ELECTROPHORETIC CHARACTERIZATION OF BASIC PROTEINS IN ACID EXTRACTS OF CENTRAL NERVOUS SYSTEM TISSUE

A technique has been outlined for identification of myelin basic proteins in mixtures of CNS proteins. Myelin basic proteins can be recognized easily by high cathodic mobility at low pH, a unique electrophoretic pattern exhibited at high pH and a characteristic colour when complexed with Amido black. The major protein extracted at pH 3·0 from either brain or spinal cord is myelin basic protein. In the low pH electrophoretic pattern of these extracts it is the most conspicuous component and the component migrating farthest cathodically; it does not appear in comparable electrophoretic patterns of liver extracts. Guinea pig myelin basic protein appears as a single dense blue-green band in low pH electrophoretic patterns, in contrast to the other proteins which are stained greyish-blue or greyish-purple by Amido black. The pattern of rat myelin basic protein is similar except that it consists of a pair of dense blue-green bands. A third characteristic which facilitates the identification of myelin basic proteins in mixtures is a considerable cathodic mobility and electrophoretic heterogeneity at pH 10·6. Most other basic CNS proteins barely penetrate the gel at this pH. We have also examined in detail the behaviour of two other components of pH 3·0 extracts which migrate close to myelin basic protein at low pH. Both are present in pH 3·0 extracts of liver and brain but not of spinal cord, and both stain grey instead of blue-green, a characteristic which readily distinguishes them from myelin basic protein. Neither of these components affects the characteristic pattern of microheterogeneity observed in high pH electrophoretograms of myelin basic proteins. One of these components has been purified and tentatively identified as lysine-rich histone F1.     

Proteomic analysis identifies in vivo candidate matrix metalloproteinase‐9 substrates in the left ventricle post‐myocardial infarction

Matrix metalloproteinase‐9 (MMP‐9) deletion has been shown to improve remodeling of the left ventricle post‐myocardial infarction (MI), but the mechanisms to explain this improvement have not been fully elucidated. MMP‐9 has a broad range of in vitro substrates, but relevant in vivo substrates are incompletely defined. Accordingly, we evaluated the infarct regions of wild‐type (wt) and MMP‐9 null (null) mice using a proteomic strategy. Wt and null groups showed similar infarct sizes (48±3 in wt and 45±3% in null), indicating that both groups received an equal injury stimulus. Left ventricle infarct tissue was homogenized and analyzed by 2‐DE and MS. Of 31 spot intensity differences, the intensities of 9 spots were higher and 22 spots were lower in null mice compared to wt (all p<0.05). Several extracellular matrix proteins were identified in these spots by MS, including fibronectin, tenascin‐C, thrombospondin‐1, and laminin. Fibronectin was observed on the gels at a lower than expected molecular weight in the wt group, which suggested substrate cleavage, and the lower molecular weight spot was observed at lower intensity in the MMP‐9 null group, which suggested cleavage by MMP‐9. Immunoblotting confirmed the presence of fibronectin cleavage products in the wt samples and lower levels in the absence of MMP‐9. In conclusion, examining infarct tissue from wt and MMP‐9 null mice by proteomic analysis provides a powerful and unique method to identify in vivo candidate MMP substrates.     

Geographical distribution of schistosomiasis and soil-transmitted helminths among school children in informal settlements in Kisumu City, Western Kenya

This cross-sectional study determined the prevalence and distribution of schistosome and soil-transmitted helminth (STH) infections among 1,308 children aged 10-18 years in 34 primary schools in 8 informal urban settlements in Kisumu City, western Kenya. Stool samples were collected and examined for eggs of Schistosoma mansoni and STH (Hookworms, Ascaris lumbricoides and Trichuris trichiura) using the Kato-Katz technique. Haematuria was used as a proxy indicator of urinary schistosomiasis. Schools and water bodies were mapped using a geographical information system. Overall, 34% of children were infected with one or more helminth species whereas 16·2% of children were infected with one or more STH species. Schools in closest proximity to Lake Victoria and River Nyamasaria had the highest S. mansoni prevalence while schools with STH were more homogenously distributed. Mean school prevalence of S. mansoni infection was 21% (range=0-69·7%), S. haematobium 3·6% (range=0-12%), hookworms 6·1% (range=0-20%), A. lumbricoides 4·9% (range=0-18·4%), and T. trichiura 7·7% (range=0-18·6%). Helminth-related morbidities were not associated with infection. Our study demonstrates that schistosomiasis and STH are important health priorities among schools in informal settlements of Kisumu City, and highlights the need for routine deworming in similar settings.     

Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues

Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.     

The coiled-coil domain of Escherichia coli FtsLB is a structurally detuned element critical for modulating its activation in bacterial cell division

The FtsLB complex is a key regulator of bacterial cell division, existing in either an off state or an on state, which supports the activation of septal peptidoglycan synthesis. In Escherichia coli, residues known to be critical for this activation are located in a region near the C-terminal end of the periplasmic coiled-coil domain of FtsLB, raising questions about the precise role of this conserved domain in the activation mechanism. Here, we investigate an unusual cluster of polar amino acids found within the core of the FtsLB coiled coil. We hypothesized that these amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is a “detuned” structural element. In addition, we identified suppressor mutations within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. We propose a revised structural model of the tetrameric FtsLB (named the “Y-model”) in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the hydrophilic terminal moieties of the polar amino acids remain more favorably exposed to water than in the original four-helix bundle model (“I-model”). We propose that a shift in this architecture, dependent on its marginal stability, is involved in activating the FtsLB complex and triggering septal cell wall reconstruction.     

Effect of Poor Access to Water and Sanitation As Risk Factors for Soil-Transmitted Helminth Infection: Selectiveness by the Infective Route

BackgroundSoil-transmitted helminth (STH) infections are a public health problem in resource-limited settings worldwide. Chronic STH infection impairs optimum learning and productivity, contributing to the perpetuation of the poverty-disease cycle. Regular massive drug administration (MDA) is the cardinal recommendation for its control; along with water, sanitation and hygiene (WASH) interventions. The impact of joint WASH interventions on STH infections has been reported; studies on the independent effect of WASH components are needed to contribute with the improvement of current recommendations for the control of STH. The aim of this study is to assess the association of lacking access to water and sanitation with STH infections, taking into account the differences in route of infection among species and the availability of adequate water and sanitation at home.ConclusionsLack of safe water and proper sanitation pose a risk of STH infections that is distinct according to the route of entry to the human host used by each of the STH species. Interventions aimed to improve water and sanitation access should be highlighted in the recommendations for the control of STH.     

In vitro antimicrobial effect of metallic nanoparticles on phytopathogenic strains of crop plants

In this research, the in vitro antimicrobial effect of zinc oxide (ZnO), copper oxide (CuO) and iron oxide (Fe₂O₃) nanoparticles (NPs)—with average sizes of 20, 46 and 30 nm, respectively—on the root rot disease caused by the fungus Fusarium oxysporum and on blight disease caused by the fungus Alternaria solani were studied. Also, bacterial diseases caused by Clavibacter michiganensis and Pseudomonas syringae that infects a wide range of plant species were assessed. Different concentrations of NPs (0, 100, 250, 500, 700 and 1,000 mg/L) were prepared on PDA agar or King's B medium in a complete randomized design with four replicates. According to the results, ZnO NPs exhibited an outstanding inhibitory effect against fungi and bacteria strains. The above results were associated with the smaller particle size. Fungi strains showed a differential sensitivity depending on the kind of NPs used. A. solani showed the highest sensitivity to ZnO NPs at 1,000 mg/L (99%), followed by CuO NPs at the same dose (95%). Fe₂O₃ NPs at all evaluated doses had no inhibitory effects on the mycelia growth of this strain, although F. oxysporum revealed greater effectiveness of the CuO NPs (96%) compared with ZnO NPs since it only inhibited 91% of the mycelial growth. The antibacterial activity was studied through optical density. C. michiganensis was found to be more sensitive to ZnO NPs because a lesser dose (700 mg/L) was required to reduce the bacterial growth (90%); in comparison, P. syringae required a dose of 1,000 mg/L to inhibit its growth (67%). CuO NPs displayed the smallest growth inhibition against the bacteria strains analysed. The antimicrobial effect of the metallic NPs that were assayed increased with higher doses.     

Isolation and Characterization of Thermophilic Bacterial Strains with Inulinase Activity

Nine bacterial strains growing on inulin as the sole carbon and energy source were isolated from soil samples by enrichment culture on a mineral medium. Four of the strains were thermophilic and belong to the genus Bacillus. The thermophilic strains synthesized a β-fructosidase that was active on both inulin and sucrose. The presence of inulin in the culture medium is necessary for enzyme synthesis. Most of the activity on inulin was recovered in the culture medium, and the enzyme was synthesized during cell growth.