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B Guerra

Publications and source records attributed to B Guerra.

At least 19 recordsLinked to original sources
PubMed2005

Validation of protein kinase CK2 as oncological target.

Protein kinase CK2 is a highly conserved enzyme composed of two catalytic subunits alpha and/or alpha' and two regulatory subunits beta whose activity is elevated in diverse tumour types as well as in highly proliferating tissues. Several results suggest that the overexpression of either CK2 catalytic subunits or the CK2 holoenzyme contributes to cellular transformation. In a similar vein, experiments performed compromising the intracellular expression of CK2 has led to somehow contradictory results with respect to the ability of this enzyme to control survival and apoptosis. To better elucidate the role of CK2 in programmed cell death, we have depleted cells of CK2 catalytic subunits by the application of antisense oligodeoxynucleotides and siRNAs techniques, respectively. Our results indicate that protein kinase CK2 is characterized by an extremely high stability that might be due to its association with other intracellular proteins, enhanced half-life or lower vulnerability towards proteolytic degradation. In addition, we show that despite the effectiveness of the methods applied in lowering CK2 kinase activity in all cells investigated, CK2 might not by itself be sufficient to trigger enhanced drug-induced apoptosis in cells.

Apoptosis↗
PubMed2005

Genetic basis of antimicrobial drug resistance in clinical isolates of Salmonella enterica serotype Hadar from a Spanish region.

The genetic bases of antimicrobial drug resistance (R) of 79 Salmonella enterica serotype Hadar clinical isolates (recovered during 1995-2001 in a Spanish region) was investigated. The isolates showed a limited genomic variation, as demonstrated by PFGE analysis using XbaI (three profiles, S>or=0.77) and BlnI (seven profiles, S>or=0.49; with 95% of the isolates falling into two clusters, S>or=0.75). Thirteen R-profiles, ranging from susceptible to multidrug resistant, were recognized. All susceptible isolates (14%) were recovered before or during 1998, when multidrug resistance (MDR) was still uncommon (20% from 1995-1998). In later years, the percentage of MDR increased considerably (92% in 2001). Resistance to nalidixic acid, tetracycline, streptomycin and ampicillin-cefalotin, encoded by gyrA-Asp87/Asn, tet(A), strA/B, and bla (TEM) genes, respectively, were the most common, appearing together in 38% of the isolates. In all tetracycline- and streptomycin-resistant isolates, strA/B and tet(A) were chromosomally located, whereas bla (TEM) was plasmid-born. Five different bla (TEM) plasmids (pUO-ShR1 to pUO-ShR5, of about 9.4, 23, 30, 45, and 95 kb, respectively) were identified. pUO-ShR3 and pUO-ShR5 harbored additional R-genes: [dfrA1] and [acc(3)IV-strA/B], respectively. pUO-Sh2, pUO-Sh3, pUO-ShR4, and pUO-Sh5 were self-transferable, and the latter could also mobilize pUOShR1. The reported data constitute a useful background for further epidemiological studies of MDR in S. Hadar.

Drug Resistance, Multiple, Bacterial↗
PubMed2005

Pulsed-field gel electrophoresis, plasmid profiles and phage types for the human isolates of Salmonella enterica serovar Enteritidis obtained over 13 years in Taiwan.

AIMS: Plasmid profile, phage typing, and pulsed-field gel electrophoresis (PFGE) patterns of 124 Salmonella Enteritidis strains isolated in 1998-2002 in Taiwan were analysed and the results were compared with those of the 63 strains obtained in 1991-1997, so that molecular subtypes and epidemic strains for Salmonella Enteritidis over a 13-year period (1991-2002) could be elucidated. METHODS AND RESULTS: A total of 124 strains of Salmonella Enteritidis isolated from human in Taiwan between 1998 and 2002 were analysed by PFGE, plasmid analysis and phage typing. The results obtained were compared with those of the 63 strains obtained in 1991-1997, so that the clonal relationships for a total of 187 strains obtained over 13 years could be elucidated. For PFGE, restriction enzymes XbaI, SpeI and NotI were used for chromosomal DNA digestion. Results showed 28 PFGE pattern combinations for the 187 Salmonella strains. Of them, pattern X3S3N3 was the major subtype as 130 strains isolated from different locations during 1991-2002 showed this PFGE pattern. For all these 187 strains, the genetic similarity was higher than 80%. Plasmid analysis showed 17 distinct types, which consist of one to four plasmids and the predominant phage type of those strains was PT4 (71.6%) and PT6a (13.4%). The three methods identified different degrees of polymorphism in the following order: plasmid profile (18 types, D = 0.659) > PFGE (28 types, D = 0.512) > phage typing (13 types, D = 0.438). As PFGE patterns, phage type and plasmid profile were combined for subtyping, the 187 strains could be grouped into 46 subtypes and the discriminatory index was raised to 0.795. For these 46 subtypes, the predominant one was X3S3N3/P1/PT4, which contained 77 (41%) isolates. CONCLUSIONS: Most of the Salmonella Enteritidis strains from sporadic cases were with pattern X3S3N3. They were the prevalent and may be the epidemic strains found in Taiwan during 1991-2002. The present study suggested that the several variants were derived from a single clonal line and the genome for strains of Salmonella Enteritidis are highly conserved over a 13-year period (1991-2002). SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here are useful for epidemiolgical study of salmonellosis caused by Salmonella Enteritidis in Taiwan. Comparing the data of the present study with those obtained for strains from other countries, the major subtypes for Salmonella Enteritidis infection in the world can be elucidated.

Bacteriophage Typing↗
PubMed2004

Characterization and localization of drug resistance determinants in multidrug-resistant, integron-carrying Salmonella enterica serotype Typhimurium strains.

The genetic background of the antimicrobial resistance of 10 selected multiresistant Salmonella serotype Typhimurium (S. Typhimurium) strains (including the emerging monophasic variant [4,5,12:i:- ]) was investigated. All strains shared class 1 integrons (with seven types of variable regions) and belonged to different lineages (L1-L6) according to their phage types, DNA polymorphisms by XbaI-pulsed-field gel electrophoresis (PFGE), integrons, and/or resistance patterns. The strains were screened for the presence and localization (chromosomal or plasmid) of 32 DNA sequences representing integron-, Tn21-like transposon-, resistance-, and virulence-plasmid genes. Strains belonging to lineage L1 (definitive phage type DT104) carried the 90-kb Salmonella virulence plasmid together with the complete or partial chromosomally located Salmonella Genomic Island 1 (SGI1). All strains belonging to the other five lineages carried their resistance determinants on various resistance plasmids. Two of these strains showed complex plasmid profiles, which included a 95 kb virulence plasmid together with two or four resistance plasmids. Two strains carried a resistance plasmid that lacked the virulence-plasmid-encoding sequences. The remaining two strains carried two different hybrid virulence-resistance plasmids. Twenty-three of the DNA sequences could be assigned to distinct XbaI genomic restriction patterns (PFGE profiles). In this way, the influence of the resistance and virulence plasmids on the PFGE profiles was determined, and several groups of resistance genes could be identified. The data obtained represent a useful epidemiological tool for tracing the emergence and distribution of multiresistant S. Typhimurium worldwide.

Base Sequence↗
PubMed2003

Incidence of quinolone resistance in strains of Salmonella isolated from poultry, cattle and pigs in Germany between 1998 and 2001.

This paper reports the susceptibility to the quinolone nalidixic acid and the fluoroquinolone ciprofloxacin of 14,514 strains of Salmonella isolated in Germany from poultry, cattle and pigs between 1998 and 2001. Quinolone-resistant salmonellae were most frequently isolated from poultry, with a prevalence of 10.2 to 16.8 per cent. Poultry-associated serotypes, such as Salmonella Paratyphi B (d-tartrate positive), Salmonella Hadar and Salmonella Virchow, had the highest prevalence of quinolone resistance, ranging between 35 and 74 per cent. All the nalidixic acid-resistant strains also had a reduced susceptibility to ciprofloxacin, with minimum inhibitory concentrations (MICS) of 0.125 to 2 microg/ml. A comparison of the MICS for ciprofloxacin of the strains of these poultry-associated serotypes and Salmonella Enteritidis phage type 4 isolated in 1998/99 and 2000/01 indicated that there had been a shift towards higher MIC values of up to 2 microg/ml. The quinolone resistance-determining region (QRDR) of the gyrA gene and the homologue region of the parC gene of 31 selected strains were sequenced. Several different amino acid changes were observed in gyrA of the quinolone-resistant isolates at positions 83 and 87, but no substitutions were observed in parC.

Amino Acid Sequence↗
PubMed2003

Estradiol prevents amyloid-beta peptide-induced cell death in a cholinergic cell line via modulation of a classical estrogen receptor.

The pathology of Alzheimer's disease includes amyloid-beta peptide aggregation that contributes to degeneration of cholinergic neurons. Even though the underlying molecular mechanisms remain unclear, recent in vitro evidence supports a protective role for estrogens against several neurotoxic agents. Here we report that, in a murine cholinergic cell line (SN56), the massive cell death induced by 1-40 fragment of amyloid-beta peptide was prevented by 17beta-estradiol through a mechanism that may involve estrogen receptor activation. The protective effect of estradiol was observed in a dose-dependent manner, and was completely blocked by the pure antiestrogen ICI 182,780. In contrast, the inactive isomer 17alpha-estradiol consistently showed weaker neuroprotection than the native hormone that was unaffected by ICI 182,780 treatment. In addition, equivalent concentrations of 17beta-estradiol enhanced luciferase activity in cells transfected with a luciferase reporter gene driven by tandem estrogen response elements. Estrogen-induced luciferase activity was blocked by ICI 182,780, indicating estrogen receptor-dependent transcriptional activity. We also observed by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry that increasing concentrations of 17beta-estradiol enhanced the expression of estrogen receptor alpha mRNA and protein during amyloid-beta-induced toxicity. Under these conditions, it was found by confocal microscopy that the localization of estrogen receptor alpha in the absence of hormone was mainly extranuclear. However, the receptor was consistently observed also at the nuclear region after estrogen exposure. Overall, these data suggest that estrogen may exert neuroprotective effects against amyloid-beta-induced toxicity by activation of estrogen receptor-mediated pathways. In addition, intracellular estrogen receptors are up-regulated by their cognate hormone even during exposure to neurotoxic agents.

Acetylcholine↗
PubMed2003

Epitope analysis of the MAb 1AD9 antibody detection site in human protein kinase CK2alpha-subunit.

1AD9 is a murine monoclonal antibody (MAb) that was produced against the recombinant human alpha-subunit of protein kinase CK2, a pleiotropic and constitutively active serine/threonine protein kinase. We have further characterized this antibody, which is suitable for Western blot, immunoprecipitation and enzyme-linked immunosorbant assay tests. Using an overlapping peptide library, we have identified the epitope targeted by MAb1AD9 characterized by the following sequence: (319)MEHPYF(324).

Amino Acid Sequence↗
PubMed2003

An ICI 182,780-sensitive, membrane-related estrogen receptor contributes to estrogenic neuroprotective actions against amyloid-beta toxicity.

Although estrogen (E2)-related neuroprotection has been repeatedly demonstrated in different models, the involvement of non-classical estrogen receptors (ERs) in this activity remains unclear. Using SN56 murine cholinergic cell line from the basal forebrain, we present evidence indicating that an ER associated with the plasma membrane participates in estrogen-dependent reduction of neuronal death induced by amyloid-beta peptide (Abeta) toxicity. Exposure to either E2 or estradiol-horseradish peroxidase (E-HRP) for 15 min significantly reduced Abeta-induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC-20 antibody directed to a region neighboring the ligand-binding domain of ERa. Using MC-20 antibody in unpermeabilized SN56 cells, we detected a protein at the plasma membrane region. The binding of impermeant forms of E2, E-HRP, and E-BSA-FITC to specific sites of SN56 plasma membrane was blocked by pre-incubation with E2, ICI 182,780, and MC-20 antibody in a concentration-dependent manner. Thus, a membrane-related ER that shares some structural homologies with ERalpha may participate in estrogen-mediated neuroprotection.

Amyloid beta-Peptides↗
PubMed2002

[Pseudomonas aeruginosa bacteremia: clinical and epidemiological patterns and treatment].

Pseudomonas aeruginosa bacteremia has the highest mortality among Gram-negative infections. We studied its clinical and epidemiological patterns in order to achieve an early and accurate diagnosis and treatment. We prospectively studied 211 cases of P. aeruginosa bacteremia admitted to our hospital from 1991 to 1998. All patients had at least one positive blood culture for P. aeruginosa and clinical symptoms of bacteremia. The prognosis of the underlying disease, past illnesses, foci location, initial clinical status, type of bacteremia, complications, and the type of antibiotic treatment were analyzed. Clinical and microbiological controls were carried out at baseline, at the end of the treatment period, and 1 month later. Of the 211 cases, 73% were men with a mean age of 56.5 years. Overall, 45.5% were hospitalized in the ICU, 28% in the surgical ward, and 26% in the medical service, with a mean length of stay of 40.4 days. As risk factors we identified a length of stay more than 14 days (76.7%), previous manipulations (87.6%), and surgery (44.5%). A total of 43.1% had had an infection in the last 6 weeks, and 46.4% were treated with antibiotics, usually broad-spectrum antibiotics without activity against P. aeruginosa. The most frequent underlying diseases were neoplasia (25.5%), postsurgical cardiopathy (24.1%), nephrosis (22.7%), chronic obstructive pulmonary disease (17.5%) and diabetes (13.2%). The most frequent foci were pulmonary (29.3%), urinary (21.8%), and undetermined (38.3%). The mortality rate was 27.9% and was considered related to P. aeruginosa bacteremia in 76.2% of the cases. The antibiotic treatment was considered adequate in 89% of the cases. It was concluded that knowledge of the clinical and epidemiological factors of patients with P. aeruginosa bacteremia may help to optimize its management and to reduce its morbidity and mortality.

Bacteremia↗
PubMed2001

Outbreaks and sporadic cases of Salmonella serovar panama studied by DNA fingerprinting and antimicrobial resistance.

In the Principality of Asturias (PA), Spain. three Salmonella serovar Panama outbreaks were registered in August 1998. In order to achieve an accurate identification of the strains implicated in the outbreaks and to study the molecular epidemiology of this serovar in the PA, the isolates collected over 1990-1999 were examined by DNA fingerprinting and antimicrobial resistance analysis. The origin of the isolates was: human (65, of which 20 were associated with the three outbreaks), octopus (2), beef (2), eggs (7), poultry faeces (2), sea water (5), sewage (2) and unknown (1). Sixteen lineages were defined by ribotyping, enterobacterial repetitive intergenic consensus sequences analysis, and randomly amplified polymorphic DNA segment analysis. One lineage was endemic in the PA and was also represented by isolates from other Spanish regions. The organisms of this lineage can be differentiated (by resistance-, plasmid- and integron-profiles) into 19 types. The three outbreaks were caused by organisms falling into a single type (nalidixic acid-resistant, plasmid- and integron-free) belonging to the endemic lineage, which was associated with poultry as the reservoir. Isolates showing drug-resistance (71%) fell into six lineages and 23 types. Ten multidrug-resistant types carried class I integrons with three types of variable regions containing resistance gene cassettes.

Animals↗
PubMed2001

Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme.

The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.

Amino Acid Sequence↗
PubMed2001

Characterization of basal nitric oxide production in living cells.

Nitric oxide (NO) is an important modulator of immune, endocrine and neuronal functions; however, measuring physiological levels of NO in cell cultures is generally difficult because of the lack of suitable methodologies. We have selected three cell lines from different origins: the neuroblastoma-derived Neuro2A (N2A), the cholinergic SN56 and the non-neuronal COS-1. We first demonstrated the presence of NADPH-diaphoretic activity, a potential marker of the NO-synthesizing (NOS) enzyme. By immunocytochemistry, using specific antibodies for each NOS subtype, we observed that subtype I was present in all cell lines and that subtype II was present in COS-1 and N2A cell lines. The presence of these NOS subtypes was further verified by Western blot analysis. Control cells treated with DAF-2 DA exhibited significant fluorescent levels corresponding to basal NO production. The subcellular distribution of the synthesizing enzyme was consistent with the NO-fluorescence signal; whereas, fixation affected the subcellular pattern of NO fluorescence signal. Addition of NOS inhibitors or NO scavengers to the incubation medium reduced the intensity of the NO fluorescence signal in a concentration-dependent manner. Conversely, increasing concentrations of a NO donor, or incident light, increased the fluorescence intensity. Our observation of NO production and distribution using the DAF-2 method has a direct impact on studies using these cell lines.

Animals↗
PubMed2001

Estrogen modulates norepinephrine-induced accumulation of adenosine cyclic monophosphate in a subpopulation of immortalized luteinizing hormone-releasing hormone secreting neurons from the mouse hypothalamus.

A subpopulation of luteinizing hormone-releasing hormone (LHRH)-producing cells that express the intermediate filament protein vimentin and the neuronal marker neurofilament 145, but not neurofilament 200 nor glial fibrillary acidic protein, has been isolated from GT1-7 cultures. These cells express the mRNA encoding estrogen receptor alpha (ERalpha) and respond to physiological concentrations of 17beta-estradiol (E2) by reducing the accumulation of cyclic adenosine monophosphate induced by norepinephrine, but not that induced by direct activation of adenylate cyclase. These results indicate that the activity of LHRH-producing neurons may be directly modulated by estrogen. In addition, they are suggestive of an estrogen-dependent desensitization of the beta-adrenoceptor in these cells.

Animals↗
PubMed2001

The amount of estrogen receptor alpha increases after heat shock in a cholinergic cell line from the basal forebrain.

Estrogen exerts a neuroprotective action in response to a variety of cell stresses. However, to what extent intracellular estrogen receptors are involved in these functions remains to be determined. We have found that SN56 cells, a neuronal-derived cholinergic cell line which produces luteinizing hormone-releasing hormone and contains the mRNAs encoding estrogen and progesterone receptors, also express estrogen receptor alpha, as well as the heat shock protein 90. Exposure of these cells to drastic temperature elevation for 1 h immediately increased the intracellular levels of these two proteins, whereas it rapidly reduced the content of estrogen receptor alpha mRNA. In addition, the amount of estrogen receptor alpha-heat shock protein 90 complexes was increased in response to thermal stress. Pre-treatment with geldanamycin, a potent inhibitor of heat shock protein 90, decreased the amount of estrogen receptor alpha, suggesting that its elevation after the heat insult may be related to its association with heat shock protein 90. In contrast, exposure of heat-shocked cells to 17beta-estradiol reduced the number of estrogen receptor alpha-heat shock protein 90 complexes, suggesting that the receptor conserves the affinity for its cognate ligand under these conditions. Therefore, the interaction of the estrogen receptor with heat shock protein 90 may serve to prevent its degradation during the thermal insult, as well as to maintain it in a high-affinity hormone-binding conformation. Since neuroprotective estrogen effects have been described in a variety of cytotoxic situations, these findings may be suggestive of an integrated neuronal response to injury, which includes the protection of available estrogen receptors through their association with heat shock protein 90.

Benzoquinones↗
PubMed2001

Phosphorylation of the Fas associated factor FAF1 by protein kinase CK2 and identification of serines 289 and 291 as the in vitro phosphorylation sites.

We previously identified the human Fas associated factor (FAF1) as one of the interacting partners of protein kinase CK2 beta subunit. Since FAF1 is a phosphoprotein we investigated whether it is a substrate for CK2. Here, we report the full length human FAF1 cDNA sequence, expression of FAF1 in Escherichia coli and purification and characterization of FAF1 as a substrate for CK2. FAF1 as well as an N-terminal 40 kDa degradation product serve as substrates for both the recombinant CK2 holoenzyme (km 100 microM) and the isolated catalytic alpha subunit (km 200 microM). Despite the high k(m) values, we obtained evidence that CK2 is the major cellular kinase responsible for FAF1 phosphorylation, using tissue extracts as kinase sources. By MALDI-MS we identified the two serine residues at positions 289 and 291 as the major in vitro CK2 phosphorylation sites. These data may help us elucidate the functions of FAF1 and the involvement of CK2 mediated phosphorylation in processes such as apoptotic signaling, ubiquitination, nuclear translocation and embryonic development.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection.

Hepatitis C virus (HCV) infections were evaluated in chimpanzees that had previously cleared HCV and were rechallenged. Animals that had previously cleared HCV infection rapidly cleared homologous and heterologous virus upon rechallenge, indicative of a strong protective immunity. In one animal, sterilizing immunity was observed with regard to viremia, although viral RNA was transiently detected in the liver. Accelerated viral clearance following rechallenge with HCV was observed in animals that had not been exposed to HCV for over 16 years, suggesting that long-lasting protective immunity may be possible. The ability of peripheral blood mononuclear cells (PBMC) to recognize HCV proteins was evaluated during the course of the rechallenge experiments. A very early and strong in vitro recall response to HCV nonstructural proteins appeared to be associated with viral clearance. In contrast, proliferative responses to HCV proteins were not observed in 4 persistently infected chimpanzees, and a weak proliferative response was observed in 1 of 2 animals during acute resolving infection. The results suggest that a strong T-cell proliferative response is induced upon rechallenge of chimpanzees with HCV and that this response is associated with rapid viral clearance. The antibody response to HCV proteins increased by over 1,000-fold in all animals following rechallenge as well. A more complete understanding of the role of the cellular immune response in the clearance of HCV and the nature of the protective immune response following viral clearance may aid in the generation of therapies and vaccines.

Acute Disease↗
PubMed2001

Multidrug resistance is mediated by large plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype [4,5,12:i:-].

A multidrug-resistant Salmonella enterica serotype [4,5,12:i:-] clone carried a class 1 integron harboring dfrA12 and aadA2 gene cassettes and bla(TEM-1), aac(3)-IV, cmlA1, and tetA genes located in large plasmids of about 140 kb (carrying spv) or 120 kb (lacking spv). Several segregants, lacking multidrug resistance, contained a plasmid smaller than the parental one and no longer hybridized with probes for the lost resistances. The genes mediating resistance to ampicillin, chloramphenicol, and tetracycline in the [4,5,12:i:-] clone are different from those found in the pentadrug-resistant serotype Typhimurium DT104 clone.

Ampicillin Resistance↗