Biomedical subjects

S L Gomes

Publications and source records attributed to S L Gomes.

At least 19 recordsLinked to original sources

PubMed2001

Investigation of 5-nitrofuran derivatives: synthesis, antibacterial activity, and quantitative structure-activity relationships.

Three sets of antibacterial nitrofuran derivatives [set I, 5-R-substituted (Z)-2-(5-nitrofuran-2-ylmethylene)-3(2H)-benzofuranones (R = OCH(3), H, CH(3), C(2)H(5), nC(3)H(7), Cl, Br, CN, and NO(2)) and their 2-hydroxyphenyl and 2-acetoxyphenyl analogues; set II, 5-R-substituted (E)-1-(2-hydroxyphenyl)-3-(5-nitrofuryl)-2-propen-1-ones (R = H, CH(3), C(2)H(5), Cl, and NO(2)); and set III, 5-R-substituted (E)-1-(2-acetoxyphenyl)-3-(5-nitrofuryl)-2-propen-1-ones (R = H, CH(3); C(2)H(5), Cl, and NO(2))] were prepared and tested against a Gram-positive (Staphylococcus aureus, strain ATCC-25923) and a Gram-negative bacterium (Caulobacter crescentus, strain NA 1000). QSAR equations derived for the IC(50) values against both bacteria show negative contributions of two terms: an electronic one, expressed either by sigma, the Hammett substituent constant, or by E, the cyclic voltametric reduction potential. Another term described by an indicator variable, I(abs), is assigned the value of 0 for set I compounds and the value of 1 for sets II and III. No important contribution of the hydrophobic factor was found. For the three sets, the QSAR regressions suggest that the same structural features describe the activities for both bacteria and that, although reduction is a necessary step, it should not be the determining one. These results agree with those found for the QSAR of 5-nitroimidazole analogues.

Anti-Bacterial Agents↗

PubMed2001

Genetic organization of plasmid pXF51 from the plant pathogen Xylella fastidiosa.

The sequence of plasmid pXF51 from the plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, has been analyzed. This plasmid codes for 65 open reading frames (ORFs), organized into four main regions, containing genes related to replication, mobilization, and conjugative transfer. Twenty-five ORFs have no counterparts in the public sequence databases, and 7 are similar to conserved hypothetical proteins from other bacteria. A pXF51 incompatibility group has not been determined, as we could not find a typical replication origin. One cluster of conjugation-related genes (trb) seems to be incomplete in pXF51, and a copy of this sequence is found in the chromosome, suggesting it was generated by a duplication event. A second cluster (tra) contains all genes necessary for conjugation transfer to occur, showing a conserved organization with other conjugative plasmids. An identifiable origin of transfer similar to oriT from IncP plasmids is found adjacent to genes encoding two mobilization proteins. None of the ORFs with putative assigned function could be predicted as having a role in pathogenesis, except for a virulence-associated protein D homolog. These results indicate that even though pXF51 appears not to have a direct role in Xylella pathogenesis, it is a conjugative plasmid that could be important for lateral gene transfer in this bacterium. This property may be of great importance for future development of transformation techniques in X. fastidiosa.

Amino Acid Sequence↗

PubMed2001

Structure, expression, and functional analysis of the gene coding for calmodulin in the chytridiomycete Blastocladiella emersonii.

The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM. B. emersonii CaM has been expressed in Escherichia coli as a fusion protein with gluthatione S-transferase (GST) and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease. In the presence of Ca(2+), B. emersonii CaM exhibited a shift in apparent molecular mass similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in B. emersonii, with CaM mRNA and protein concentrations increasing during sporulation to maximum levels observed just prior to the release of the zoospores into the medium. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists compound 48/80, calmidazolium, and W7 were shown to completely inhibit B. emersonii sporulation when added to the cultures at least 120, 150, and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca(2+) channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that the Ca(2+)-CaM complex and CaMKII play an important role during growth and sporulation in B. emersonii.

Base Sequence↗

PubMed2000

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Bacterial Adhesion↗

PubMed2000

PEST sequences in cAMP-dependent protein kinase subunits of the aquatic fungus Blastocladiella emersonii are necessary for in vitro degradation by endogenous proteases.

During Blastocladiella emersonii germination, the regulatory (R) and the catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) are rapidly and concurrently degraded, after PKA activation in response to a transient increase in intracellular cAMP levels. The possibility that PEST sequences could be acting as proteolytic recognition signals in this process was investigated, and high score PEST sequences were found in both B. emersonii R and C subunits. Deletions in the PEST sequences were obtained by site-directed mutagenesis and the different PKA subunits were independently expressed in Escherichia coli. Proteolysis assays of the various R and C recombinant forms, using B. emersonii cell extracts as the source of proteases, showed a strong correlation between the presence of high score PEST sequences and susceptibility to degradation. Furthermore, the amino-terminal sequence of the proteolytic fragments indicated that the cleavage sites in both subunits are located at or near the PEST regions. The PEST sequence in B. emersonii C subunit, which when deleted or disrupted leads to resistance to proteolysis, is entirely contained in the 72-amino-acid extension located in the N-terminus of the protein. C subunit mutants carrying deletions in this region displayed little difference in their kinetic properties or enzyme thermostability. These results suggest that the N-terminal extension may only play a role in C subunit degradation.

Amino Acid Sequence↗

PubMed2000

Particulate bone grafting of osteolytic femoral lesions around stable cementless stems.

The purpose of the current study was to evaluate the effect of particulate grafting for proximal femoral osteolysis in the presence of a well-fixed cementless femoral stem at the time of acetabular liner change or revision. Sixteen patients (17 hips) who averaged 51 years of age underwent curettage and packing of proximal femoral osteolytic lesions with cancellous allograft. Modular acetabular liners were changed in 11 patients, acetabular revisions were performed in six patients, and femoral heads were exchanged in all patients. The femoral component was retained in all patients. The majority of patients were asymptomatic before revision surgery. The size of the femoral osteolytic lesions was measured preoperatively and postoperatively with anteroposterior and Lauenstein lateral radiographs of the hip. Preoperatively, the average lesion was 41 x 16 mm on the anteroposterior view and 18 x 7 mm on the lateral view. The average clinical and radiographic followup was 39 and 32 months, respectively, with a minimum followup of 24 months. All but one patient remained asymptomatic during the followup period and no femoral stem showed evidence of loosening. The size of the femoral osteolytic lesion averaged 16 x 6 mm on the anteroposterior view and 6 x 2 mm on the lateral view at most recent followup. In 15 of 17 patients, the size of the femoral lesion had regressed. This technique seems to be a viable means of preventing progressive osteolysis and femoral loosening while preserving bone stock for future reconstruction.

Acetabulum↗

PubMed1999

Characterization and submitochondrial localization of the alpha subunit of the mitochondrial processing peptidase from the aquatic fungus Blastocladiella emersonii.

In an effort to investigate the molecular mechanisms responsible for the drastic morphological changes the mitochondria go through during the life cycle of the aquatic fungus Blastocladiella emersonii, the gene encoding the alpha subunit of the mitochondrial processing peptidase (alpha-MPP) was isolated. Nucleotide sequence analysis revealed that the predicted alpha-MPP polypeptide comprises 474 amino acids with a calculated molecular mass of 51,900 Da, presenting a characteristic mitochondrial signal sequence. Northern blot analysis indicated a single 1.4-kb transcript encoding the B. emersonii alpha-MPP, whose levels decrease during sporulation, becoming very low in the zoospore, and increase again during germination. Despite these variations in mRNA concentration, B. emersonii alpha-MPP protein levels do not change significantly during the life cycle of the fungus, as observed in Western blots. Experiments to investigate the submitochondrial localization of B. emersonii alpha-MPP and beta-MPP were also carried out, and the results indicated that both subunits are associated with the mitochondrial inner membrane, possibly as part of the bc1 complex, as described for plants.

Amino Acid Sequence↗

PubMed1998

A P-type ATPase from the aquatic fungus Blastocladiella emersonii similar to animal Na,K-ATPases.

We have cloned a P-type ATPase gene from the aquatic fungus Blastocladiella emersonii (BePAT1) using a probe obtained with degenerate oligonucleotides, corresponding to two amino acid sequences highly conserved among all P-type ATPase isoforms, and the polymerase chain reaction technique. Nucleotide sequence analysis revealed a 3.4 kb open reading frame encoding a putative peptide of 1080 amino acid residues with a calculated molecular mass of 119 kDa, which presents all diagnostic features of P-type transporting ATPases. Comparison to other members of the family and phylogenetic analyses have shown that the BePAT1 protein belongs to the subfamily of Na,K- and H,K-ATPases, indicating that the divergence between the alpha-subunit of the Na,K-ATPase and other members of the P-type ATPase family has occurred before the divergence between the animal and fungal lineages in evolution.

Adenosine Triphosphatases↗

PubMed1998

Intermediate clinical follow-up of a dual-radius acetabular component.

In this study, 92 primary total hip arthroplasties were performed in 83 patients using a porous-coated, dual-radius, cementless, acetabular component. All hips underwent line-to-line dome reaming with press-fit implantation that was judged to have complete bone contact. This acetabular shell provides a 1-mm oversized peripheral rim, which adds excellent initial stability while allowing complete bone contact in all hips. No fractures occurred. In 83% of hips, adjunctive screw fixation was not necessary. At a minimum of 4 years, follow-up, there were no revisions, no acetabular migration, one case of acetabular erosion consistent with osteolysis, and the average Harris Hip Score was 95. The design features of this new acetabular component have provided excellent fixation with complete initial bone contact, resulting in satisfactory intermediate clinical and radiographic results. The design provides excellent peripheral stability and complete bone contact.

Acetabulum↗

PubMed1998

Heat shock alters poly(A) tail length of Dictyostelium discoideum hsp32 RNA.

Hsp32 is a heat shock gene in D. discoideum. We have previously observed that heat stress-induced change produces a broad band on Northern blots, suggesting that more than one population of mRNA is present under those conditions. This was not the result of a defect in the splicing of the hsp32 mRNA, nor did it result from the use of a different transcription start site under heat shock conditions. Here, we show that the broad banding pattern reflects the appearance of a transcript with a poly(A) tail that is approximately 100 nt longer than that seen in unstressed cells. Experiments indicated that this tail was not a property of newly synthesized mRNA but rather a response to heat stress. This response appeared to be specific to the hsp32 transcript and did not result in the retention of the RNA in the nucleus. These results document a relatively unusual heat shock response and also indicate that the nature of the response differs among RNAs and has selective consequences.

Animals↗

PubMed1998

The CIRCE element and its putative repressor control cell cycle expression of the Caulobacter crescentus groESL operon.

The groESL operon is under complex regulation in Caulobacter crescentus. In addition to strong induction after exposure to heat shock, under physiological growth conditions, its expression is subject to cell cycle control. Transcription and translation of the groE genes occur primarily in predivisional cells, with very low levels of expression in stalked cells. The regulatory region of groESL contains both a sigma32-like promoter and a CIRCE element. Overexpression of C. crescentus sigma32 gives rise to higher levels of GroEL and increased levels of the groESL transcript coming from the sigma32-like promoter. Site-directed mutagenesis in CIRCE has indicated a negative role for this cis-acting element in the expression of groESL only at normal growth temperatures, with a minor effect on heat shock induction. Furthermore, groESL-lacZ transcription fusions carrying mutations in CIRCE are no longer cell cycle regulated. Analysis of an hrcA null strain, carrying a disruption in the gene encoding the putative repressor that binds to the CIRCE element, shows constitutive synthesis of GroEL throughout the Caulobacter cell cycle. These results indicate a negative role for the hrcA gene product and the CIRCE element in the temporal control of the groESL operon.

Bacterial Proteins↗

PubMed1997

Cloning, structural analysis and expression of the gene encoding Hsp32 from Dictyostelium discoideum.

We have isolated and characterized genomic clones encoding a novel small heat shock (HS) protein (Hsp32) from Dictyostelium discoideum that is not homologous to the alpha-crystallin family Hsps. Besides its induction by HS, this gene is also regulated during the life cycle of the organism. At physiologic temperatures hsp32 is expressed at high levels in growing cells and at low levels in cells starved to initiate their developmental programme. However, in both cases the gene can be induced by HS. A DNA fragment containing the upstream region of hsp32 was shown to confer HS induction to a cat reporter gene, indicating a transcriptional regulation for this gene. A single transcription start site, located at position -152 relative to the initiator Met, 17 nucleotides downstream from a putative TATA box, was determined both in vegetative cells and cells starved for 6 h. This site was unchanged when either vegetative or starved cells were submitted to HS at 30 degrees C for 30 min. Despite HS induction, a perfect HSE element was not found in the 5' regulatory region of the gene. The hsp32 coding region is interrupted by a single intron located near its 5' end, which is properly spliced even under HS conditions.

Amino Acid Sequence↗

PubMed1997

Protein factors in Blastocladiella emersonii cell extracts recognize similar sequence elements in the promoters of the genes encoding cAMP-dependent protein kinase subunits.

Blastocladiella emersonii contains a single cAMP-dependent protein kinase (PKA), which is similar to the mammalian type II isoforms. Its activity is regulated during development by changes in the levels of the catalytic (C) and regulatory (R) subunits, which occur in parallel with changes in levels of the corresponding mRNAs, suggesting coordinate transcriptional control of the expression of both subunits. Both R and C mRNA levels are low in vegetative cells, rise sharply during sporulation and decrease to basal levels again after germination. To investigate sequence elements common to both Blastocladiella R and C gene promoters, which might be involved in the coordinate regulation of these genes, their 5'-flanking regions were analyzed by gel mobility shift and DNase I footprinting assays. We determined that different DNA-protein complexes are generated when fragments of the R and C gene promoters are incubated with extracts from cells expressing (sporulating cells) or not expressing (vegetative cells) both subunits, and competition experiments suggested that similar protein factors bind to both promoters. DNase I footprinting experiments have indicated that a sequence common to both R and C promoters, and similar to mammalian E-boxes, binds factors present in extracts from vegetative and sporulating cells, whereas sequences flanking the E-boxes in both promoters showed a change in the pattern of DNase I digestion only when the vegetative cell extract was used. This result suggests that the composition of the protein complexes binding to these regions changes during sporulation.

Base Sequence↗

PubMed1997

An alkB gene homolog is differentially transcribed during the Caulobacter crescentus cell cycle.

A Caulobacter crescentus alkB gene homolog was identified in a clone previously shown to contain the heat shock genes dnaK and dnaJ; the homolog is located upstream of dnaK and is transcribed in the opposite orientation. An analysis of the alkB gene has shown that the deduced amino acid sequence is that of a 21-kDa protein, which is 42% identical and 78% similar to Escherichia coli AlkB. Furthermore, an alkB-null mutant was constructed by gene disruption and was shown to be highly sensitive to the alkylating agent methyl methanesulfonate (MMS). However, the alkB gene of C. crescentus, unlike its E. coli counterpart, is not located downstream of the ada gene, and its transcription is not induced by alkylating agents. In addition, no acquired enhanced resistance to MMS toxicity by treatment with low MMS doses was observed, suggesting that no adaptive response occurs in C. crescentus. Nevertheless, transcription of the alkB gene is cell cycle controlled, with a pattern of expression similar to that of several Caulobacter genes involved in DNA replication.

Adaptation, Physiological↗

PubMed1997

A gene coding for a putative sigma 54 activator is developmentally regulated in Caulobacter crescentus.

In Caulobacter crescentus, the alternative sigma factor sigma54 plays an important role in the expression of late flagellar genes. Sigma54-dependent genes are temporally and spatially controlled, being expressed only in the swarmer pole of the predivisional cell. The only sigma54 activator described so far is the FlbD protein, which is involved in activation of the class III and IV flagellar genes and repression of the fliF promoter. To identify new roles for sigma54 in the metabolism and differentiation of C. crescentus, we cloned and characterized a gene encoding a putative sigma54 activator, named tacA. The deduced amino acid sequence from tacA has high similarity to the proteins from the NtrC family of transcriptional activators, including the aspartate residues that are phosphorylated by histidine kinases in other activators. The promoter region of the tacA gene contains a conserved sequence element present in the promoters of class II flagellar genes, and tacA shows a temporal pattern of expression similar to the patterns of these genes. We constructed an insertional mutant that is disrupted in tacA (strain SP2016), and an analysis of this strain showed that it has all polar structures, such as pili, stalk, and flagellum, and displays a motile phenotype, indicating that tacA is not involved in the flagellar biogenesis pathway. However, this strain has a high percentage of filamentous cells and shows a clear-plaque phenotype when infected with phage phiCb5. These results suggest that the TacA protein could mediate the effect of sigma54 on a different pathway in C. crescentus.

Amino Acid Sequence↗

PubMed1996

Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers.

1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated. 2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii. 3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h. 4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals.

Animals↗

PubMed1996

Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE.

In response to elevated temperature, both prokaryotic and eukaryotic cells increase expression of a small family of chaperones. The regulatory network that functions to control the transcription of the heat shock genes in bacteria includes unique structural motifs in the promoter region of these genes and the expression of alternate sigma factors. One of the conserved structural motifs, the inverted repeat CIRCE element, is found in the 5' region of many heat shock operons, including the Caulobacter crescentus groESL operon. We report the identification of another C. crescentus heat shock operon containing two genes, hrcA (hrc for heat shock regulation at CIRCE elements) and a grpE homolog. Disruption of the hrcA gene, homologs of which are also found upstream of grpE in other bacteria, increased transcription of the groESL operon, and this effect was dependent on the presence of an intact CIRCE element. This suggests a role for HrcA in negative regulation of heat shock gene expression. We identified a major promoter transcribing both hrcA and grpE and a minor promoter located within the hrcA coding sequence just upstream of grpE. Both promoters were heat shock inducible, with maximal expression 10 to 20 min after heat shock. Both promoters were also expressed constitutively throughout the cell cycle under physiological conditions. C. crescentus GrpE, shown to be essential for viability at low and high temperatures, complemented an Escherichia coli delta grpE strain in spite of significant differences in the N- and C-terminal regions of these two proteins, demonstrating functional conservation of this important stress protein.

Amino Acid Sequence↗

PubMed1995

Cloning of a cDNA encoding a novel heat-shock protein from Dictyostelium discoideum.

We have cloned, from Dictyostelium discoideum, a cDNA encoding a new heat-shock (HS) protein (Hsp) with a predicted molecular mass of 31,447 Da. Outside of its low molecular mass, this Hsp does not share any similarity with the small Hsp currently identified or with alpha-crystallins. Northern blot analysis indicates that this HS-inducible gene is also developmentally regulated .

Amino Acid Sequence↗