Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

Identification of soil quality indicators for assessing the effect of different tillage practices through a soil quality index in a semi-arid environment

Tillage is known to potentially affect soil quality in various ways. In this study, a soil quality index (SQI) was developed by quantifying several soil attributes either sensitive or insensitive to physical disturbance, using factor analysis as a dimension reduction technique, in order to discriminate different tillage systems. Soil properties including physical (MWD), chemical (pH, organic C, total N, available P and POM contents) and microbial (MBC, MBN, PCM, PNM and three enzymes) parameters were measured to establish a minimum data set (MDS) for the assessment of overall SQI. The soil attributes were determined on samples (0–20 cm depth) collected under moldboard (MP) and disk (DP) plows as conventional tillage (CT), and rotary (RP) and chisel (CP) plows as reduced tillage (RT) systems with a similar plant C input rate and cover crop over a period of six years (2005–2011) in a semi-arid calcareous soil (Calcixerepts) from Central Iran. Results indicated a clear difference in soil quality among the tillage systems with a significant increase of SQI under RT over time, particularly under CP practices. Although RT improved most soil microbial attributes, not all attributes contributed to SQI because of their close interrelationship. The final SQI consisted only of geometric mean of microbial activity (GMA, the square root of the product of PCM and PNM) and geometric mean of enzyme activity (GME, the cube root of the product of enzyme activities). Soil GME and GMA were found to be as key indicators contributing 55% and 36% to SQI, respectively. Therefore, the GME and GMA were the most important indicators effectively discriminating tillage systems, and could be used to monitor the enhancement of soil quality under RT in this semiarid environment. The influence of tillage year on SQI was greater than that of tillage practices. In conclusion, RT systems were characterized by a higher value of SQI, suggesting a good recovery of soil capacity and functions after abandoning CT in the studied area. Smallholder farmers should therefore be aware of the potential for high soil quality in future as a result of continuing RT systems, especially with surface tillage using CP practices.     

A genetic and spatial Bayesian analysis of mastitis resistance

A nationwide health card recording system for dairy cattle was introduced in Norway in 1975 (the Norwegian Cattle Health Services). The data base holds information on mastitis occurrences on an individual cow basis. A reduction in mastitis frequency across the population is desired, and for this purpose risk factors are investigated. In this paper a Bayesian proportional hazards model is used for modelling the time to first veterinary treatment of clinical mastitis, including both genetic and environmental covariates. Sire effects were modelled as shared random components, and veterinary district was included as an environmental effect with prior spatial smoothing. A non-informative smoothing prior was assumed for the baseline hazard, and Markov chain Monte Carlo methods (MCMC) were used for inference. We propose a new measure of quality for sires, in terms of their posterior probability of being among the, say 10% best sires. The probability is an easily interpretable measure that can be directly used to rank sires. Estimating these complex probabilities is straightforward in an MCMC setting. The results indicate considerable differences between sires with regards to their daughters disease resistance. A regional effect was also discovered with the lowest risk of disease in the south-eastern parts of Norway.     

Uncertainty and sensitivity analysis of West Java Water Sustainability Index – A case study on Citarum catchment in Indonesia

Water sustainability indices have been recently used to measure the sustainability of water resources within a catchment. Developing a sustainability index involves various steps, some of which have uncertainties associated with them. For the recently developed West Java Water Sustainability Index (WJWSI), three sources of uncertainties were identified, namely uncertainties in the thresholds of non-categorical indicators and sub-indicators, in the weighting schemes, and in the aggregation methods. This paper presents the uncertainty and sensitivity analysis of WJWSI, based on the application of WJWSI to Citarum catchment in West Java, Indonesia. The results of the uncertainty analysis, measured by the coefficient of variation of the thresholds and the sub-indices, indicates that minimum thresholds of Land Use Changes, Coverage, Education, Poverty, Health Impact and Sanitation, and the maximum threshold of Water Quality have higher variation when compared to variation of the other thresholds. The results of the sensitivity analysis, measured by the correlation coefficients between the final index and the thresholds, indicate that changes in the thresholds of WJWSI indicators have not significantly affected the sub-index values of most indicators and sub-indicators. The sensitivity analysis also concluded that either the equal or non-equal weighting scheme can be used for future use of the aggregation of WJWSI indicators and sub-indicators, as changes from equal to non-equal weighting scheme did not significantly affect the final index. However, it was found that the final index values were most sensitive to the aggregation method used (i.e. arithmetic and geometric methods), shown by the significant changes in the final index value when the aggregation method was changed from arithmetic to geometric. The uncertainty and sensitivity analysis presented in this study will not just assist in the efficient use of the WJWSI, but will also help undertake similar analysis for other indices.     

A novel galactosyl-binding lectin from the plasma of the blood clam,Anadara granosa (L) and a study of its combining site

Control Analysis has been carried out in the first steps of a rat liver glycolytic system. Attention has been focused on the effect of several glucose concentrations on the control, particularly regarding the role of glucokinase. From kinetic studies of the whole metabolic system we have obtained information on the flux variation under different glucose concentrations. This information together with the kinetics of glucokinase has allowed us to calculate Flux Control and Elasticity Coefficients for glucokinase and the Response Coefficient of the system with respect to glucose. The changes in of the value of Flux Control Coefficients demonstrates that in conditions of low glucose concentration, glucokinase is the main enzyme in controlling the flux through the pathway, but at high glucose concentration the control moves to phosphofructokinase. Next, we have compared our results with those obtained with the shortening and titration method, previously described (Torres, N.V., Mateo, F., Mélendez-Hevia, E. and Kacser, H., (1986) Biochem. J. 234, 169–174; Torres, N.V. and Meléndez-Hevia, E. 1991. Molec. Cell. Biochem. 101, 1–10). Furthermore, from knowledge of the enzyme kinetics of the system we have been able to build a model of the pathway that allows us computer similation of its behavior and calculation of the Flux Control Coefficient profile at different glucose concentrations. By the three methods the results correlate, supporting the use of the pathway substrate as external modulator of the metabolic system as a tool for practical application of Control Analysis.     

Bird sensitivity to disturbance as an indicator of forest patch conditions: An issue in environmental assessments

An Environmental Assessment (EA) is one of the steps within the Environmental Impact Assessment process. Birds are often used in EA to help decision makers evaluate potential human impacts from proposed development activities. A “sensitivity to human disturbance” index, created by Parker III et al. (1996) for all Neotropical species, is commonly considered an ecological indicator. However, this parameter was created subjectively and, for most species, there have been no rigorous field test to validate its effectiveness as such. Therefore, in this study, we aim to: (1) evaluate if, at the local scale, birds from forest patches in a human-modified landscape (HML) may differ in sensitivity from Parker's sensitivity classification; (2) evaluate the effectiveness of the species richness value at each sensitivity level as an ecological indicator; (3) gather information on how often and in which manner Parker's classification has been used in EA. To do so, bird sampling was performed in eight forest patches in a HML over one year. Then, we created a local sensitivity to disturbance using information about threat, endemism, spatial distribution and relative abundance of all species in the study area. We found that 37% of the forest birds showed different local sensitivity levels when compared with Parker's classification. Our results show that only the richness of high-sensitivity species from our local classification fitted the ecological indicator assumptions helping the environmental conditions evaluation of the studied patches. We conclude that species richness of each Parker's bird sensitivity levels do not necessarily perform as an ecological indicator at the local scale, and particularly in HML. Nevertheless, Parker's Neotropical bird sensitivity classification was used in 50% of EA we reviewed. In these, 76% assumed that it was an accurate ecological indicator of the local forest conditions for birds. The lack of clear criteria used in Parker's classification allows diverse interpretations by ornithologists, and there is no agreement about the ecological meaning of each sensitivity level and what environmental conditions each level may indicate of. Therefore, the use of Parker's classification in EA may jeopardize accurate interpretations of proposed anthropogenic impacts. Furthermore, because a bird species’ sensitivity often varies between locations, we argue that Parker's generalized classification of bird sensitivity should not be used as an indicator of forest environmental conditions in EA throughout HMLs in Neotropics. Rather, local bird ecological indices should be explored, otherwise, erroneous predictions of the anthropogenic impacts will continue to be common.     

A novel approach for assessing factors affecting biodiversity based on networks analysis

We propose in this work a novel approach aiming at assessing cause and effect relationships between variables that can affect target biodiversity issues. These cause–effect relations are used to build a network whose nodes represent variables linked by directed arcs. The arcs have associated a value that represents trends of cause–effect relations. An important novelty of this approach is the use of product and addition operations between trends of cause–effect relations for assessing factors that can affect target variables. For the analysis of the network we use the concept of paths. Paths are defined as sequences of cause–effect relations from source variables to target variables. For example, the path from population increment that causes effects on the increment of transport routes, which in turn causes effects on the loss of vegetation cover. This approach was applied to the assessment of vegetation cover in the Morelos State, México during the period 2000–2010. The results show a promising practical alternative to assess the potential effects on biodiversity issues based on the analysis of the paths represented in the network.     

Effect of thiostrepton and 3′-terminal fragments of aminoacyl-tRNA on EF-Tu and ribosome-dependent GTP hydrolysis

The effect of the antibiotics thiostrepton and micrococcin on EF-T_u-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3′-terminus of aminoacyl-tRNA)(System I) or by methanol (‘uncoupled GTPase’, System II) was investigated. In System I, thiostrepton increases the binding affinities of the effectors to the EF-T_u·GTP·70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the K^GTP_m is virtually unchanged. Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases V^GTP_max, whereas K^GTP_m remains unaffected. Micrococcin is without any effect in both systems. The ‘uncoupled GTPase’ (in System II) is also strongly inhibited by C-A-Phe. The results indicate the crucial role of the EF-T_u site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis. It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-T_u for GTP.     

Discovery of new low-molecular-weight p53–Mdmx disruptors and their anti-cancer activities

Although several p53–Mdm2-binding disruptors have been identified to date, few studies have been published on p53–Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200–300 selectively inhibited the p53–Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53–Mdmx interaction over the p53–Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53–Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.