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Biomedical subjects

I Lasa

Publications and source records attributed to I Lasa.

18 recordsLinked to original sources
PubMed2001

Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli.

An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC ) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as "nudix" hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli.

Adenosine Diphosphate↗
PubMed2001

The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation.

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

Bacterial Adhesion↗
PubMed2001

Bap, a Staphylococcus aureus surface protein involved in biofilm formation.

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection.

Adhesins, Bacterial↗
PubMed1999

Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity.

A bacteriocin-producing strain of Bacillus cereus was identified and isolated from a soil sample. The bacteriocin could be purified by a two-step procedure: ammonium sulfate precipitation of culture supernatants followed by a butanol extraction step, the antibiotic was recovered from the organic phase. The peptidic nature of the bacteriocin was proven by its sensitivity to proteolytic enzymes; its molecular mass, determined by mass spectrometry, was 3940 Da; and its amino-terminal sequence (GWGDVL) is unique in the databases. The compound was active against most Gram-positive but not Gram-negative bacteria. This is to our knowledge the first bacteriocin with these characteristics reported to be produced by B. cereus.

Bacillus cereus↗
PubMed1999

A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conorii.

Listeria monocytogenes, Shigella flexneri, and Rickettsia conorii are three bacterial pathogens that are able to polymerize actin into 'comet tail' structures and move within the cytosol of infected cells. The actin-based motilities of L. monocytogenes and S. flexneri are known to require the bacterial proteins ActA and IcsA, respectively, and several mammalian cytoskeleton proteins including the Arp2/3 complex and VASP (vasodilator-stimulated phosphoprotein) for L. monocytogenes and vinculin and N-WASP (the neural Wiskott-Aldrich syndrome protein) for S. flexneri. In contrast, little is known about the motility of R. conorii. In the present study, we have analysed the actin-based motility of this bacterium in comparison to that of L. monocytogenes and S. flexneri. Rickettsia moved at least three times more slowly than Listeria and Shigella in both infected cells and Xenopus laevis egg extracts. Decoration of actin with the S1 subfragment of myosin in infected cells showed that the comet tails of Rickettsia have a structure strikingly different from those of L. monocytogenes or S. flexneri. In Listeria and Shigella tails, actin filaments form a branching network while Rickettsia tails display longer and not cross-linked actin filaments. Immunofluorescence studies revealed that the two host proteins, VASP and (&agr;)-actinin colocalized with actin in the tails of Rickettsia but neither the Arp2/3 complex which we detected in the Shigella actin tails, nor N-WASP, were detected in Rickettsia actin tails. Taken together, these results suggest that R. conorii may use a different mechanism of actin polymerization.

Actins↗
PubMed1998

Characterization of a plasmid replicative origin from an extreme thermophile.

The minimal replicative origin from a 16-kbp natural plasmid of Thermus sp. ATCC 27737 is described. The 1798-bp sequence encodes a single protein (RepA) whose expression is required for replication. This requirement is extended to 200 bp upstream the coding region, which contains promoter-like sequences transcriptionally active in E. coli. Despite the absence of typical iterons, we show that an N-terminal fragment of this protein binds to a DNA fragment internal to its own coding sequence. As this DNA fragment contains an unusually A + T rich sequence surrounded by C + G rich stretches, the possible location of the replicative origin within the region encoding RepA is proposed.

Amino Acid Sequence↗
PubMed1998

Identification, characterization, and In situ detection of a fruit-body-specific hydrophobin of Pleurotus ostreatus.

Hydrophobins are small (length, about 100 +/- 25 amino acids), cysteine-rich, hydrophobic proteins that are present in large amounts in fungal cell walls, where they form part of the outermost layer (rodlet layer); sometimes, they can also be secreted into the medium. Different hydrophobins are associated with different developmental stages of a fungus, and their biological functions include protection of the hyphae against desiccation and attack by either bacterial or fungal parasites, hyphal adherence, and the lowering of surface tension of the culture medium to permit aerial growth of the hyphae. We identified and isolated a hydrophobin (fruit body hydrophobin 1 [Fbh1]) present in fruit bodies but absent in both monokaryotic and dikaryotic mycelia of the edible mushroom Pleurotus ostreatus. In order to study the temporal and spatial expression of the fbh1 gene, we determined the N-terminal amino acid sequence of Fbh1. We also synthesized and cloned the double-stranded cDNA corresponding to the full-length mRNA of Fbh1 to use it as a probe in both Northern blot and in situ hybridization experiments. Fbh1 mRNA is detectable in specific parts of the fruit body, and it is absent in other developmental stages.

Agaricales↗
PubMed1997

ActA is a dimer.

ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various L. monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97-126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by L. monocytogenes, involving the ActA dimer, is presented.

Bacterial Proteins↗
PubMed1997

Identification of two regions in the N-terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes.

The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface. The continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts. Here, by fusing the N-terminus of ActA (residues 1-234) to the omega fragment of beta-galactosidase, we present the first evidence that this domain contains all the necessary elements for actin tail formation. A detailed analysis of ActA variants, in which small fragments of the N-terminal region were deleted, allowed the identification of two critical regions. Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process. The first region (region T, amino acids 117-121) is critical for filament elongation, as shown by the absence of actin tail in a 117-121 deletion mutant or when motility assays are performed in the presence of anti-region T antibodies. The second region (region C, amino acids 21-97), is more specifically involved in maintenance of the continuity of the process, probably by F-actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21-97) leading to 'discontinuous' actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33-74 can interact with F-actin. Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N-terminal domain of ActA.

Actins↗
PubMed1997

Medical factors in the morbidity and mortality associated with emergency colorectal cancer surgery.

OBJECTIVE: To review the results of the analysis and quantification of the influence of medical factors on the morbidity and mortality associated with emergency surgery for colorectal cancer. PATIENTS AND METHODS: We analyze retrospectively the data for 51 patients who underwent emergency surgery: 34 (67%) to treat acute obstruction and 17 (33%) for perforation. The median follow-up period was 18.5 months (3 to 62 months). RESULTS: The main tumor was located in the right colon in 13 patients (25%), in the left colon in 35 (69%) and in, the rectum in 3 (6%). Primary resection was performed in 94% of the patients: without anastomosis in 35% (18 of 51) and with primary anastomosis in 59% (30 of 51); 6% of the tumors were not resectable. The postoperative morbidity was 41%: 29% (10 of 34) occurring in obstructions and 65% (11 of 17) in perforations. The mortality rate was 14% (9% and 23%, respectively). There were statistically significant associations between mortality and the American Society of Anesthesia grading (p < 0.01) and between both the mortality and morbidity and the score for the acute physiology component of APACHE II (p = 0.01, respectively) and the total APACHE II score (p < 0.01 in both cases). The rate of actuarial disease-free survival was 26% at 36 months. Overall survival was 15% at 62 months. A recurrence rate of 48% (14 of 29) has been recorded. CONCLUSIONS: Emergency surgery for colorectal cancer is associated with a high postoperative morbidity and mortality rate, which correlate with the medical status and, particularly, with the acute physiology score of the APACHE II risk stratification system.

APACHE↗
PubMed1996

Differential domain accessibility to monoclonal antibodies in three different morphological assemblies built up by the S-layer protein of Thermus thermophilus HB8.

A collection of 27 monoclonal antibodies (MAbs) against the S-layer protein (P100) of Thermus thermophilus HB8 has been obtained. They have been classified according to their ability to recognize S-layer regions expressed in E. coli from plasmids containing different fragments of its coding gene, slpA. The accessibility of the binding sites in hexagonal, trigonal, or tetragonal assemblies of P100 was analyzed by enzyme-linked immunosorbent assays with six of these MAbs and their respective Fab fragments. When packed hexagonally as the native S-layer (S1 assemblies), only a small region located near the amino terminus of the P1OO was accessible. However, when P1OO was assembled into trigonal (pS2 assemblies) or tetragonal (S2 assemblies) arrays, most of the protein domains analyzed were easily detected, thus suggesting that P1OO is assembled in S2 and pS2 in a similar way and that these two arrangements are quite different from the S1 assembly. Relationships between accessibility and sequence predictions are discussed.

Antibodies, Bacterial↗
PubMed1995

The amino-terminal part of ActA is critical for the actin-based motility of Listeria monocytogenes; the central proline-rich region acts as a stimulator.

The intracellular bacterial pathogen Listeria monocytogenes moves inside the host-cell cytoplasm propelled by continuous actin assembly at one pole of the bacterium. This process requires expression of the bacterial surface protein ActA. Recently, in order to identify the regions of ActA which are required for actin assembly, we and others have expressed different domains of ActA by transfection in eukaryotic cells. As this type of approach cannot address the role of ActA in the actin-driven bacterial propulsion, we have now generated several L. monocytogenes strains expressing different domains of ActA and analysed the ability of the different domains to trigger actin assembly and bacterial movement in both infected cells and cytoplasmic extracts. We show here that the amino-terminal part is critical for F-actin assembly and movement. The internal proline-rich repeats and the carboxy-terminal domains are not essential. However, in vitro motility assays have demonstrated that mutants lacking the proline-rich repeats domain of ActA moved two times slower (6+/-2 micrometers min(-1)) than the wild type (13 +/-3 micrometers min(-1)). In addition, phosphatase treatment of protein extracts of cells infected with the L. monocytogenes strains expressing the ActA variants suggested that phosphorylation may not be essential for ActA activity.

Actins↗
PubMed1995

Horizontal transference of S-layer genes within Thermus thermophilus.

The S-layers of Thermus thermophilus HB27 and T. thermophilus HB8 are composed of protein units of 95 kDa (P95) and 100 kDa (P100), respectively. We have selected S-layer deletion mutants from both strains by complete replacement of the slpA gene. Mutants of the two strains showed similar defects in growth and morphology and overproduced an external cell envelope inside of which cells remained after division. However, the nature of this external layer is strain specific, being easily stained and regular in the HB8 delta slpA derivative and amorphous and poorly stained in the HB27 delta slpA strain. The addition of chromosomic DNA from T. thermophilus HB8 to growing cultures of T. thermophilus HB27 delta slpA led to the selection of a new strain, HB27C8, which expressed a functional S-layer composed of the P100 protein. Conversely, the addition of chromosomic DNA from T. thermophilus HB27 to growing cultures of T. thermophilus HB8 delta slpA allowed the isolation of strain HB8C27, which expressed a functional S-layer composed of the P95 protein. The driving force which selected the transference of the S-layer genes in these experiments was the difference in growth rates, one of the main factors leading to selection in natural environments.

Bacterial Outer Membrane Proteins↗
PubMed1993

Thermophilic enzymes and their biotechnological potential.

The ability of many microorganisms to grow at high temperatures has held a particular fascination for microbiologists and biochemists since a long time. As any of their cellular components, their proteins are inherently more stable to heat than those of conventional organisms. This thermal stability is not due to any specific characteristic, but results a consequence of various changes which contribute to the whole stability of the protein in an additive manner. These enzymes are not only more thermostable, but also more resistant to chemical agents than their mesophilic homologous, what makes them extremely interesting for industrial processes. Despite this, most of the enzymes used at present in industrial processes have been isolated from mesophiles due to the limited knowledge and difficulties to grow thermophiles in high scale. The objective of this review is to consider briefly the importance of the thermostability in order to apply enzymes in the industry, and to overview the most recent advances in the identification of new thermophilic organisms and enzymes. Furthermore, the recent development of genetic model systems for moderate and extreme thermophiles are referred.

Bacteria↗
PubMed1992

Insertional mutagenesis in the extreme thermophilic eubacteria Thermus thermophilus HB8.

The transcription and translation signals of the S-layer gene (slpA) from Thermus thermophilus HB8 have been used to express a thermostable kanamycin adenyl transferase gene in this organism. The chimaeric resistance gene was inserted in vitro into slpA to produce different inactive forms of the gene, which were used to transform T. thermophilus HB8. After 48 hours of incubation at 70 degrees C, only two constructions that contained the kat gene flanked by Thermus sequences from both sides of slpA were able to produce protein layer (P100)-defective mutants. The mutants obtained with both constructions showed identical protein patterns, in which a major 50 kDa protein and two other minor proteins were tentatively identified as P100 fragments, expressed from the extreme 5' end of slpA. They also exhibited important phenotypic defects, such as slow growth in liquid broth, a tendency to aggregate as 'rotund bodies', a twisted filamentous shape, and an extreme sensitivity to lysozyme, suggesting protective and shaping roles for the S-layer in T. thermophilus HB8. These results also demonstrate for the first time the feasibility of using selective antibiotic-resistance markers in extreme thermophiles.

Amino Acid Sequence↗
PubMed1992

Development of Thermus-Escherichia shuttle vectors and their use for expression of the Clostridium thermocellum celA gene in Thermus thermophilus.

We describe the self-selection of replication origins of undescribed cryptic plasmids from Thermus aquaticus Y-VII-51B (ATCC 25105) and a Thermus sp. strain (ATCC 27737) by random insertion of a thermostable kanamycin adenyltransferase cartridge. Once selected, these autonomous replication origins were cloned into the Escherichia coli vector pUC9 or pUC19. The bifunctional plasmids were analyzed for their sizes, relationships, and properties as shuttle vectors for Thermus-Escherichia cloning. Seven different vectors with diverse kanamycin resistance levels, stabilities, transformation efficiencies, and copy numbers were obtained. As a general rule, those from T. aquaticus (pLU1 to pLU4) were more stable than those from the Thermus sp. (pMY1 to pMY3). To probe their usefulness, we used one of the plasmids (pMY1) to clone in E. coli a modified form of the cellulase gene (celA) from Clostridium thermocellum in which the native signal peptide was replaced in vitro by that from the S-layer gene of T. thermophilus HB8. The hybrid product was expressed and exported by E. coli. When the gene was transferred by transformation into T. thermophilus, the cellulase protein was also expressed and secreted at 70 degrees C.

Amino Acid Sequence↗