Biomedical subjects

Manuel T Silva

Publications and source records attributed to Manuel T Silva.

16 recordsLinked to original sources

PubMed2006

Evidence for an intramacrophage growth phase of Mycobacterium ulcerans.

Mycobacterium ulcerans is the etiologic agent of Buruli ulcer (BU), an emerging tropical skin disease. Virulent M. ulcerans secretes mycolactone, a cytotoxic exotoxin with a key pathogenic role. M. ulcerans in biopsy specimens has been described as an extracellular bacillus. In vitro assays have suggested a mycolactone-induced inhibition of M. ulcerans uptake by macrophages in which its proliferation has not been demonstrated. Therefore, and uniquely for a mycobacterium, M. ulcerans has been classified as an extracellular pathogen. In specimens from patients and in mouse footpad lesions, extracellular bacilli were concentrated in central necrotic acellular areas; however, we found bacilli within macrophages in surrounding inflammatory infiltrates. We demonstrated that mycolactone-producing M. ulcerans isolates are efficiently phagocytosed by murine macrophages, indicating that the extracellular location of M. ulcerans is not a result of inhibition of phagocytosis. Additionally, we found that M. ulcerans multiplies inside cultured mouse macrophages when low multiplicities of infection are used to prevent early mycolactone-associated cytotoxicity. Following the proliferation phase within macrophages, M. ulcerans induces the lysis of the infected host cells, becoming extracellular. Our data show that M. ulcerans, like M. tuberculosis, is an intracellular parasite with phases of intramacrophage and extracellular multiplication. The occurrence of an intramacrophage phase is in accordance with the development of cell-mediated and delayed-type hypersensitivity responses in BU patients.

Animals↗

PubMed2006

First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia.

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924bp cDNA of sea bass caspase-9 was obtained, consisting of 1308bp open reading frame coding for 435 amino acids, 199bp of the 5'-UTR and 417bp of the 3'-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp(121), Asp(325) and Asp(343). The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida (Phdp) strain PP3, showed increased expression from 0 to 12h returning to control levels at 24h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48h post-infection, when the fish were with advanced septicaemia.

Amino Acid Sequence↗

PubMed2006

Molecular cloning and characterisation of sea bass (Dicentrarchus labrax L.) caspase-3 gene.

Caspase-3 is one of the major caspases operating in apoptosis, cleaving and inactivating a number of molecules and largely contributing to the apoptotic phenotype and the dismantling of the apoptoting cell. The opening reading frame of sea bass (Dicentrarchus labrax L.) caspase-3 has 281 amino acids. The complete sequence of caspase-3 shows a very close homology to the correspondent sequence from other vertebrates, in particularly with that of Takifugu rubripes and Oryzias latipes, with 87.7 and 87.9% of similarity, respectively. Furthermore, the sea bass caspase-3 sequence retains the motifs that are functionally important, such as the pentapeptide active-site motif (QACRG) and the putative cleavage sites at the aspartic acids. In the sea bass genome, the caspase-3 gene exists as a single copy gene and is organised in six exons and five introns. A very low expression of caspase-3 was detected by RT-PCR in various organs of non-stimulated sea bass, with slightly higher levels in thymus and heart and was increased in head kidneys of Photobacterium damselae ssp. piscicida infected sea bass. This increased expression was accompanied by the occurrence of high numbers of apoptoting cells with activated caspase-3.

Amino Acid Sequence↗

PubMed2005

Truepera radiovictrix gen. nov., sp. nov., a new radiation resistant species and the proposal of Trueperaceae fam. nov.

Two isolates, belonging to a new species of a novel genus of the Phylum "Deinococcus/Thermus ", were recovered from hot spring runoffs on the Island of São Miguel in the Azores. Strains RQ-24(T) and TU-8 are the first cultured representatives of a distinct phylogenetic lineage within this phylum. These strains form orange/red colonies, spherical-shaped cells, have an optimum growth temperature of about 50 degrees C, an optimum pH for growth between about 7.5 and 9.5, and do not grow at pH below 6.5 or above pH 11.2. These organisms grow in complex media without added NaCl, but have a maximum growth rate in media with 1.0% NaCl and grow in media containing up to 6.0% NaCl. The organisms are extremely ionizing radiation resistant; 60% of the cells survive 5.0 kGy. These strains are chemoorganotrophic and aerobic; do not grow in Thermus medium under anaerobic conditions with or without nitrate as electron acceptor and glucose as a source of carbon and energy, but ferment glucose to D-lactate without formation of gas. The organisms assimilate a large variety of sugars, organic acids and amino acids. Fatty acids are predominantly iso- and anteiso-branched; long chain 1,2 diols were also found in low relative proportions; menaquinone 8 (MK-8) is the primary respiratory quinone. Peptidoglycan was not detected. Based on 16S rRNA gene sequence analysis, physiological, biochemical and chemical analysis we describe a new species of one novel genus represented by strain RQ-24(T) (CIP 108686(T)=LMG 22925(T)=DSM 17093(T)) for which we propose the name Truepera radiovictrix. We also propose the family Trueperaceae fam. nov. to accommodate this new genus.

Amino Acids↗

PubMed2005

Hyperosmotic stress induces metacaspase- and mitochondria-dependent apoptosis in Saccharomyces cerevisiae.

During the last years, several reports described an apoptosis-like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress caused by high glucose or sorbitol concentrations in culture medium induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1Deltacyc7Delta and cyc3Delta, both lacking mature cytochrome c, displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis-like programmed cell death that is mediated by a caspase-dependent mitochondrial pathway partially dependent on cytochrome c.

Apoptosis↗

PubMed2005

AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils.

A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.

Animals↗

PubMed2005

Infection with Mycobacterium ulcerans induces persistent inflammatory responses in mice.

Buruli ulcer (BU) is a devastating, necrotizing, tropical skin disease caused by infections with Mycobacterium ulcerans. In contrast to other mycobacterioses, BU has been associated with minimal or absent inflammation. However, here we show that in the mouse M. ulcerans induces persistent inflammatory responses with virulence-dependent patterns. Mycolactone-positive, cytotoxic strains are virulent for mice and multiply progressively, inducing both early and persistent acute inflammatory responses. The cytotoxicity of these strains leads to progressive destruction of the inflammatory infiltrates by postapoptotic secondary necrosis, generating necrotic acellular areas with extracellular bacilli released by the lysis of infected phagocytes. The necrotic areas, always surrounded by acute inflammatory infiltrates, expand through the progressive invasion of healthy tissues around the initial necrotic lesions by bacteria and by newly recruited acute inflammatory cells. Our observations show that the lack of inflammatory infiltrates in the extensive areas of necrosis seen in advanced infections results from the destruction of continuously produced inflammatory infiltrates and not from M. ulcerans-induced local or systemic immunosuppression. Whether this is the mechanism behind the predominance of minimal or absent inflammatory responses in BU biopsies remains to be elucidated.

Animals↗

PubMed2003

Apoptosis of sea bass (Dicentrarchus labrax L.) neutrophils and macrophages induced by experimental infection with Photobacterium damselae subsp. piscicida.

The infection of sea bass (Dicentrarchus labrax L.) by intraperitoneal (i.p.) injection of the agent of fish pasteurellosis Photobacterium damselae subsp. piscicida resulted in the apoptosis of peritoneal neutrophils and macrophages. All the eight virulent and none of the two non-virulent strains tested exhibited apoptogenic activity. A secreted bacterial protein(s) is a likely candidate as the factor(s) responsible for this activity, since no apoptosis was induced by i.p. injected UV-killed virulent strains and the virulent culture supernatants exhibited a thermo-labile apoptogenic activity identical to that of live bacteria. The apoptotic process was characterized by the occurrence of DNA fragmentation detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining and DNA electrophoresis, and of typical ultrastructural alterations namely cell shrinkage, chromatin condensation, nuclear fragmentation and production of blebs with shedding of apoptotic bodies. In the apoptotic process induced by lethal doses of virulent bacteria or culture supernatants both peritoneal macrophages and neutrophils were extensively affected, the majority of these cells being apoptotic and reaching values around 10(7)per peritoneal cavity for each cell type at 24h post-injection. Moreover, the number of non-apoptotic macrophages was always below the initial number in the resting peritoneal cavity. Since macrophages are key cells in the elimination of both bacteria and apoptotic moribund cells and apoptotic bodies, the induction by Ph. damselae subsp. piscicida of simultaneous macrophage and neutrophil apoptosis results, on the one hand, in the destruction of the two phagocytic cell types involved in the restriction of multiplication of the bacteria and, on the other hand, in the uncontrolled progression of the apoptotic process towards secondary necrosis and eventual lysis of high numbers of moribund neutrophils and of neutrophilic apoptotic bodies, with the consequent extensive release of their highly cytotoxic components. Abundant apoptotic cells were also seen in sections of head-kidney from fish dying from experimental pasteurellosis. In contrast, no apoptosis was seen in vitro after the treatment with virulent culture supernatants of sea bass head-kidney macrophage cultures or after the treatment ex vivo of peritoneal exudate leukocytes with virulent bacteria or culture supernatants. The apoptotic process described here appears as a novel and very powerful microbial pathogenic strategy.

Animals↗

PubMed2003

Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii.

Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.

Acetic Acid↗

PubMed2003

Porphyrobacter cryptus sp. nov., a novel slightly thermophilic, aerobic, bacteriochlorophyll a-containing species.

Two strains of a novel aerobic, bacteriochlorophyll a-containing species of the alpha-4 subclass of the Proteobacteria were isolated from the hot spring at Alcafache in central Portugal. 16S rRNA gene sequence-based phylogenetic analyses showed the two novel isolates to be phylogenetically related to members of the genera Erythrobacter, Erythromicrobium and Porphyrobacter. The strains produce reddish-orange-pigmented colonies, have an optimum growth temperature of about 50 degrees C and could be distinguished from the species Porphyrobacter tepidarius, which also has a high growth temperature, primarily on the basis of the fatty acid composition. The novel species does not grow anaerobically in the presence or absence of a light source. The strains of the novel species utilize several single carbon sources for growth, most of which are also used by P. tepidarius. The species status of strains ALC-2T and ALC-3 was confirmed by low reassociation values of the DNA with species of the genera Erythrobacter, Erythromicrobium and Porphyrobacter. Phenotypic characteristics and 16S rRNA gene sequence analyses also show that strains ALC-2T (=DSM 12079T =ATCC BAA-386T) and ALC-3 (=DSM 12080) represent a novel species, for which the name Porphyrobacter cryptus sp. nov. is proposed.

Aerobiosis↗

PubMed2002

Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction.

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.

Animals↗

PubMed2002

Increased hepatic iron in mice lacking classical MHC class I molecules.

Iron accumulation in the liver in hereditary hemochromatosis (HH) has been shown to be highly variable. Some studies point to the importance of major histocompatibility complex (MHC) class I (MHC-I) and CD8(+) cells as modifiers of iron overload. In this report, using mice knockout for H2K(b-/-) and H2D(b-/-) genes, it is demonstrated that lack of classical MHC-I molecules results in a spontaneous increase of nonheme iron content in the liver (mainly located in the hepatocytes) when compared to wild-type mice. In CD8(-/-) and Rag2(-/-) mice, no spontaneous hepatic iron accumulation was observed. These results demonstrate for the first time that classical MHC-I molecules could be involved in the regulation of iron metabolism and contribute to the established genotype/phenotype discrepancies seen in HH.

Animals↗

PubMed2002

The professional phagocytes of sea bass (Dicentrarchus labrax L.): cytochemical characterisation of neutrophils and macrophages in the normal and inflamed peritoneal cavity.

In order to identify the phagocytic cells of sea bass, the peritoneal leucocyte population of fish injected intraperitoneally with Photobacterium damselae subspecies piscicida was studied by light microscopy using cytocentrifuge preparations stained by the Antonow technique for peroxidase detection. Among the leucocytes present in the peritoneal exudate of the infected fish (macrophages, neutrophils, eosinophilic granular cells, lymphocytes and thrombocytes), macrophages and neutrophils were the only phagocytic cells. Neutrophils were easily distinguished from macrophages in Antonow stained preparations by the pattern of peroxidase positivity. Using ultrastructural cytochemistry, neutrophils were found to have abundant cytoplasmic granules positive for peroxidase and arylsulphatase and were negative for alpha-naphthyl butyrate (ANB) esterase. In contrast, ANB esterase activity was detected in macrophages. These leucocytes were typically negative for peroxidase, but ocasionally, some macrophages with peroxidase or arylsulphatase-positive vacuoles were observed. Both phagocytes had cytoplasmic granules positive for acid phosphatase. Glycogen particles were found in the cytoplasm of the two phagocytic cells, but they were much more abundant in neutrophils. Macrophages were much more abundant than neutrophils in the peritoneal cavity of non-injected sea bass but early after the intraperitoneal injection of bacteria, the number of neutrophils increased quickly and extensively. Higher numbers of intraperitoneally injected bacteria were found inside macrophages as compared to neutrophils because macrophages strongly predominated in the peritoneal population at the time of injection. However, when the bacteria were injected into peritoneal cavities with high numbers of neutrophils (attracted by a previous injection of 12% casein), the percentage of neutrophils with phagocytosed bacteria increased, approaching that of infected macrophages. Taken together, these results show that in sea bass, as in many other organisms, in addition to macrophages, neutrophils are important phagocytic cells, the relative participation of each of the two phagocytes in defense mechanisms against infection depending on the opportunity to encounter the invading infectious agents.

Animals↗

PubMed2002

Cytochrome c release and mitochondria involvement in programmed cell death induced by acetic acid in Saccharomyces cerevisiae.

Evidence is presented that mitochondria are implicated in the previously described programmed cell death (PCD) process induced by acetic acid in Saccharomyces cerevisiae. In yeast cells undergoing a PCD process induced by acetic acid, translocation of cytochrome c (CytC) to the cytosol and reactive oxygen species production, two events known to be proapoptotic in mammals, were observed. Associated with these events, reduction in oxygen consumption and in mitochondrial membrane potential was found. Enzymatic assays showed that the activity of complex bc(1) was normal, whereas that of cytochrome c oxidase (COX) was strongly decreased. This decrease is in accordance with the observed reduction in the amounts of COX II subunit and of cytochromes a+a(3). The acetic acid-induced PCD process was found to be independent of oxidative phosphorylation because it was not inhibited by oligomycin treatment. The inability of S. cerevisiae mutant strains (lacking mitochondrial DNA, heme lyase, or ATPase) to undergo acetic acid-induced PCD and in the ATPase mutant (knockout in ATP10) the absence of CytC release provides further evidence that the process is mediated by a mitochondria-dependent apoptotic pathway. The understanding of the involvement of a mitochondria-dependent apoptotic pathway in S. cerevisiae PCD process will be most useful in the further elucidation of an ancestral pathway common to PCD in metazoans.

Acetic Acid↗

PubMed2002

Albidovulum inexpectatum gen. nov., sp. nov., a nonphotosynthetic and slightly thermophilic bacterium from a marine hot spring that is very closely related to members of the photosynthetic genus Rhodovulum.

Several bacterial isolates, with an optimum growth temperature of about 50 degrees C, were recovered from the marine hot spring at Ferraria on the island of São Miguel in the Azores. The geothermal water emerged from a porous lava flow and rapidly cooled in contact with seawater except at low tide. The bacterial species represented by strains FRR-10(T) and FRR-11 was nonpigmented, strictly aerobic, and organotrophic. Several genes, bchZ, pufB, pufA, pufL, or pufM, encoding the photosynthetic reaction center proteins and the core light-harvesting complexes were not detected in these strains. The organism oxidized thiosulfate to sulfate with enhancement of growth. The organism did not require additional NaCl in the culture medium for growth, but NaCl at 1.0% enhanced growth. Phylogenetic analyses using the 16S rRNA gene sequence of strain FRR-10(T) indicated that the new organism represented a new species of the alpha-3 subclass of the Proteobacteria and that it branches within the species of the genus Rhodovulum. The contradiction of classifying an organism which branches within the radiation of the genus Rhodovulum but does not possess the hallmark characteristics of this genus is discussed. However, the absence of several of these characteristics, namely, the lack of photosynthesis and pigmentation, which could be related to colonization of dark environments, and growth at high temperatures, leads to our proposal that strains FRR-10(T) and FRR-11 should be classified as a new species of a novel genus, Albidovulum inexpectatum, representing, at present, the most thermophilic organism within the alpha-3 subclass of the Proteobacteria.

Fatty Acids↗

PubMed2001

Saccharomyces cerevisiae commits to a programmed cell death process in response to acetic acid.

Recent evidence has revealed the occurrence of an apoptotic phenotype in Saccharomyces cerevisiae that is inducible with oxidative stress. Here, exposure of S. cerevisiae to 20-200 mM acetic acid for 200 min at pH 3.0 resulted in cell death. Yeast mortality induced by 120-200 mM acid was not inhibited by cycloheximide and was accompanied by ultrastructural alterations typical of necrosis. In contrast, alterations associated with cell death induced by 20-80 mM acetic acid included: (i) cycloheximide-inhibitable chromatin condensation along the nuclear envelope; (ii) exposure of phosphatidylserine on the surface of the cytoplasmic membrane, revealed by the FITC-annexin V reaction; and (iii) the occurrence of DNA strand breaks, demonstrated by the TUNEL assay. These results show that a programmed cell death process sharing common features with an apoptotic phenotype can be induced by acetic acid in S. cerevisiae. This observation raises the possibility of this mode of cell death being more generalized in yeasts than previously considered and extended to cell death induced by other stress agents.

Acetic Acid↗