targets of oxidative damage inside cells after AP and the role of endogenous nitric oxide (NO) in it. METHODS: A model of cerulein-induced AP in rats was used and N-nitro-l-arginine methyl ester (l-NAME) was administered as an NO production inhibitor. After pancreatitis induction, indicative parameters of lipid peroxidation and protein oxidation together with some enzymatic and nonenzymatic endogenous free radical scavengers were assessed in serum and pancreatic subcellular fractions. CONCLUSIONS: In pancreatitic rats, malondialdehyde and protein carbonyl group concentrations were significantly increased (P < 0.05) in serum and some fractions. The increases were higher in l-NAME-treated rats (P < 0.05). Superoxide dismutase and catalase activities were also increased (P < 0.05) but were decreased (P < 0.05) with l-NAME. The alpha-tocopherol concentration diminished (P < 0.05) in serum and all the studied subcellular fractions and the decrease was stronger in l-NAME-treated rats. Our data suggest that microsomes followed by lysosomal + mitochondrial are the fractions most susceptible to oxidative damage in AP. Endogenous NO plays a protective role against oxidative damage to subcellular fractions.

Acute Disease↗

PubMed2004

Sialidase, receptor-binding and fusion-promotion activities of Newcastle disease virus haemagglutinin-neuraminidase glycoprotein: a mutational and kinetic study.

Mutations were generated in residues at the putative catalytic site of the haemagglutinin-neuraminidase (HN) protein of Newcastle disease virus Clone 30 strain (Arg498, Glu258, Tyr262, Tyr317 and Ser418) and their effects on its three associated activities were studied. Expression of the mutant proteins at the surface of HeLa cells was similar to that of the wild-type. Sialidase, receptor-binding and fusion-promotion activities were affected to different degrees for all mutants studied. Mutant Arg498Lys lost most of its sialidase activity, although it retained most of the receptor-binding activity, suggesting that, for the former activity, besides the presence of a basic residue, the proximity to the substrate molecule is also important, as Lys is shorter than Arg. Proximity also seems to be important in substrate recognition, since Tyr262Phe retained most of its sialidase activity while Tyr262Ser lost most of it. Also, Ser418Ala displayed most of the wild-type sialidase activity. However, a kinetic and thermodynamic study of the sialidase activity of the Tyr262Ser and Ser418Ala mutants was performed and revealed that the hydroxyl group of these residues also plays an important role in catalysis, since such activity was much less effective than that of the wild-type and these mutations modified their activation energy for sialidase catalysis. The discrepancy of the modifications in sialidase and receptor-binding activities in the mutants analysed does not account for the topological coincidence of the two sites. These results also suggest that the globular head of HN protein may play a role in fusion-promotion activity.

Amino Acid Substitution↗

PubMed2004

Conformational changes of Newcastle disease virus envelope glycoproteins triggered by gangliosides.

We have investigated the conformational changes of Newcastle disease virus (NDV) glycoproteins in response to receptor binding, using 1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid (bis-ANS) as a hydrophobicity-sensitive probe. Temperature- and pH-dependent conformational changes were detected in the presence of free bovine gangliosides. The fluorescence of bis-ANS was maximal at pH 5. The binding of bis-ANS to NDV was not affected by chemicals that denature the fusion glycoprotein, such as reducing agents, nor by the presence of neuraminidase inhibitors such as N-acetyl neuramicic acid. Gangliosides partially inhibited fusion and hemadsorption, but not neuraminidase hemagglutinin-neuraminidase glycoprotein (HN) activity. A conformational intermediate of HN, triggered by the presence of gangliosides acting as receptor mimics, was detected. Our results indicate that, upon binding to free gangliosides, HN undergoes a certain conformational change that does not affect the fusion glycoprotein.

Anilino Naphthalenesulfonates↗

PubMed2002

Acute pancreatitis decreases pancreas phospholipid levels and increases susceptibility to lipid peroxidation in rat pancreas.

The objective of this study was to analyze whether acute pancreatitis leads to changes in the lipid composition and susceptibility to lipid peroxidation of pancreatic membranes. Total lipids, cholesterol, phospholipids, FA, and lipid peroxidation were determined in the pancreatic tissue of rats treated with cerulein and of control rats. In pancreatitic rats, significant decreases in membrane total phospholipid contents (P < 0.05) and in choline and ethanolamine glycerophospholipid levels (P < 0.05 and P < 0.01, respectively), with concomitant significantly higher values of their lysoderivative forms, were found. The cholesterol/phospholipid molar ratio increased by 26%. The unsaturation index of the FA profile decreased significantly (P < 0.01) as a consequence of a decrease in the arachidonic acid content. Incubation of membranes with xanthine oxidase/hypoxanthine-Fe2+/ADP resulted in an increase in the production of TBARS in pancreatitic rats compared to controls. In summary, acute pancreatitis causes changes in the lipid composition of rat pancreatic crude membranes and a greater susceptibility of these membranes to lipid peroxidation.

Animals↗