DNA polymerase alpha-primase complex from the silk glands of the non-mulberry silkworm Philosamia ricini.

The DNA content in the silk glands of the non-mulberry silkworm Philosamia ricini increases continuously during the fourth and fifth instars of larval development indicating high levels of DNA replication in this terminally differentiated tissue. Concomitantly, the DNA polymerase alpha activity also increases in the middle and the posterior silk glands during development, reaching maximal levels in the middle of the fifth larval instar. A comparable level of DNA polymerase delta/epsilon was also observed in this highly replicative tissue. The DNA polymerase alpha-primase complex from the silk glands of P. ricini has been purified to homogeneity by conventional column chromatography as well as by immunoaffinity techniques. The molecular mass of the native enzyme is 560 kDa and the enzyme comprises six non-identical subunits. The identity of the enzyme as DNA polymerase alpha has been established by its sensitivity to inhibitors such as aphidicolin, N-ethylmaleimide, butylphenyl-dGTP, butylanilino-dATP and antibodies to polymerase alpha. The enzyme possesses primase activity capable of initiating DNA synthesis on single-stranded DNA templates. The tight association of polymerase and primase activities at a constant ratio of 6:1 is observed through all the purification steps. The 180 kDa subunit harbours the polymerase activity, while the primase activity is associated with the 45 kDa subunit.

Simultaneous purification of biotin-binding proteins-I and -II from chicken egg yolk and their characterization

Chicken egg yolk biotin-binding protein-I (BBP-I) has been purified to homogeneity along with the tetrameric BBP-II by a common protocol. The purification includes delipidation of egg yolk by butanol extraction, DEAE-Sephacel chromatography, treatment with guanidinium chloride and biotin-aminohexyl-Sepharose affinity chromatography. The identity of purified BBP-I was ascertained by its physicochemical properties as well as by its immunological cross-reactivity and precursor-product relationship with BBP-II.

Affinity-Chromatographic Procedure For The Purification Of The Enzyme From Mung-Bean (Phaseolus, Aureus) Seeds And Conformational-Changes On Its Interaction With Ortho-Phosphate

Flavokinase was purified, for the first time from a plant source [mung bean (Phaseolus aureus)] by affinity chromatography in the presence of orthophosphate and by using C-8 ATP-agarose (ATP linked through the C-8 position to beaded agarose), Cibacron Blue and riboflavin--Sepharoses. An altered substrates-saturation pattern was observed in the presence of K2HPO4. The conformational changes of the enzyme in the presence of K2HPO4 were monitored by fluorescence spectroscopy. These results highlight the regulatory nature of this enzyme.

Probabilistic Shared Cache Management (PriSM)

Effective sharing of the last level cache has a significant influence on the overall performance of a multicore system. We observe that existing solutions control cache occupancy at a coarser granularity, do not scale well to large core counts and in some cases lack the flexibility to support a variety of performance goals. In this paper, we propose Probabilistic Shared Cache Management (PriSM), a framework to manage the cache occupancy of different cores at cache block granularity by controlling their eviction probabilities. The proposed framework requires only simple hardware changes to implement, can scale to larger core count and is flexible enough to support a variety of performance goals. We demonstrate the flexibility of PriSM, by computing the eviction probabilities needed to achieve goals like hit-maximization, fairness and QOS. PriSM-HitMax improves performance by 18.7% over LRU and 11.8% over previously proposed schemes in a sixteen core machine. PriSM-Fairness improves fairness over existing solutions by 23.3% along with a performance improvement of 19.0%. PriSM-QOS successfully achieves the desired QOS targets.

Regulation of S100A2 expression by TGF-beta-induced MEK/ERK signalling and its role in cell migration/invasion

S100A2, an EF hand calcium-binding protein, is a potential biomarker in several cancers and is also a TGF-beta (transforming growth factor-beta)-regulated gene in melanoma and lung cancer cells. However, the mechanism of S100A2 regulation by TGF-beta and its significance in cancer progression remains largely unknown. In the present study we report the mechanism of S100A2 regulation by TGF-beta and its possible role in TGF-beta-mediated tumour promotion. Characterization of the S100A2 promoter revealed an AP-1 (activator protein-1) element at positions -1161 to -1151 as being the most critical factor for the TGF-beta 1 response. Chromatin immunoprecipitation and electrophoretic mobility-shift assays confirmed the functional binding of the AP-1 complex, predominantly JunB, to the S100A2 promoter in response to TGF-beta 1 in HaCaT keratinocytes. JunB overexpression markedly stimulated the S100A2 promoter which was blocked by the dominant-negative JunB and MEK1 MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1] inhibitor, PD98059. Intriguingly, despite the presence of a putative SMAD-binding element, S100A2 regulation by TGF-beta 1 was found to be SMAD3 independent. Interestingly, p53 protein and TGF-beta 1 show synergistic regulation of the S100A2 promoter. Finally, knockdown of S100A2 expression compromised TGF-beta 1-induced cell migration and invasion of Hep3B cells. Together our findings highlight an important link between the TGF-beta 1-induced MAPK and p53 signalling pathways in the regulation of S100A2 expression and pro-tumorigenic actions.

Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation

The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is similar to 10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch 1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.

Joint pitch-analysis formant-synthesis framework for CS recovery of speech

A joint analysis-synthesis framework is developed for the compressive sensing (CS) recovery of speech signals. The signal is assumed to be sparse in the residual domain with the linear prediction filter used as the sparse transformation. Importantly this transform is not known apriori, since estimating the predictor filter requires the knowledge of the signal. Two prediction filters, one comb filter for pitch and another all pole formant filter are needed to induce maximum sparsity. An iterative method is proposed for the estimation of both the prediction filters and the signal itself. Formant prediction filter is used as the synthesis transform, while the pitch filter is used to model the periodicity in the residual excitation signal, in the analysis mode. Significant improvement in the LLR measure is seen over the previously reported formant filter estimation.

Automatic speech segmentation using probabilistic latent component modeling

Latent variable methods, such as PLCA (Probabilistic Latent Component Analysis) have been successfully used for analysis of non-negative signal representations. In this paper, we formulate PLCS (Probabilistic Latent Component Segmentation), which models each time frame of a spectrogram as a spectral distribution. Given the signal spectrogram, the segmentation boundaries are estimated using a maximum-likelihood approach. For an efficient solution, the algorithm imposes a hard constraint that each segment is modelled by a single latent component. The hard constraint facilitates the solution of ML boundary estimation using dynamic programming. The PLCS framework does not impose a parametric assumption unlike earlier ML segmentation techniques. PLCS can be naturally extended to model coarticulation between successive phones. Experiments on the TIMIT corpus show that the proposed technique is promising compared to most state of the art speech segmentation algorithms.

Crystal structures of SCP2-thiolases of Trypanosomatidae, human pathogens causing widespread tropical diseases: the importance for catalysis of the cysteine of the unique HDCF loop

Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites.

Sequestration of the abrin A chain to the nucleus by BASP1 increases the resistance of cells to abrin toxicity

Abrin, a type II ribosome-inactivating protein, comprises A and B subunits wherein the A subunit harbours toxin activity and the B subunit has a galactose-specific lectin activity. The entry of the protein inside the cell is through the binding of the B chain to cell surface glycoproteins followed by receptor-mediated endocytosis and retrograde transport. A previous study from our laboratory showed that different cell lines exhibited differences of as great as similar to 200-fold in abrin toxicity, prompting the present study to compare the trafficking of the toxin within cells. Observations made in this regard revealed that the abrin A chain, after being released into the cytosol, is sequestered into the nucleus through interaction with a cellular protein of similar to 25 kDa, BASP1 (brain acid-soluble protein 1). The nuclear localization of the A chain is seen predominantly in cells that are less sensitive to abrin toxicity and dependent on the levels of BASP1 in cells. The sequestration by BASP1 renders cells increasingly resistant to the inhibition of protein synthesis by abrin and the nucleus act as a sink to overcome cellular stress induced

Synthesis and Characterization of Fly Ash Geopolymer Sand

Geopolymers are an alternative binder to portland cement in the manufacture of mortars and concrete, as its three-dimensional aluminosilicate network imparts excellent mechanical properties. Use of geopolymers in place of ordinary portland cement is favored owing to the possible energy and carbon dioxide savings. River sand is another construction industry material that needs development of a sustainable alternate in India. Geopolymerization of fly ash amorphous silica mixtures is employed to produce fine aggregates as a possible replacement to river sand. Geopolymerization of fly ash amorphous silica mixtures in 10M NaOH solution at 100 degrees C for 7days produced fine aggregates termed fly ash geopolymer sand (FAPS)] that had comparable grain size distribution, specific gravity, and improved frictional resistance with river sand. The FAPS particles exhibited more alkaline pH (12.5) and higher total dissolved solids (TDS) concentration (TDS=747 mg/L) in comparison to the river sand specimen (pH=7.9 and TDS=32.5 mg/L). However, when used as fine aggregate in mortar, FAPS-mortar specimens develop similar pH, lower TDS, similar compressive strength, and modulus in relation to river sand-mortar specimens. The experimental results suggest that FAPS particles have the potential to replace river sand in the manufacture of mortar and concrete.

Low-Cost Cloud-Based Design of Smart Rural Energy Device in Microgrid: Perspective India An Energy-On-Demand Service for Rural India

In this paper we present a combination of technologies to provide an Energy-on-Demand (EoD) service to enable low cost innovation suitable for microgrid networks. The system is designed around the low cost and simple Rural Energy Device (RED) Box which in combination with Short Message Service (SMS) communication methodology serves as an elementary proxy for Smart meters which are typically used in urban settings. Further, customer behavior and familiarity in using such devices based on mobile experience has been incorporated into the design philosophy. Customers are incentivized to interact with the system thus providing valuable behavioral and usage data to the Utility Service Provider (USP). Data that is collected over time can be used by the USP for analytics envisioned by using remote computing services known as cloud computing service. Cloud computing allows for a sharing of computational resources at the virtual level across several networks. The customer-system interaction is facilitated by a third party Telecom Service provider (TSP). The approximate cost of the RED Box is envisaged to be under USD 10 on production scale.

Retrieving clay minerals from stabilised soil compacts

Stabilised soil products such as stabilised soil blocks, rammed earth and stabilised adobe are being used for building construction since the last 6-7 decades. Major advantages of stabilised soil products include low embodied carbon, use of local materials, decentralized production, and easy to adjust the strength, texture, size and shape. Portland cement and lime represent the most commonly used stabilisers for stabilised soil products. The mechanism of strength development in cement and lime stabilised soils is distinctly different. The paper presents results of scientific investigations pertaining to the status of clay minerals in the 28 day cured cement and lime stabilised soil compacts. XRD, SEM imaging, grain size distribution and Atterberg's limits of the ground stabilised soil products and the natural soil were determined. Results reveal that clay minerals can be retrieved from cement stabilised soil products, whereas in lime stabilised soil products clay minerals get consumed in the lime-clay reactions and negligible percentage of clay minerals are left in the stabilised soil compacts. The results of the present investigation clearly demonstrate that cement stabilisation is superior to lime stabilisation in retrieving the clay minerals from the stabilised soil compacts. (C) 2014 Elsevier B.V. All rights reserved.

Boundary Tracking using a Hybrid Cyclic Pursuit Scheme

There has been a lot of work in the literature, related to the mapping of boundaries of regions, using multiple agents. Most of these are based on optimization techniques or rely on potential fields to drive the agents towards the boundary and then retain them there while they space out evenly along the perimeter or surface (in two-dimensional and three-dimensional cases, respectively). In this paper an algorithm to track the boundary of a region in space is provided based on the cyclic pursuit scheme. This enables the agents to constantly move along the perimeter in a cluster, thereby tracking a dynamically changing boundary. The trajectories of the agents provide a sketch of the boundary. The use of multiple agents may facilitate minimization of tracking error by providing accurate estimates of points on the boundary, besides providing redundancy. Simulation results are provided to highlight the performance of the proposed scheme.