Biomedical subjects

J Machado

Publications and source records attributed to J Machado.

At least 37 records · Page 2Linked to original sources

PubMed2000

The action of Cd, Cu, Cr, Zn, and Pb on fluid composition of Anodonta cygnea (L.): organic components.

The heavy metals, Cd, Cu, Cr, Zn, and Pb, were used to incubate healthy specimens of the freshwater mussel species, Anodonta cygnea. Afterwards, their biological fluids, either haemolymph or extrapallial fluid were analyzed for the presence of several organic constituents, known to be important for biomineralization, such as proteins, glycosaminoglycans (GAGs) and glucosamine. Proteins were subjected to further study, namely through the total amino acid determination after acid hydrolysis. The most disturbing pollutants tested seem to be Pb, Zn, and Cr, which caused highly decreased overall compositions, namely with respect to protein, and glucosamine, in comparison to the control group. This suggests that this group contributes to a decrease of the metabolic activity, and thus mineralization, in the exposed animals.

Animals↗

PubMed2000

Organic compounds in the extrapalial fluid and haemolymph of Anodonta cygnea (L.) with emphasis on the seasonal biomineralization process.

Bivalve mollusks, such as the freshwater mussel Anodonta cygnea, show seasonal changes in calcification. This cycle of calcification must either be a cause or a consequence for seasonal fluctuations in the organic composition of the animal's fluids, haemolymph and extrapallial fluid, the liquid media for biomineralization. We monitored the fluids of A. cygnea, throughout a 1-year cycle, for the presence of organic constituents, known to be important for biomineralization, such as proteins, glycosaminoglycans (GAGs) and hexosamines. Proteins were subjected to further study, namely through the total amino acid determination and fraction separation by agarose gel electrophoresis. GAG levels were fairly constant throughout the year, with a maximum concentration in July and a minimum in January, a feature also detected for glucosamine, although with higher fluctuations. Proteins showed highly increased concentrations during June and July, both in total amounts and individual fractions. All fractions showed similar trends throughout the year, with lowest general levels in October, the starting month of a period when some fractions were not detectable at all. All fractions ended this low period in May, when a sometimes-important increase could be detected. As to the total amino acid composition of the fluids, the general trend followed that of proteins, except for ornithine (Orn), a non-proteic amino acid. The overall fluctuations detected in the biological fluids of A. cygnea suggest that the main variation related to the calcification cycle must be quantitative, since no different compounds appear in specific periods, to achieve also specific results.

Amino Acids↗

PubMed2000

Identification of Escherichia coli flagellar types by restriction of the amplified fliC gene.

A total of 182 strains of Escherichia coli (133 reference strains, 22 clinical strains, nine nonmotile strains and 18 strains derived from K-12) were characterized by HhaI restriction of the amplified flagellin gene (fliC). The amplified fliC product was a single band between 0.9 and 2.6 kbp. With the collection of reference strains which represented 48 flagellar types (H-types), a total of 62 patterns (F-types) were observed after HhaI restriction. A single F-type was associated with each of 39 H-types and more than one F-type was associated with the other nine H-types. Antigenically related H-types 12 and 45 gave a single F-type. The determination of HhaI-fliC F-types could allow deduction of all H-types and subdivision of some of these. Application of this identification system to 22 E. coli clinical isolates yielded nine F-patterns and the deduced H-types were confirmed by serotyping in all cases. Nine nonmotile strains were studied and their F-types were also identified. The proposed determination of fliC restriction patterns should be helpful for epidemiological studies.

Bacterial Typing Techniques↗

PubMed2000

Characterisation of NF-kappa B complexes in Theileria parva-transformedT cells.

Transformation of T cells by the intracellular parasite Theileria parva is accompanied by constitutive I-kappa B degradation and NF-kappa B activation, a process which is essential to prevent the spontaneous apoptosis of these parasite-transformed cells. NF-kappa B-mediated responses are regulated by selective combinations of NF-kappa B proteins as homo- or heterodimers and by distinct kappa B motifs. We characterised the NF-kappa B complexes induced by T. parva infection in TpM(803) T cells. By western blot, we demonstrated that all members of the NF-kappa B/Rel family of proteins translocate to the nucleus of infected cells. Using two different kappa B oligonucleotides (kappa B-1 and kappa B-2), both containing the decameric consensus kappa B motif (GGGACTTTCC), clearly distinct patterns of DNA binding activities could be demonstrated in electrophoretic mobility shift assays. Supershift analysis and UV cross-linking assays showed that complexes binding to kappa B-1 consisted of p50, p65 and RelB homo and/or heterodimers. We could also detect an association of ATF-2 and c-Fos with one of the complexes. The HIV-derived kappa B-2 oligo only bound p50 and p65. Additionally, several agents known to inhibit a wide range of NF-kappa B activation pathways had no inhibitory effect on the activation of NF-kappa B DNA binding in TpM(803) T cells.

Activating Transcription Factor 2↗

PubMed1999

Interference by the intracellular parasite Theileria parva with T-cell signal transduction pathways induces transformation and protection against apoptosis.

The intracellular parasite Theileria parva transforms bovine T-lymphocytes, inducing uncontrolled proliferation. Upon infection, cells cease to require antigenic stimulation and exogenous growth factors to proliferate. Earlier studies have shown that pathways triggered via stimulation of the T-cell receptor are silent in transformed cells. This is reflected by a lack of phosphorylation of key signalling molecules and the fact that proliferation is not inhibited by immunosuppressants such as cyclosporin and ascomycin that target calcineurin. This suggests that the parasite bypasses the normal T-cells activation pathways to induce proliferation. Among the MAP-kinase pathways, ERK and p38 are silent, and only Jun N-terminal kinase is activated. This appears to suffice to induce constitutive activation of the transcription factor AP-1. More recently, it could be shown that the presence of the parasite in the host cell cytoplasm also induces constitutive activation of NF-kappaB, a transcription factor involved in proliferation and protection against apoptosis. Activation is effectuated by parasite-induced degradation of IkappaBs, the cytoplasmic inhibitors which sequester NF-kappaB in the cytoplasm. NF-kappaB activation is resistant to the antioxidant N-acetyl cysteine and a range of other reagents, suggesting that activation might occur in an unorthodox manner. Studies using inhibitors and dominant negative mutants demonstrate that the parasite activates a NF-kappaB-dependent anti-apoptotic mechanism that protects the transformed cell form spontaneous apoptosis and is essential for maintaining the transformed state of the parasitised cell.

Animals↗

PubMed1999

The intracellular parasite Theileria parva protects infected T cells from apoptosis.

Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells.

Animals↗

PubMed1999

N-acetylcysteine blocks apoptosis induced by N-alpha-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells.

The serine protease inhibitor N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, N-acetylcysteine (NAC), as well as L- or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-kappaB activation in T. parva-transformed T-cells, with phosphorylation of IkappaBalpha and IkappaBbeta being inhibited with different kinetics. TPCK-mediated inhibition of IkappaB phosphorylation, NF-kappaB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-kappaB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-kappaB activation pathway(s).

Acetylcysteine↗

PubMed1999

The inhibition of NF-kappaB activation pathways and the induction of apoptosis by dithiocarbamates in T cells are blocked by the glutathione precursor N-acetyl-L-cysteine.

Nuclear factor-kappaB regulates genes that control immune and inflammatory responses and are involved in the pathogenesis of several diseases, including AIDS and cancer. It has been proposed that reactive oxygen intermediates participate in NF-kappaB activation pathways, and compounds with putative antioxidant activity such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) have been used interchangeably to demonstrate this point. We examined their effects, separately and combined, on different stages of the NF-kappaB activation pathway, in primary and in transformed T cells. We show that NAC, contrary to its reported role as an NF-kappaB inhibitor, can actually enhance rather than inhibit IkappaB degradation and, most importantly, show that in all cases NAC exerts a dominant antagonistic effect on PDTC-mediated NF-kappaB inhibition. This was observed at the level of IkappaB degradation, NF-kappaB DNA binding, and HIV-LTR-driven reporter gene expression. NAC also counteracted growth arrest and apoptosis induced by dithiocarbamates. Antagonistic effects were further observed at the level of jun-NH2-terminal kinase, p38 and ATF-2 activation. Our findings argue against the widely accepted assumption that NAC inhibits all NF-kappaB activation pathways and shows that two compounds, previously thought to function through a common inhibitory mechanism, can also have antagonistic effects.

Acetylcysteine↗

PubMed1999

A unified framework for data modeling on medical information systems.

Medical Information Systems (MIS) are seen as a way of optimizing the use of existing health-care infrastructure, without resorting to new and costly hospital (re)construction. The qualitative (re)design of such an environment requires a basic understanding of patient and doctors related characteristics and capabilities. Patient care, patient education, medical education, and clinical research need to be considered to meet the basic requirements on the level of services desirable, determined on the basis of the patient's length of stay; i.e., used for modeling the significant entities of such a world. The aim is to extract conclusions for the level of services provided to the users. One's concept will capture, as well as will integrate, the basic design principles under which MIS may be set.

Artificial Intelligence↗

PubMed1999

[Infective endocarditis].

Two different series of patients with Infectious Endocarditis (IE), hospitalized in the same ward of Curry Cabral Hospital in Lisbon, are presented and compared. The two series were separated by a ten-year period--the first included patients observed from 1970 to 1976 and the second from 1988 to 1998. In the first series (20 patients), IE occurred mainly in patients with previous cardiac valvular lesion, the course was subacute and Streptococcus viridans was the predominant agent. In the last series (65 patients), most of the patients were young and had no previous valvular lesions. Right side endocarditis predominated and the course was acute. Most of patients were drug addicts and had HIV infection. Staphylococcus aureus was the predominant agent. The ages of the two series were significantly different (p < 0.005). The diagnostic value of transthoracic and transesophageal echocardiography is stressed. The details of the evolution of patients with HIV infection are presented according to the values of CD4+ lymphocyte counts. The relatively good prognosis of IE in drug addicts with antibiotic treatment, even with HIV infection, is emphasized.

Adult↗

PubMed1998

Inhibition by the anti-mitotic drug doxorubicin of platelet-activating-factor-induced late eosinophil accumulation in rats.

Platelet-activating factor (PAF) has been shown, in the rat model of pleural inflammation, to induce the generation of an intermediate proteic factor able to cause eosinophil proliferation in vitro. This study was undertaken to investigate the effect of the anti-mitotic compound doxorubicin on PAF-induced eosinophilia in rats, in order to evaluate the contribution of local cell proliferation to this phenomenon. The late eosinophil infiltration caused by another chemoattractant leukotriene B4 was used for comparison. We observed that local treatment with doxorubicin (20 and 40 microg/cavity), given 6 h after PAF (1 microg/cavity), suppressed the eosinophil accumulation within 24 h, whilst only the higher dose was effective when the drug was given 12 h post-PAF. An effect on chemotaxis was ruled out, since local doxorubicin (40 microg/cavity) failed to modify the eosinophil migration noted 24 h after leukotriene B4 (0.5 microg/cavity) and the neutrophil/eosinophil infiltration noted at 6 h after PAF injection. Transfer of the pleural fluids collected 6 h after PAF from donors to recipient rats caused significant eosinophil accumulation in the recipient rats, an effect which was inhibited by the co-administration of doxorubicin (40 microg/cavity). No inhibitory effect was noted when the drug was given 6 h after the pleural fluids were transferred. We also found no change in the number of blood or bone marrow eosinophils after PAF stimulation. We conclude that doxorubicin selectively impaired the late eosinophil accumulation triggered by PAF in the pleural cavity of rats, clearly indicating that local cell proliferation seems to contribute to the development of this inflammatory response.

Animals↗

PubMed1998

Food borne microbial pathogens of cultured aquatic species.

This article provides a broad overview of microbial pathogens associated with marine and fresh water aquatic animals, including Vibrio species, Escherichia coli, Streptococcus iniae, Salmonella species, and Edwardsiella tarda. Historically, cultured fish were not considered important vectors of human pathogens. This situation is changing, partly due to increasing animal densities as a consequence of a rapidly growing industry and partly due to increasing awareness by health care providers of pathogens in aquatic species that may result in human illness. Concerns facing the industry are also discussed along with possible solutions.

Aeromonas↗

PubMed1998

Computer identification of Escherichia coli rRNA gene restriction patterns.

A total of 191 strains of Escherichia coli comprising 164 serovar reference strains and 28 clinical strains were characterized by rRNA gene restriction patterns (ribotypes) generated after cleavage of total DNA with MluI, ClaI or HindIII restriction endonucleases and hybridization of fragments with acetylaminofluorene-labelled 16 + 23S rRNA. A wide diversity of ribotypes was observed with endonucleases MluI (104 patterns), ClaI (90 patterns) and HindIII (98 patterns). When MluI was used, 85% of patterns (11 to 15 fragments) shared five fragments 17.09, 3.94, 3.06, 2.23 and 1.76 kb in size. When these fragments were used as internal standards, the percent errors in fragment length determination was half of that obtained with an external standard. Two fragment size databases of MluI and ClaI ribotypes were built. Automatic identification was obtained after setting the percent fragment size variation tolerance (error) at 5%. MluI ribotyping is recommended as a primary epidemiological marker. Strains with similar MluI ribotype should then be submitted to ClaI ribotyping. Ribotyping with HindIII can only be the third choice, since the patterns were often uncertain due to the frequent occurrence of faint bands. Most of the studied serovars gave discrete patterns and these data provide the basis for a molecular typing system for E. coli which could possibly substitute for serotyping when the latter is not available.

2-Acetylaminofluorene↗

PubMed1998

Somatic mutation analysis of the APP and Presenilin 1 and 2 genes in Alzheimer's disease brains.

The molecular basis for sporadic Alzheimer disease (AD) remains largely unknown. We hypothesized that in some cases of sporadic AD, a somatic mutation in an embryonic cell committed to neuronal development within the amyloid precursor protein (APP), the presenilin 1 (PS-1) or the presenilin 2 (PS-2) genes (genes known to be involved in familial AD) may result in AD phenotype. Using PCR, denaturing gradient gel electrophoresis (DGGE), restriction enzyme digest and direct DNA sequencing, we analyzed these genes in 99 brain tissues from patients with histopathologically proven AD. One brain sample showed a mutation within the PS-1 gene, His163 Arg, later shown to be a germline mutation. No other migration abnormalities were demonstrated in any sample in exon 16 or 17 of the APP gene or the coding exons of the PS-1 gene. Restriction digest pattern was normal with regard to the predominant PS-2 gene mutation (N141I). A known mutation in the APP gene, as well as novel mutations within the PS-1 gene were easily detected by DGGE (Reznick Wolf et al. manuscript submitted). We conclude that the genes that are involved in familial AD do not display somatic mutations in the brains of sporadic AD patients, and that other molecular mechanisms are probably involved in the pathogenesis of sporadic AD.

Alzheimer Disease↗

PubMed1997

Parasite-mediated nuclear factor kappaB regulation in lymphoproliferation caused by Theileria parva infection.

Infection of cattle with the protozoan Theileria parva results in uncontrolled T lymphocyte proliferation resulting in lesions resembling multicentric lymphoma. Parasitized cells exhibit autocrine growth characterized by persistent translocation of the transcriptional regulatory factor nuclear factor kappaB (NFkappaB) to the nucleus and consequent enhanced expression of interleukin 2 and the interleukin 2 receptor. How T. parva induces persistent NFkappaB activation, required for T cell activation and proliferation, is unknown. We hypothesized that the parasite induces degradation of the IkappaB molecules which normally sequester NFkappaB in the cytoplasm and that continuous degradation requires viable parasites. Using T. parva-infected T cells, we showed that the parasite mediates continuous phosphorylation and proteolysis of IkappaBalpha. However, IkappaBalpha reaccumulated to high levels in parasitized cells, which indicated that T. parva did not alter the normal NFkappaB-mediated positive feedback loop which restores cytoplasmic IkappaBalpha. In contrast, T. parva mediated continuous degradation of IkappaBbeta resulting in persistently low cytoplasmic IkappaBbeta levels. Normal IkappaBbeta levels were only restored following T. parva killing, indicating that viable parasites are required for IkappaBbeta degradation. Treatment of T. parva-infected cells with pyrrolidine dithiocarbamate, a metal chelator, blocked both IkappaB degradation and consequent enhanced expression of NFkappaB dependent genes. However treatment using the antioxidant N-acetylcysteine had no effect on either IkappaB levels or NFkappaB activation, indicating that the parasite subverts the normal IkappaB regulatory pathway downstream of the requirement for reactive oxygen intermediates. Identification of the critical points regulated by T. parva may provide new approaches for disease control as well as increase our understanding of normal T cell function.

Animals↗

PubMed1997

Antimicrobial activity of Terminalia macroptera root.

Terminalia macroptera Guill et Perr. (Combretaceae) is a medicinal plant used in Guinea-Bissau and other West African countries to treat infectious diseases. The ethanol extract from T. macroptera decorticated root and their liquid-liquid partition fractions, were screened for antimicrobial activity, by the twofold serial microdilution assay against seven reference bacterial strains and against Candida albicans. The extract and fractions showed some activity against at least one of the test microorganisms. The best results were obtained against Shigella dysenteriae and Vibrio cholerae. The minimum inhibitory concentrations (MIC) of T. macroptera ethanol extract were also determined for about 100 clinical strains of Campylobacter sp., Escherichia coli, Salmonella sp., Shigella sp. and Vibrio cholerae. The ethanol extract activity against Campylobacter strains is similar to co-trimoxazole, higher than sulfamethoxazole but lower than tetracycline, erythromycin, ampicillin and streptomycin. Ellagitannins are the major compounds in the extract and active fractions. The obtained results suggest a potential importance of this medicinal plant in the treatment of enteric diseases, particularly in Campylobacter infections.

Anti-Bacterial Agents↗

PubMed1997

Caprine arthritis encephalitis virus dysregulates the expression of cytokines in macrophages.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues, in particular, the radiocarpal joints. Cells of the monocyte/macrophage lineage are the major host cells of CAEV in vivo. We have shown that infection of cultured goat macrophages with CAEV results in an alteration of cytokine expression in vitro. Constitutive expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) was increased in infected macrophages, whereas transforming growth factor beta1 (TGF-beta1) mRNA was down-regulated. When macrophages were infected with a CAEV clone lacking the trans-acting nuclear regulatory gene tat, IL-8 and MCP-1 were also increased. No significant differences from cells infected with the wild-type clone were observed, suggesting that Tat is not required for the increased expression of IL-8 and MCP-1 in infected macrophages. Furthermore, infection with CAEV led to an altered pattern of cytokine expression in response to lipopolysaccharide (LPS), heat-killed Listeria monocytogenes plus gamma interferon, or fixed cells of Staphylococcus aureus Cowan I. In infected macrophages, tumor necrosis factor alpha, IL-1beta, IL-6, and IL-12 p40 mRNA expression was reduced in response to all stimuli tested whereas changes in expression of granulocyte-macrophage colony-stimulating factor depended on the stimulating agent. Electrophoretic mobility shift assays demonstrated that, in contrast to effects of human immunodeficiency virus infection of macrophages, CAEV infection had no effect on the level of constitutive nuclear factor-kappaB (NF-kappaB) activity or on the level of LPS-stimulated NF-kappaB activity, suggesting that NF-kappaB is not involved in altered regulation of cytokine expression in CAEV-infected cells. In contrast, activator protein 1 (AP-1) binding activity was decreased in infected macrophages. These data show that CAEV infection may result in a dysregulation of expression of cytokines in macrophages. This finding suggests that CAEV may modulate the accessory functions of infected macrophages and the antiviral immune response in vivo.

Animals↗

PubMed1995

[Cerebral metastasis as the form of presentation of a basaloid carcinoma of the esophagus].

A case of oesophageal basaloid carcinoma is reported. The disease was revealed as a brain metastasis and was found ad initium to be in an advanced stage, with evidence of brain, lung and liver metastasis. The treatment performed was palliative subtotal oesophagectomy followed by roentgen therapy. A year and a half later, the patient is still alive and in home care follow up. The authors end stressing this uncommon presentation for an oesophageal neoplasm and the rarity of its histologic type. They also stress the role of surgery, although palliative, in long survival, very unlike the rapid evolution of the few published cases.

Brain Neoplasms↗