ATP hydrolysis is required for DNA cleavage by EcoPI restriction enzyme

The type III restriction endonuclease EcoPI, coded by bacteriophage Fl, cleaves unmodified DNA in the presence of ATP and magnesium ions. We show that purified EcoPI restriction enzyme fails to cleave DNA in the presence of non-hydrolyzable ATP analogs. More importantly, this study demonstrates that EcoPI restriction enzyme has an inherent ATPase activity, and ATP hydrolysis is necessary for DNA cleavage. Furthermore, we show that the progress curve of the reaction with Eco PI restriction enzyme exhibits a lag which is dependent on the enzyme concentration. Kinetic analysis of the progress curves of the reaction suggest slow transitions that can occur during the reaction, characteristic of hysteretic enzymes. The role of ATP in the cleavage mechanism of type III restriction enzymes is discussed.

Machine learning modelling for predicting soil liquefaction susceptibility

This study describes two machine learning techniques applied to predict liquefaction susceptibility of soil based on the standard penetration test (SPT) data from the 1999 Chi-Chi, Taiwan earthquake. The first machine learning technique which uses Artificial Neural Network (ANN) based on multi-layer perceptions (MLP) that are trained with Levenberg-Marquardt backpropagation algorithm. The second machine learning technique uses the Support Vector machine (SVM) that is firmly based on the theory of statistical learning theory, uses classification technique. ANN and SVM have been developed to predict liquefaction susceptibility using corrected SPT (N-1)(60)] and cyclic stress ratio (CSR). Further, an attempt has been made to simplify the models, requiring only the two parameters (N-1)(60) and peck ground acceleration (a(max)/g)], for the prediction of liquefaction susceptibility. The developed ANN and SVM models have also been applied to different case histories available globally. The paper also highlights the capability of the SVM over the ANN models.

Engineered dimer interface mutants of triosephosphate isomerase: the role of inter-subunit interactions in enzyme function and stability

The role of inter-subunit interactions in maintaining optimal catalytic activity in triosephosphate isomerase (TIM) has been probed, using the Plasmodium falciparum enzyme as a model. Examination of subunit interface contacts in the crystal structures suggests that residue 75 (Thr, conserved) and residue 13 (Cys, variable) make the largest number of inter-subunit contacts. The mutants Cys13Asp (C13D) and Cys13Glu (C13E) have been constructed and display significant reduction in catalytic activity when compared with wild-type (WT) enzyme (similar to 7.4-fold decrease in k(cat) for the C13D and similar to 3.3-fold for the C13E mutants). Analytical gel filtration demonstrates that the C13D mutant dissociates at concentrations < 1.25 mu M, whereas the WT and the C13E enzymes retain the dimeric structure. The order of stability of the mutants in the presence of chemical denaturants, like urea and guanidium chloride, is WT > Cys13Glu > Cys13Asp. Irreversible thermal precipitation temperatures follow the same order as well. Modeling studies establish that the Cys13Asp mutation is likely to cause a significantly greater structural perturbation than Cys13Glu. Analysis of sequence and structural data for TIMs from diverse sources suggests that residues 13 and 82 form a pair of proximal sites, in which a limited number of residue pairs may be accommodated.

Permeability and Compressibility Behavior of Bentonite-Sand/Soil Mixes

As a seepage barrier slurry trench material should have a relatively low coefficient of permeability, in the range of 10(-7) cm/s, and at the same time should be compatible with surrounding material with regard to compressibility. Although bentonite-sand/soil mixes are used widely, there is no specific engineering approach to proportion these mixes that satisfies the above practical requirements. In this paper, a generalized approach is presented for predicting the permeability and compressibility characteristics of mixes with minimum input parameters. This approach will be helpful in proportioning mixes and predicting corresponding changes in engineering behavior. It is possible to proportion a mix to arrive at the required compressibility without affecting the permeability. This is explained using an illustrative example.

Characteristic water contents of a fine-grained soil-water system

This paper critically appraises the limitations of the liquid-limit water content of clayey soils determined conventionally either by percussion cup or by the cone penetration method. It is shown that the conventional liquid limit and plastic limit are arbitrary, strength-based water contents and that they cannot represent the plasticity limits, and that the state of the soil-water system at the conventional liquid limit does not correspond to a stress-free reference state. The present investigation identifies three characteristic limiting water contents for a soil-water system which have well-defined, unique mechanisms controlling them, namely the free swell limit, settling limit and shrinkage limit. Simple procedures for the determination of the free swell limit and settling limit of natural soils are presented. The settling limit is shown to be the 'real liquid limit' of any clayey soil. With a number of experimental illustrations, it is clearly shown that the settling limit represents the maximum water-holding capacity of clayey soils and that it corresponds to the stress-free reference state.

Mutations in the Res subunit of the EcoPI restriction enzyme that affect ATP-dependent reactions

The Res subunits of the type III restriction-modification enzymes share a statistically significant amino acid sequence similarity with several RNA and DNA helicases of the so-called DEAD family. It was postulated that in type III restriction enzymes a DNA helicase activity may be required for local unwinding at the cleavage site. The members of this family share seven conserved motifs, all of which are found in the Res subunit of the type III restriction enzymes. To determine the contribution, if any, of these motifs in DNA cleavage by EcoPI, a type III restriction enzyme, we have made changes in motifs I and II. While mutations in motif I (GTGKT) clearly affected ATP hydrolysis and resulted in loss of DNA cleavage activity, mutation in motif II (DEPH) significantly decreased ATP hydrolysis but had no effect on DNA cleavage. The double mutant R.EcoPIK90R-H229K showed no significant ATPase or DNA restriction activity though ATP binding was not affected. These results imply that there are at least two ATPase reaction centres in EcoPI restriction enzyme. Motif I appears to be involved in coupling DNA restriction to ATP hydrolysis. Our results indicate that EcoPI restriction enzyme does not have a strand separation activity. We suggest that these motifs play a role in the ATP-dependent translocation that has been proposed to occur in the type III restriction enzymes. (C) 1997 Academic Press Limited.

Relative performances of textural models in estimating soil moisture characteristic

The soil moisture characteristic (SMC) forms an important input to mathematical models of water and solute transport in the unsaturated-soil zone. Owing to their simplicity and ease of use, texture-based regression models are commonly used to estimate the SMC from basic soil properties. In this study, the performances of six such regression models were evaluated on three soils. Moisture characteristics generated by the regression models were statistically compared with the characteristics developed independently from laboratory and in-situ retention data of the soil profiles. Results of the statistical performance evaluation, while providing useful information on the errors involved in estimating the SMC, also highlighted the importance of the nature of the data set underlying the regression models. Among the models evaluated, the one possessing an underlying data set of in-situ measurements was found to be the best estimator of the in-situ SMC for all the soils. Considerable errors arose when a textural model based on laboratory data was used to estimate the field retention characteristics of unsaturated soils.

Comparison of electrical resistivity by geophysical method and neutron probe logging for soil moisture monitoring in a forested watershed

Geophysical methods are becoming more popular nowadays in the field of hydrology due to their time and space efficiency. So an attempt has been made here to relate electrical resistivity with soil moisture content in the field. The experiments were carried out in an experimental watershed `Mulehole' in southern India, which is a forested watershed with approximately 80% red soil. Five auger holes were drilled to perform the soil moisture and electrical resistivity measurements in a toposequence having red and black soils, with sandy weathered soil at the bottom. Soil moisture was measured using neutron probe and electrical resistivity was measured using electrical logging tool. The results indicate that electrical resistivity measurements can be used to measure soil moisture content for red soils only.

Triaxial A-value versus swell or collapse for compacted soil - Discussion

A discussion of a technical note with the aforementioned title by Day and Marsh, published in this journal (Volume 121, Number 7, July 1995), is presented. Discussers Robinson and Allam assert that the authors' application of the pore-pressure parameter A to predict and quantify swell or collapse of compacted soils is hard to use because the authors visualize the collapse-swell phenomenon to occur in compacted soils broadly classified as sands and clays. The literature demonstrates that mineralogy has an important role in the volume change behavior of fine-grained soils. Robinson and Allam state that the A-value measurements may not completely predict the type of volume change anticipated in compacted soils on soaking without soil clay mineralogy details. Discussion is followed by closure from the authors.

California bearing ratio behavior of cement-stabilized fly ash-soil mixes

Thermal power stations using pulverized coal as fuel generate large quantities of fly ash as a byproduct, which has created environmental and disposal problems. Using fly ash for gainful applications will solve these problems. Among the various possible uses for fly ash, the most massive and effective utilization is in geotechnical engineering applications like backfill material, construction of embankments, as a subbase material, etc. A proper understanding of fly ash-soil mixes is likely to provide viable solutions for its large-scale utilization. Earlier studies initiated in the laboratory have resulted in a good understanding of the California Bearing Ratio (CBR) behavior of fly ash-soil mixes. Subsequently, in order to increase the CBR value, cement has been tried as an additive to fly ash-soil mixes. This paper reports the results.

Collapse behaviour of a residual soil

Residually derived red soils occur in Bangalore District of Karnataka State, India. The porous and unsaturated nature of the red soils makes them susceptible to collapse on wetting under load. The present study analyses the collapse behaviour of an unsaturated bonded (undisturbed) red soil from Bangalore referenced to tests on samples in an unbonded (remoulded) state. A filter paper method was used to determine the matric suction of the bonded and unbonded specimens, and mercury intrusion porosimetry (MIP) was used to determine their soil structure. Analysis of the experimental results shows that bonding plays an important role in the collapse behaviour of the unsaturated residual soil. The results of the study also provide insight into the volume change behaviour of unsaturated bonded soils on wetting within and beyond the yield locus.

Effect of peptide-based captopril analogues on angiotensin converting enzyme activity and peroxynitrite-mediated tyrosine nitration

Angiotensin converting enzyme (ACE) regulates the blood pressure by converting angiotensin I to angiotensin II and bradykinin to bradykinin 1-7. These two reactions elevate the blood pressure as angiotensin II and bradykinin are vasoconstrictory and vasodilatory hormones, respectively. Therefore, inhibition of ACE is an important strategy for the treatment of hypertension. The natural substrates of ACE, i.e., angiotensin II and bradykinin, contain a Pro-Phe motif near the site of hydrolysis. Therefore, there may be a Pro-Phe binding pocket at the active site of ACE, which may facilitate the substrate binding. In view of this, we have synthesized a series of thiol-and selenol-containing dipeptides and captopril analogues and studied their ACE inhibition activities. This study reveals that both the selenol or thiol moiety and proline residues are essential for ACE inhibition. Although the introduction of a Phe residue to captopril and its selenium analogue considerably reduces the inhibitory effect, there appears to be a Phe binding pocket at the active site of ACE.

Thermodynamic analysis of enzyme catalysed reactions: new insights into the Michaelis-Menten equation

A simple thermodynamic analysis of the well-known Michaelis-Menten equation (MME) of enzyme catalysis is proposed that employs the chemical potential mu to follow the Gibbs free energy changes attending the formation of the enzyme-substrate complex and its turnover to the product. The main conclusion from the above analysis is that low values of the Michaelis constant KM and high values of the turnover number k(cat) are advantageous: this supports a simple algebraic analysis of the MME, although at variance with current thinking. Available data apparently support the above findings. It is argued that transition state stabilisation - rather than substrate distortion or proximity - is the key to enzyme catalysis.

Reliability Based Design Optimization for Internal Stability of Reinforced Soil Structures Using Pseudo‐Static Method

Seismic design of reinforced soil structures involves many uncertainties that arise from the backfill soil properties and tensile strength of the reinforcement which is not addressed in current design guidelines. This paper highlights the significance of variability in the internal stability assessment of reinforced soil structures. Reliability analysis is applied to estimate probability of failure and pseudo‐static approach has been used for the calculation of the tensile strength and length of the reinforcement needed to maintain the internal stability against tension and pullout failures. Logarithmic spiral failure surface has been considered in conjunction with the limit equilibrium method. Two modes of failure namely, tension failure and pullout failure have been considered. The influence of variations of the backfill soil friction angle, the tensile strength of reinforcement, horizontal seismic acceleration on the reliability index against tension failure and pullout failure of reinforced earth structure have been discussed.