Pathophysiology of L-dopa-induced motor and non-motor complications in Parkinson's disease.

Involuntary movements, or dyskinesia, represent a debilitating complication of levodopa (L-dopa) therapy for Parkinson's disease (PD). L-dopa-induced dyskinesia (LID) are ultimately experienced by the vast majority of patients. In addition, psychiatric conditions often manifested as compulsive behaviours, are emerging as a serious problem in the management of L-dopa therapy. The present review attempts to provide an overview of our current understanding of dyskinesia and other L-dopa-induced dysfunctions, a field that dramatically evolved in the past twenty years. In view of the extensive literature on LID, there appeared a critical need to re-frame the concepts, to highlight the most suitable models, to review the central nervous system (CNS) circuitry that may be involved, and to propose a pathophysiological framework was timely and necessary. An updated review to clarify our understanding of LID and other L-dopa-related side effects was therefore timely and necessary. This review should help in the development of novel therapeutic strategies aimed at preventing the generation of dyskinetic symptoms.

Evaluation of the effects of erythritol on gene expression in Brucella abortus.

Bacteria of the genus Brucella have the unusual capability to catabolize erythritol and this property has been associated with their virulence mainly because of the presence of erythritol in bovine foetal tissues and because the attenuated S19 vaccine strain is the only Brucella strain unable to oxydize erythritol. In this work we have analyzed the transcriptional changes produced in Brucella by erythritol by means of two high throughput approaches: RNA hybridization against a microarray containing most of Brucella ORF's constructed from the Brucella ORFeome and next generation sequencing of Brucella mRNA in an Illumina GAIIx platform. The results obtained showed the overexpression of a group of genes, many of them in a single cluster around the ery operon, able to co-ordinately mediate the transport and degradation of erythritol into three carbon atoms intermediates that will be then converted into fructose-6P (F6P) by gluconeogenesis. Other induced genes participating in the nonoxidative branch of the pentose phosphate shunt and the TCA may collaborate with the ery genes to conform an efficient degradation of sugars by this route. On the other hand, several routes of amino acid and nucleotide biosynthesis are up-regulated whilst amino acid transport and catabolism genes are down-regulated. These results corroborate previous descriptions indicating that in the presence of erythritol, this sugar was used preferentially over other compounds and provides a neat explanation of the the reported stimulation of growth induced by erythritol.

Analysis of genes encoding penicillin-binding proteins in clinical isolates of Acinetobacter baumannii.

There is limited information on the role of penicillin-binding proteins (PBPs) in the resistance of Acinetobacter baumannii to ß-lactams. This study presents an analysis of the allelic variations of PBP genes in A. baumannii isolates. Twenty-six A. baumannii clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals in Spain were included. The antimicrobial susceptibility profile, clonal pattern, and genomic species identification were also evaluated. Based on the six complete genomes of A. baumannii, the PBP genes were identified, and primers were designed for each gene. The nucleotide sequences of the genes identified that encode PBPs and the corresponding amino acid sequences were compared with those of ATCC 17978. Seven PBP genes and one monofunctional transglycosylase (MGT) gene were identified in the six genomes, encoding (i) four high-molecular-mass proteins (two of class A, PBP1a [ponA] and PBP1b [mrcB], and two of class B, PBP2 [pbpA or mrdA] and PBP3 [ftsI]), (ii) three low-molecular-mass proteins (two of type 5, PBP5/6 [dacC] and PBP6b [dacD], and one of type 7 (PBP7/8 [pbpG]), and (iii) a monofunctional enzyme (MtgA [mtgA]). Hot spot mutation regions were observed, although most of the allelic changes found translated into silent mutations. The amino acid consensus sequences corresponding to the PBP genes in the genomes and the clinical isolates were highly conserved. The changes found in amino acid sequences were associated with concrete clonal patterns but were not directly related to susceptibility or resistance to ß-lactams. An insertion sequence disrupting the gene encoding PBP6b was identified in an endemic carbapenem-resistant clone in one of the participant hospitals.

A leaky mutation in CD3D differentially affects αß and γδ T cells and leads to a Tαß-Tγδ+B+NK+ human SCID.

T cells recognize antigens via their cell surface TCR and are classified as either αß or γδ depending on the variable chains in their TCR, α and ß or γ and δ, respectively. Both αß and γδ TCRs also contain several invariant chains, including CD3δ, which support surface TCR expression and transduce the TCR signal. Mutations in variable chains would be expected to affect a single T cell lineage, while mutations in the invariant chains would affect all T cells. Consistent with this, all CD3δ-deficient patients described to date showed a complete block in T cell development. However, CD3δ-KO mice have an αß T cell-specific defect. Here, we report 2 unrelated cases of SCID with a selective block in αß but not in γδ T cell development, associated with a new splicing mutation in the CD3D gene. The patients' T cells showed reduced CD3D transcripts, CD3δ proteins, surface TCR, and early TCR signaling. Their lymph nodes showed severe T cell depletion, recent thymus emigrants in peripheral blood were strongly decreased, and the scant αß T cells were oligoclonal. T cell-dependent B cell functions were also impaired, despite the presence of normal B cell numbers. Strikingly, despite the specific loss of αß T cells, surface TCR expression was more reduced in γδ than in αß T cells. Analysis of individuals with this CD3D mutation thus demonstrates the contrasting CD3δ requirements for αß versus γδ T cell development and TCR expression in humans and highlights the diagnostic and clinical relevance of studying both TCR isotypes when a T cell defect is suspected.

Caracterización molecular de aislados clínicos de Pseudomonas aeruginosa productores de la metalobetalactamasa VIM-2 en Cantabria, España / Molecular characterization of Pseudomonas aeruginosa isolates in Cantabria, Spain, producing VIM-2 metallo-beta-lactamase

Introducción En España el aislamiento de cepas de Pseudomonas aeruginosa productoras de metalobetalactamasas (MBL) es poco frecuente. En este artículo se describe la caracterización de 9 aislados clínicos de P. aeruginosa multirresistentes clonalmente relacionados, aislados en Cantabria (España) que poseen el gen (..) (AU)

Introduction Pseudomonas aeruginosa strains producing (..) (AU)

[Molecular characterization of Pseudomonas aeruginosa isolates in Cantabria, Spain, producing VIM-2 metallo-beta-lactamase]. / Caracterización molecular de aislados clínicos de Pseudomonas aeruginosa productores de la metalobetalactamasa VIM-2 en Cantabria, España.

INTRODUCTION: Pseudomonas aeruginosa strains producing metallo-beta-lactamases (MbetaL) are uncommon in Spain. This study describes the characterization of 9 new clonally related multiresistant P. aeruginosa isolates possessing the bla(VIM-2) gene in Cantabria (Northern Spain). METHODS: P. aeruginosa clinical strains (1 per patient) were isolated in the Microbiology Service of Marqués de Valdecilla University Hospital between January 2004 and December 2006. Identification and preliminary susceptibility studies were performed with the MicroScan WalkAway system (Dade Behring, Sacramento, CA) and results were verified by a microdilution reference method. RESULTS: MICs of imipenem and meropenem for the 9 isolates ranged from 32 to 128 and 16 to 64 microg/mL, respectively. Nine isolates had a single Rep-PCR pattern and were intermediate or resistant to ceftazidime, cefepime, gentamicin, tobramycin, amikacin and ciprofloxacin. Eight of the 9 isolates were susceptible to aztreonam. Hydrolysis activity of imipenem in MbetaL-positive isolates ranged from 162+/-18 to 235+/-28 pmol/min/microg protein and was abolished in the presence of 5 mM EDTA. All isolates possessed an integron with genes aac(6')32, bla(VIM-2) and a putative transposase-encoding gene, flanked by the conserved 5'CS and 3'CS regions. CONCLUSION: In the clinical isolates studied, the presence of MbetaL VIM-2 sufficed to explain their resistance to carbapenems.

Emergence of imipenem resistance in clinical Escherichia coli during therapy.

The molecular epidemiology and the mechanisms of resistance of Escherichia coli isolated from two patients infected by imipenem-resistant strains are reported in this study. From one patient, three closely related consecutive isolates of E. coli were recovered; the first was carbapenem-susceptible but acquired imipenem resistance after treatment with ertapenem, and the third isolate was again imipenem-susceptible. An additional imipenem-resistant isolate was recovered from another patient who received imipenem. The genetic relatedness of the E. coli isolates was determined by pulsed-field gel electrophoresis (PFGE) after digestion with XbaI. Standard polymerase chain reaction (PCR) conditions were used to amplify several beta-lactamase genes coding for carbapenemases, extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC; the E. coli ampC gene promoter was also amplified and sequenced. Primers OmpF-F/OmpF-R and OmpC-F/OmpC-R were used to amplify the ompF and ompC genes. The outer membrane protein (OMP) profiles were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Imipenem-resistant E. coli isolates did not produce carbapenemases but lacked the two major OMPs OmpF and OmpC and had ampC promoter mutations; in addition, one of the imipenem-resistant isolates produced the CMY-2 cephalosporinase, whilst the other produced the new CTX-M-67 ESBL. Carbapenem resistance in this study was associated with lack of expression of OmpF and OmpC porins. Additional mechanisms of beta-lactam resistance, such as plasmid-mediated AmpC and ESBL production, were also found. Development of carbapenem resistance in a CTX-M-67-producing E. coli is first described in this study.

Identification of novel non-pathogenic mutation in SH3 domain of Btk in an XLA patient.

A first report of an XLA patient with a polymorphism in Btk SH3 domain has been identified after sequencing of the entire gene. SH3 domain variants might not be detected due to well characterized mutations outside the domain.

Characterization of the urease operon of Brucella abortus and assessment of its role in virulence of the bacterium.

Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.

Bruton's tyrosine kinase is not essential for LPS-induced activation of human monocytes.

BACKGROUND: X-linked agammaglobulinemia (XLA) is characterized by impaired B-cell differentiation caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The natural disease model, the X-linked immunodeficiency mouse, shows a less severe phenotype, indicating a different requirement of Btk in human and mouse B cells. Btk is also expressed in the myeloid line and participates in LPS signaling. Deficient oxidative burst and myeloid differentiation have been reported in the X-linked immunodeficiency mouse, but the precise mechanism and relevance of Btk activity in human monocytes is poorly understood. OBJECTIVE: The apparent absence in XLA of clinical manifestations of myeloid deficiency prompted us to explore the relevance of complete Btk absence in human myeloid cells. METHODS: Seven patients with XLA with BTK mutations conditioning a null protein expression were included in the study. Monocyte LPS-induced mitogen-activated protein kinase activation, TNF-alpha and IL-6 production in monocytes, and oxidative burst in monocytes and granulocytes were analyzed by means of flow cytometry. RESULTS: We show that in response to LPS, Btk-null monocytes from patients with XLA induce early mitogen-activated protein kinase activation and intracellular TNF-alpha and IL-6 production with the same intensity as cells from age- and sex-matched control subjects. In addition, the oxidative burst in response to LPS and other stimulants was completely normal in Btk-null monocytes and neutrophils. CONCLUSION: Our results indicate that Btk is not essential for early LPS signaling in human monocytes and that different Btk dependency might exist between human and mouse myeloid cells. CLINICAL IMPLICATIONS: These findings provide a better understanding of XLA, and they show the differences between human XLA and murine Xid models.

A genotype-phenotype correlation study in a group of 54 patients with X-linked agammaglobulinemia.

BACKGROUND: X-linked (Bruton's) agammaglobulinemia (XLA) is a rare immunodeficiency caused by a block in B-cell development caused by mutations in the Bruton's tyrosine kinase (BTK) gene. Many aspects of XLA and BTK function remain unresolved; atypical presentations have been reported, and no clear genotype-phenotype correlation has been established. OBJECTIVES: We sought to contribute to the understanding of XLA through the phenotypic and biochemical characterization of a large group of Spanish patients with agammaglobulinemia. We also sought to classify the mutations according to their severity to analyze a genotype-phenotype correlation. METHODS: Clinical and analytic data were collected from the clinical records. We studied the BTK gene, protein expression, and function, and the findings were correlated with the phenotypic information. RESULTS: Fifty-four patients were given diagnoses of XLA. We identified 38 different mutations in BTK, 26 not described in other patients, and several uncommon clinical phenotypes or analytic characteristics were found. The statistical analysis shows that less severe mutations or minimal detection of protein by means of flow cytometry are associated with decreased severity in clinical and analytic data, demonstrating a clear relation between the type of mutation and the disease expressivity. However, some exemptions to this rule were noted. CONCLUSIONS: XLA is a variable disease. Globally, a genotype-phenotype correlation is observed, but individual discrepancies between the severity of the mutation and the clinical and analytic phenotype suggest that other loci or ambient factors significantly influence the disease presentation and evolution.

The aquaporin gene aqpX of Brucella abortus is induced in hyperosmotic conditions.

An aquaporin gene (aqpX) was previously detected in the pathogenic bacterium Brucella abortus. Earlier studies showed that AqpX mediated rapid and large water fluxes in both directions in response to sudden osmotic up- or downshifts. Here, to study the role and the expression of the aqpX gene in B. abortus, an aqpX null mutant was constructed using an aqpX : : lacZ gene fusion. This mutant showed no significant difference in growth rate compared to the wild-type strain when grown in rich and minimal media, demonstrating that disruption of the aqpX gene was not lethal for B. abortus. The role of the B. abortus AqpX water channel was investigated by exposing the cells to hypo- and hyperosmolar conditions. While in hyperosmolar environments the growth rate of the knockout mutant was not affected, in hypo-osmolar conditions this mutant showed reduced viability after 50 h of growth. beta-Galactosidase assays and RT-PCR revealed that aqpX gene expression and the amount of aqpX mRNA were markedly increased in hyperosmolar conditions. Moreover, B. abortus aqpX expression levels were enhanced during the mid-exponential phase of growth. These results indicated that the expression of aqpX was regulated during the growth curve and induced in hyperosmolar conditions. This report is believed to be the first example of the induction of a bacterial aquaporin in hypertonic conditions.