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The main goal of this work consisted in cloning, purifying and characterizing a protein phosphatase 2C (PP2C) from promastigotes of Leishmania major. The gene was cloned and amplified by PCR using specific oligonucleotides and the recombinant protein was purified by affinity chromatography. The peak with maximal protein concentration was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and revealed a protein of 44·9 kDa with PP2C activity. This activity was dependent on divalent cations (Mg+2 and Mn+2) and was optimal at pH of 8·5, using phosphothreonine as the substrate. Sanguinarine inhibited the activity of the recombinant LmPP2C, while protein tyrosine phosphatase inhibitors had no effect. The recombinant LmPP2C was used to generate polyclonal antibodies. These antibodies recognized a protein of 44·9 kDa in different Leishmania species; the LmPP2C was localized in the flagellar pocket and the flagellum of promastigotes.
Asymptomatic colonisation of the gastrointestinal tract by carbapenemase-producing Enterobacteriaceae is an important reservoir for transmission, which may precede infection. This retrospective observational case–control study was designed to identify risk factors for developing clinical infection with OXA-48-producing Klebsiella pneumoniae in rectal carriers during hospitalisation. Case patients (n = 76) had carbapenemase-producing K. pneumoniae (CPKP) infection and positive rectal culture for CPKP. Control patients (n = 174) were those with rectal colonisation with CPKP but without CPKP infection. Multivariate analysis identified the presence of a central venous catheter (OR 4·38; 95% CI 2·27–8·42; P = 0·008), the number of transfers between hospital units (OR 1·27; 95% CI (1·06–1·52); P < 0·001) and time at risk (OR 1·02 95% CI 1·01–1·03; P = 0·01) as independent risk factors for CPKP infection in rectal carriers. Awareness of these risk factors may help to identify patients at higher risk of developing CPKP infection.
Phosphatase activity of Leishmania spp. has been shown to deregulate the signalling pathways of the host cell. We here show that Leishmania mexicana promastigotes and amastigotes secrete proteins with phosphatase activity to the culture medium, which was higher in the Promastigote Secretion Medium (PSM) as compared with the Amastigote Secretion Medium (ASM) and was not due to cell lysis, since parasite viability was not affected by the secretion process. The biochemical characterization showed that the phosphatase activity present in PSM was higher in dephosphorylating the peptide END (pY) INASL as compared with the peptide RRA (pT)VA. In contrast, the phosphatase activity in ASM showed little dephosphorylating capacity for both peptides. Inhibition assays demonstrated that the phosphatase activity of both PSM and ASM was sensible only to protein tyrosine phosphatases inhibitors. An antibody against a protein phosphatase 2C (PP2C) of Leishmania major cross-reacted with a 44·9 kDa molecule in different cellular fractions of L. mexicana promastigotes and amastigotes, however, in PSM and ASM, the antibody recognized a protein about 70 kDa. By electron microscopy, the PP2C was localized in the flagellar pocket of amastigotes. PSM and ASM induced the production of tumor necrosis factor alpha, IL-1β, IL-12p70 and IL-10 in human macrophages.
I deficiency is still a worldwide public health problem, with children being especially vulnerable. No nationwide study had been conducted to assess the I status of Spanish children, and thus an observational, multicentre and cross-sectional study was conducted in Spain to assess the I status and thyroid function in schoolchildren aged 6–7 years. The median urinary I (UI) and thyroid-stimulating hormone (TSH) levels in whole blood were used to assess the I status and thyroid function, respectively. A FFQ was used to determine the consumption of I-rich foods. A total of 1981 schoolchildren (52 % male) were included. The median UI was 173 μg/l, and 17·9 % of children showed UI<100 μg/l. The median UI was higher in males (180·8 v. 153·6 μg/l; P<0·001). Iodised salt (IS) intake at home was 69·8 %. IS consumption and intakes of ≥2 glasses of milk or 1 cup of yogurt/d were associated with significantly higher median UI. Median TSH was 0·90 mU/l and was higher in females (0·98 v. 0·83; P<0·001). In total, 0·5 % of children had known hypothyroidism (derived from the questionnaire) and 7·6 % had TSH levels above reference values. Median TSH was higher in schoolchildren with family history of hypothyroidism. I intake was adequate in Spanish schoolchildren. However, no correlation was found between TSH and median UI in any geographical area. The prevalence of TSH above reference values was high and its association with thyroid autoimmunity should be determined. Further assessment of thyroid autoimmunity in Spanish schoolchildren is desirable.
According to the reports of Z.E. Horvath et al  and Liu Yun-quan et al , carbon nanotubes can be synthesized by spray pyrolysis from different carbon sources (n-pentane, n-hexane, n-heptane, cyclohexane, toluene and acrylonitrile) and several metallocene catalysts (ferrocene, cobaltocene and nickelocene). This paper describes two different existing methods for growth of carbon nanotubes and the influence of applied parameters (oven temperature, synthesis time, catalyst concentration, carrier gas flow and solution flow) on the CNT's morphology. Also, a possible influence of number of carbons in carbon sources and structures of their compounds (linear or aromatic) on properties of formed carbon nanotubes. Transmission Electron Microscopy (TEM), Infrared Spectroscopy (FTIR) and Raman spectroscopy were applied for characterization of obtained materials.
Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. Leishmania promastigotes primarily infect macrophages in the host, where they transform into amastigotes and multiply. Lipophosphoglycan (LPG), the most abundant surface molecule of the parasite, is a virulence determinant that regulates the host immune response. Promastigotes are able to modulate this effect through LPG, creating a favourable environment for parasite survival, although the mechanisms underlying this modulation remain unknown. We analysed the participation of TLR2 and TLR4 in the production of cytokines and explored the possible phosphorylation of ERK and/or p38 MAP kinase signalling cascades in human macrophages stimulated with Leishmania mexicana LPG. The results show that LPG induced the production of TNF-α, IL-1β, IL-12p40, IL-12p70 and IL-10 and led to phosphorylation of ERK and p38 MAP kinase. Specific inhibitors of ERK or p38 MAP kinases and mAbs against TLR2 and TLR4 reduced cytokine production and phosphorylation of both kinases. Our results suggest that L. mexicana LPG binds TLR2 and TLR4 receptors in human macrophages, leading to ERK and MAP kinase phosphorylation and production of pro-inflammatory cytokines.
People detection and tracking are essential capabilities in order to achieve a natural human–robot interaction. A great portion of the research in that area has been focused on monocular techniques. However, the use of stereo vision for these purposes concentrates a great interest nowadays. This paper presents a multi-agent system that implements a basic set of perceptual-motor skills providing mobile robots with primitive interaction capabilities. The skills designed use stereo and ultrasound information to enable mobile robots to (i) detect an interested user who desires to interact with the robot, (ii) keep track of the user while they move in the environment without confusing them with other people, and (iii) follow the user along the environment avoiding obstacles in the way. The system presented has been evaluated in several real-life experiments achieving good results and real-time performance on modest computers.
PTPases have been reported as a virulence factor in different pathogens. Recent studies suggest that PTPases play a role in the pathogenesis of Leishmania infections through activation of macrophage PTPases by the parasite. We report here the presence of a membrane-bound PTPase in Leishmania major promastigotes. We detected differences in the PTPases present in the procyclic and metacyclic stages of promastigotes. In metacyclic promastigotes, the PTPase activity was totally inhibited by specific PTPase and serine/threonine inhibitors, whereas in procyclic promastigotes the PTPase activity was inhibited only with PTPase inhibitors. Two antibodies against the catalytic domains of the human placental PTPase1B and a PTPase from Trypanosoma brucei cross-reacted with a 55–60 kDa molecule present in the soluble detergent-extracted fraction of a Leishmania homogenate. Metacyclic promastigotes expressed more of this molecule than parasites in the procyclic stage. Yet the specific activity of the enzyme was lower in metacyclic than in procyclic promastigotes. Ultrastructural localization of the enzyme showed that it was more membrane-associated in metacyclic promastigotes, whereas in procyclic promastigotes it was scattered throughout the cytoplasm. This is the first demonstration of a PTPase present in Leishmania major promastigotes that differs in expression, activity and ultrastructural localization between the procyclic and metacyclic stages of the parasite's life-cycle.
Protein tyrosine phosphatases (PTPases) have been described as virulence factors in different pathogenic microorganisms. The pathogenic process by Entamoeba histolytica is a multifactorial phenomenon that occurs in 3 steps: adhesion, cytolytic and cytotoxic effect, and phagocytosis. Lytic enzymes may participate during the second part of this process. In this work, we determined that purified membrane-bound acid phosphatase (MAP) from E. histolytica trophozoites has PTPase activity. The enzyme specifically dephosphorylated O-phospho-L-tyrosine at optimum pH of 5·0, with little activity towards O-phospho-L-serine, O-phospho-L-threonine, and ATP. It was inhibited by ammonium molybdate and sodium tungstate, and trifluoperazine did not show any effect. A monoclonal antibody against the catalytic domain of the human placental PTPase 1B, cross-reacted with a 55 kDa molecule present in the solubilized fraction. The interaction of the amoebic PTPase with HeLa cells resulted in the alteration of the cell actin cytoskeleton by disruption of the actin stress fibres.
Little is known about the carcass characteristics of unimproved commercial (or ‘Criollo’) cattle although these still account for a large proportion of slaughter animals in Latin America.
Data were collected from 52 commercial cattle in a commercial slaughterhouse in Monterrey, N.L. Mexico. They included carcass weight, length, total edible meat (carcass fat was negligible), total bone and eleven typical Mexican cuts whose anatomical relations are described. The age of the animals was judged according to dentition and data were stratified using this criterion for analysis. Variations in total edible meat, which averaged 77·4%, were almost entirely accounted for by differences in body weight. Data are presented to indicate amounts of various cuts in the carcasses and some significant differences were demonstrated in values for these cuts among age groups; possible explanations for this were discussed. Prediction equations were calculated to relate total edible meat with the selected cuts and carcass weight and it was found that carcass weight gave the best prediction of total edible meat and could also be used to predict expected weights of individual joints with reasonable accuracy.
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