Locus Bonus : The Relationship of the Roman Villa to its Environment in the Vicinity of Rome

The aim of this study is to examine the relationship of the Roman villa to its environment. The villa was an important feature of the countryside intended both for agricultural production and for leisure. Manuals of Roman agriculture give instructions on how to select a location for an estate. The ideal location was a moderate slope facing east or south in a healthy area and good neighborhood, near good water resources and fertile soils. A road or a navigable river or the sea was needed for transportation of produce. A market for selling the produce, a town or a village, should have been nearby. The research area is the surroundings of the city of Rome, a key area for the development of the villa. The materials used consist of archaeological settlement sites, literary and epigraphical evidence as well as environmental data. The sites include all settlement sites from the 7th century BC to 5th century AD to examine changes in the tradition of site selection. Geographical Information Systems were used to analyze the data. Six aspects of location were examined: geology, soils, water resources, terrain, visibility/viewability and relationship to roads and habitation centers. Geology was important for finding building materials and the large villas from the 2nd century BC onwards are close to sources of building stones. Fertile soils were sought even in the period of the densest settlement. The area is rich in water, both rainfall and groundwater, and finding a water supply was fairly easy. A certain kind of terrain was sought over very long periods: a small spur or ridge shoulder facing preferably south with an open area in front of the site. The most popular villa resorts are located on the slopes visible from almost the entire Roman region. A visible villa served the social and political aspirations of the owner, whereas being in the villa created a sense of privacy. The area has a very dense road network ensuring good connectivity from almost anywhere in the region. The best visibility/viewability, dense settlement and most burials by roads coincide, creating a good neighborhood. The locations featuring the most qualities cover nearly a quarter of the area and more than half of the settlement sites are located in them. The ideal location was based on centuries of practical experience and rationalized by the literary tradition.

Use of semi-active dampers in seismic mitigation of building structures

This research provides information for providing the required seismic mitigation in building structures through the use of semi active and passive dampers. The Magneto-Rheological (MR) semi-active damper model was developed using control algorithms and integrated into seismically excited structures as a time domain function. Linear and nonlinear structure models are evaluated in real time scenarios. Research information can be used for the design and construction of earthquake safe buildings with optimally employed MR dampers and MR-passive damper combinations.

A theoretical model for the magnetic helicity of solar active regions

Active regions on the solar surface are known to possess magnetic helicity, which is predominantly negative in the northern hemisphere and positive in the southern hemisphere. Choudhuri et al. [Choudhuri, A.R. On the connection between mean field dynamo theory and flux tubes. Solar Phys. 215, 31–55, 2003] proposed that the magnetic helicity arises due to the wrapping up of the poloidal field of the convection zone around rising flux tubes which form active regions. Choudhuri [Choudhuri, A.R., Chatterjee, P., Nandy, D. Helicity of solar active regions from a dynamo model. ApJ 615, L57–L60, 2004] used this idea to calculate magnetic helicity from their solar dynamo model. Apart from getting broad agreements with observational data, they also predict that the hemispheric helicity rule may be violated at the beginning of a solar cycle. Chatterjee et al. [Chatterjee, P., Choudhuri, A.R., Petrovay, K. Development of twist in an emerging magnetic flux tube by poloidal field accretion. A&A 449, 781–789, 2006] study the penetration of the wrapped poloidal field into the rising flux tube due to turbulent diffusion using a simple 1-d model. They find that the extent of penetration of the wrapped field will depend on how weak the magnetic field inside the rising flux tube becomes before its emergence. They conclude that more detailed observational data will throw light on the physical conditions of flux tubes just before their emergence to the photosphere.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Structures and molecular-dynamics studies of three active-site mutants of bovine pancreatic phospholipase A(2)

Phospholipase A(2) hydrolyzes phospholipids at the sn-2 position to cleave the fatty-acid ester bond of L-glycerophospholipids. The catalytic dyad (Asp99 and His48) along with a nucleophilic water molecule is responsible for enzyme hydrolysis. Furthermore, the residue Asp49 in the calcium-binding loop is essential for controlling the binding of the calcium ion and the catalytic action of phospholipase A2. To elucidate the structural role of His48 and Asp49, the crystal structures of three active-site single mutants H48N, D49N and D49K have been determined at 1.9 angstrom resolution. Although the catalytically important calcium ion is present in the H48N mutant, the crystal structure shows that proton transfer is not possible from the catalytic water to the mutated residue. In the case of the Asp49 mutants, no calcium ion was found in the active site. However, the tertiary structures of the three active-site mutants are similar to that of the trigonal recombinant enzyme. Molecular-dynamics simulation studies provide a good explanation for the crystallographic results.

Amino acid sequence of the winged bean (Psophocarpus tetragonolobus) basic lectin. Adenine binding and identification of the active-site tryptophan residue

The complete amino acid sequence of winged bean basic agglutinin (WBA I) was obtained by a combination of manual and gas-phase sequencing methods. Peptide fragments for sequence analyses were obtained by enzymatic cleavages using trypsin and Staphylococcus aureus V8 endoproteinase and by chemical cleavages using iodosobenzoic acid, hydroxylamine, and formic acid. COOH-terminal sequence analysis of WBA I and other peptides was performed using carboxypeptidase Y. The primary structure of WBA I was homologous to those of other legume lectins and more so to Erythrina corallodendron. Interestingly, the sequence shows remarkable identities in the regions involved in the association of the two monomers of E. corallodendron lectin. Other conserved regions are the double metal-binding site and residues contributing to the formation of the hydrophobic cavity and the carbohydrate-binding site. Chemical modification studies both in the presence and absence of N-acetylgalactosamine together with sequence analyses of tryptophan-containing tryptic peptides demonstrate that tryptophan 133 is involved in the binding of carbohydrate ligands by the lectin. The location of tryptophan 133 at the active center of WBA I for the first time subserves to explain a role for one of the most conserved residues in legume lectins.

Cloning,overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from scherichia coli

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively. (C) 2010 Elsevier Inc. All rights reserved.

Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

Sequential Assembly of an Active RNA Polymerase Molecule at the Air-Water Interface

At the heart of understanding cellular processes lies our ability to explore the specific nature of communication between sequential information carrying biopolymers. However, the data extracted from conventional solution phase studies may not reflect the dynamics of communication between recognized partners as they occur in the crowded cellular milieu. We use the principle of immobilization of histidine-tagged biopolymers at a Ni(II)-encoded Langmuir monolayer to study sequence-specific protein-protein interactions in an artificially crowded environment The advantage of this technique lies in increasing the surface density of one of the interacting partners that allows us to study macromolecular interactions in a controlled crowded environment, but without compromising the speed of the reactions. We have taken advantage of this technique to follow the sequential assembly process of the multiprotein complex Escherichia coil RNA polymerase at the interface and also deciphered the role of one of the proteins, omega (omega), in the assembly pathway. Our reconstitution studies indicate that in the absence of molecular chaperones or other cofactors, omega (omega) plays a decisive role in refolding the largest protein beta prime (beta') and its recruitment into the multimeric assembly to reconstitute an active RNA polymerase. It was also observed that the monolayer had the ability to distinguish between sequence-specific and -nonspecific interactions despite the immobilization of one of the biomacromolecules. The technique provides a universal two-dimensional template for studying protein-ligand interactions while mimicking molecular crowding.

Analysis, Design, Modeling, and Implementation of an Active Clamp HF Link Converter

This paper deals with the system oriented analysis, design, modeling, and implementation of active clamp HF link three phase converter. The main advantage of the topology is reduced size, weight, and cost of the isolation transformer. However, violation of basic power conversion rules due to presence of the leakage inductance in the HF transformer causes over voltage stresses across the cycloconverter devices. It makes use of the snubber circuit necessary in such topologies. The conventional RCD snubbers are dissipative in nature and hence inefficient. The efficiency of the system is greatly improved by using regenerative snubber or active clamp circuit. It consists of an active switching device with an anti-parallel diode and one capacitor to absorb the energy stored in the leakage inductance of the isolation transformer and to regenerate the same without affecting circuit performance. The turn on instant and duration of the active device are selected such that it requires simple commutation requirements. The time domain expressions for circuit dynamics, design criteria of the snubber capacitor with two conflicting constrains (over voltage stress across the devices and the resonating current duration), the simulation results based on generalized circuit model and the experimental results based on laboratory prototype are presented.

The orientational relaxation of bipolar active regions

In the mean, bipolar active regions are oriented nearly toroidally, according to Hale's polarity law, with a latitude-dependent tilt known as Joy's Law. The tilt angles of individual active regions deviate from this mean behavior and change over time. It has been found that on average the change is toward the mean angle at a rate characteristic of 4.37 days (Howard, 1996). We show that this orientational relaxation is consistent with the standard model of flux tube emergence from a deep dynamo layer. Under this scenario Joy's law results from the Coriolis effect on the rising flux tube (D'Silva and Choudhuri, 1993), and departures from it result from turbulent buffeting of the tubes (Longcope and Fisher, 1996). We show that relaxation toward Joy's angle occurs because the turbulent perturbations relax on shorter time scales than the perturbations from the Coriolis force. The turbulent perturbations relax more rapidly because they are localized to the topmost portion of the convection zone while the Coriolis perturbations are more widely distributed. If a fully-developed active region remains connected to the strong toroidal magnetic field at the base of the convection zone, its tilt will eventually disappear, leaving it aligned perfectly toroidally. On the other hand, if the flux becomes disconnected from the toroidal field the bipole will assume a tilt indicative of the location of disconnection. We compare models which are connected and disconnected from the toroidal field. Only those disconnected at points very deep in the convection zone a-re consistent with observed time scale of orientational relaxation.

Fluorescence quenching of biologically active carboxamide by aniline and carbon tetrachloride in different solvents using Stern-Volmer plots

Fluorescence quenching of biologically active carboxamide namely (E)-2-(4-chlorobenzylideneamino)-N-(2-chlorophenyl)-4,5,6,7-tetrahydrobe nzo[b]thiophene-3-carboxamide [ECNCTTC] by aniline and carbon tetrachloride (CCl(4)) quenchers in different solvents using steady state method and time resolved method using only one solvent has been carried out at room temperature to understand the role of quenching mechanisms. The Stern-Volmer plot has been found to be linear for all the solvents studied. The probability of quenching per encounter p (p') was determined in all the solvents and was found to be less than unity. Further, from the studies of rate parameters and life time measurements in n-heptane and cyclohexane with aniline and carbon tetrachloride as quenchers have been shown that, the phenomenon of quenching is generally governed by the well-known Stern-Volmer (S-V) plot. The activation energy E(a) (or E(a)') of quenching was determined using the literature values of activation energy of diffusion E(d) and the experimentally determined values of p (or p'). It has been found that, the activation energy E(a) (E(a)') is greater than the activation energy for diffusion E(d) in all solvents. Hence, from the magnitudes of E(a) (or E(a)') as well as p (or p') infer that, the quenching mechanism is not solely due to the material diffusion, but there is also contribution from the activation energy. (C) 2011 Elsevier B.V. All rights reserved.

Solvent effect on the fluorescence quenching of biologically active carboxamide by aniline and carbon tetrachloride in different solvents using S-V plots

The fluorescence quenching studies of carboxamide namely (E)-N-(3-Chlorophenyl)-2-(3,4,5-trimethoxybenzylideneamino)-4,5,6,7 tetrahydrobenzob]thiophene-3-carboxamide ENCTTTC] by aniline and carbon tetrachloride in six different solvents namely toluene, cyclohexane, n-hexane, n-heptane, n-decane and n-pentane have been carried out at room temperature with a view to understand the quenching mechanisms. The Stern-Volmer (S-V) plots have been found to be nonlinear with a positive deviation for all the solvents studied. In order to interpret these results we have invoked the ground state complex formation and sphere of action static quenching models. Using these models various quenching rate parameters have been determined. The magnitudes of these parameters suggest that sphere of action static quenching model agrees well with the experimental results. Hence the positive deviation is attributed to the static and dynamic quenching. Further, with the use of Finite Sink approximation model, it was possible to check these bimolecular reactions as diffusion-limited and to estimate independently distance parameter R' and mutual diffusion coefficient D. Finally an effort has been made to correlate the values of R' and D with the values of the encounter distance R and the mutual coefficient D determined using the Edward's empirical relation and Stokes Einstein relation. (C) 2011 Elsevier B.V. All rights reserved.