Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.     

A detailed and validated three dimensional dynamic model of the patellofemoral joint

A detailed 3D anatomical model of the patellofemoral joint was developed to study the tracking, force, contact and stability characteristics of the joint. The quadriceps was considered to include six components represented by 15 force vectors. The patellar tendon was modeled using four bundles of viscoelastic tensile elements. Each of the lateral and medial retinaculum was modeled by a three-bundle nonlinear spring. The femur and patella were considered as rigid bodies with their articular cartilage layers represented by an isotropic viscoelastic material. The geometrical and tracking data needed for model simulation, as well as validation of its results, were obtained from an in vivo experiment, involving MR imaging of a normal knee while performing isometric leg press against a constant 140 N force. The model was formulated within the framework of a rigid body spring model and solved using forth-order Runge-Kutta, for knee flexion angles between zero and 50 degrees. Results indicated a good agreement between the model predictions for patellar tracking and the experimental results with RMS deviations of about 2 mm for translations (less than 0.7 mm for patellar mediolateral shift), and 4 degrees for rotations (less than 3 degrees for patellar tilt). The contact pattern predicted by the model was also consistent with the results of the experiment and the literature. The joint contact force increased linearly with progressive knee flexion from 80 N to 210 N. The medial retinaculum experienced a peak force of 18 N at full extension that decreased with knee flexion and disappeared entirely at 20 degrees flexion. Analysis of the patellar time response to the quadriceps contraction suggested that the muscle activation most affected the patellar shift and tilt. These results are consistent with the recent observations in the literature concerning the significance of retinaculum and quadriceps in the patellar stability.     

Uneven modulation of the annexin 1 system in osteoblast-like cells by dexamethasone

We tested whether glucocorticoids modulated osteoblast expression of the annexin 1 system, including the ligand and two G-coupled receptors termed formyl-peptide receptor (FPR) and FPR-like-1 (FPRL-1). In Saos-2 cells, rapid up-regulation of FPR mRNA upon cell incubation with dexamethasone (0.01-1 microM) was observed, with significant changes as early as 2h and a more marked response at 24h; annexin 1 and FPRL-1 mRNA changes were more subtle. At the protein level, dexamethasone provoked a rapid externalization of annexin 1 (maximal at 2h) followed by delayed time-dependent changes in the cell cytosol. Saos-2 cell surface expression of FPR or FPRL-1 could not be detected, even when dexamethasone was added with the bone modelling cytokines interleukin-6 or interleukin-1. The uneven modulation of the annexin 1 system (mediator and its putative receptors) in osteoblasts might lead to a better understanding of how these complex biochemical pathways become operative in bone.     

Facies analysis of a large-scale Jurassic shelf-lagoon: the Kamar-e-Mehdi Formation of east-central Iran

The Callovian–Lower Kimmeridgian Kamar-e-Mehdi Formation of the Tabas Block (east-central Iran) is an up to 1,350-m-thick, fine-grained, marly-calcareous unit containing a basal Echellon Limestone Member (up to 180 m thick) and a terminal Nar Limestone Member (up to 100 m thick). The formation was deposited in a relatively deep shelf-lagoon that was part of the large-scale carbonate system of the Esfandiar Subgroup, extending N–S for about 500 km along the strike with a width of up to 100 km. The lagoonal Kamar-e-Mehdi Formation shows sedimentation rates of 150 m/myr, twice as high as those of the shelf-edge carbonate barrier (Esfandiar Platform). The repetitive lithologies and uniform depositional environment suggest equilibrium conditions between sedimentation and subsidence, related to constant slow rotation of the Tabas fault-block around a horizontal axis, the platform sitting on the crest, and the lagoon occupying the dip-slope. Lagoonal sedimentation was dominated by suspended carbonate mud and peloids from the eastern Esfandiar Platform whereas the subordinate siliciclastic material was derived from the west (Yazd Block). The diverse macrobenthos (mainly bivalves) suggests fully marine conditions for the major part of the Kamar-e-Mehdi Formation. However, towards the upper part, biotic impoverishment and the deposition of skeletal-poor, evaporitic sediments indicate increasing restriction. The overlying Magu Gypsum Formation marks the end of an arid basin-fill cycle and possibly forms an effective seal for hydrocarbon reservoirs in that area. The Esfandiar Subgroup was a Neotethys-facing carbonate margin, forming part of a belt of carbonate systems tracking the margins of the Iran Plate during Callovian to Late Jurassic times.     

Impact of the C1' configuration of abasic sites on DNA duplex structure

Naturally occurring abasic sites in DNA exist as an equilibrium mixture of the aldehyde, the hydrated aldehyde, and the hemiacetal forms (dominant). The influence of the configuration of the C1' hydroxyl group of the hemiacetal form on duplex structure and abasic site repair has been examined using novel carbocyclic analogues. Both the alpha- and beta-forms of this novel abasic site were introduced into oligomeric DNA using the standard DMT-phosphoramidite approach in an automated solid-phase synthesizer. Solution structures of the d(CGTACXCATGC).d(GCATGAGTACG) duplex (where X is the alpha- or beta-anomer of the carbocyclic abasic site analogue) were determined by NMR spectroscopy and restrained molecular dynamics simulations. The structures were only minimally perturbed by the presence of either anomer of the abasic site. All residues adopted an anti conformation, and Watson-Crick alignments were observed on all base pairs of the duplexes. At the lesion site, the abasic residues and their partner adenines showed increased dynamic behavior but adopted intrahelical positions in the final refined structures. Incision of duplexes having the alpha- or beta-anomer of the carbocyclic abasic site by human AP endonuclease showed that the enzyme recognizes both configurations of the lesion and nicks the DNA backbone with similar efficiency. Our results challenge the suggestion that Ape1 is stereoselective and imply a plasticity at the active site of the enzyme for accommodating either anomer of the lesion.     

Heat shock protein 65-reactive T cells are involved in the pathogenesis of non-antigenic dimethyl dioctadecyl ammonium bromide-induced arthritis

Dimethyl dioctadecyl ammonium bromide (DDA) (C(38)H(80)NBr) is a nonantigenic lipoid material. DDA-induced arthritis (DIA) in the Lewis (LEW) (RT.1(l)) rat is a new experimental model for human rheumatoid arthritis (RA). DIA is a T cell-mediated autoimmune disease. However, the precise self/foreign Ags associated with the disease process in DIA are not yet known. We observed that LEW rats with DIA spontaneously raised a vigorous T cell response both to 65-kDa self (rat) heat shock protein (Rhsp65) and mycobacterial hsp65 (Bhsp65), but not to another arthritis-related Ag, bovine collagen type II. The T cell response to Rhsp65 was focused predominantly on determinant regions 120-134 and 213-227 of the self protein. Interestingly, pretreatment of adult LEW rats using either a mixture of peptides 120-134 and 213-227 of Rhsp65 or a low nonarthritogenic dose of DDA induced protection against subsequent DIA. Intriguingly, the protection induced by the latter was associated with spontaneous priming of T cells specific for peptide 213-227 of Rhsp65. Similarly, LEW rats neonatally tolerized against either Rhsp65 or Bhsp65 were significantly protected from subsequently induced DIA at adult stage, showing the disease-modulating attribute of the hsp65-specific T cells. Taken together, the above findings demonstrate that the hsp65-directed T cell repertoire is of significance in the pathogenesis of autoimmune arthritis induced by nonantigenic DDA. Like other animal models of RA involving hsp65, these first insights into the disease-associated Ags in the DIA model would pave the way for further understanding of the immunological aspects of induction and regulation of RA.     

Genetic variability of Cotton leaf curl betasatellite in Northern India

Cotton is an important crop and its production is affected by various disease pathogens. Monopartite begomovirus associated betasatellites cause Cotton leaf curl disease (CLCuD) in Northern India. In order to access the occurrence and genetic variability of Cotton leaf curl betasatellites, an extensive field survey was conducted in states of Rajasthan, Punjab and Haryana. We selected the betasatellite sequence for analysis as they are reported as important for disease severity and sequence variability. Based on the field observations, the disease incidence ranged from 30% to 80% during the survey. Full genome and DNA β were amplified from various samples while no amplicon was obtained in some samples. The nucleotide sequence homology ranged from 90.0% to 98.7% with Cotton leaf curl virus (CLCuV), 55.2–55.5% with Bhendi yellow vein mosaic virus, 55.8% with Okra leaf curl virus and 51.70% with Tomato leaf curl virus isolates. The lowest similarity (47.8%) was found in CLCuV-Sudan isolate. Phylogenetic analysis showed that analyzed isolates formed a close cluster with various CLCuV isolates reported earlier. The analysis results show sequence variation in Cotton leaf curl betasatellite which could be the result of recombination. The results obtained by genome amplification and sequence variability indicate that some new variants are circulating and causing leaf curl disease in Rajasthan, Punjab and Haryana.Abbreviations: CLCuD, Cotton leaf curl disease; CLCuV, Cotton leaf curl virus; PCR, polymerase chain reaction; SCR, satellite conserved region     

Mesenchymal stem cell therapy: A promising cell‐based therapy for treatment of myocardial infarction

For decades, mesenchymal stem (MSCs) cells have been used for cardiovascular diseases as regenerative therapy. This review is an attempt to summarize the types of MSCs involved in myocardial infarction (MI) therapy, as well as its possible mechanisms effects, especially the paracrine one in MI focusing on the studies (human and animal) conducted within the last 10 years. Recently, reports showed that MSC therapy could have infarct‐limiting effects after MI in both experimental and clinical trials. In this context, various types of MSCs can help cardiac regeneration by either revitalizing the cardiac stem cells or revascularizing the arteries and veins of the heart. Furthermore, MSCs could produce paracrine growth factors that increase the survival of nearby cardiomyocytes, as well as increase angiogenesis through recruitment of stem cell from bone marrow or inducing vessel growth from existing capillaries. Recent research suggests that the paracrine effects of MSCs could be mediated by extracellular vesicles including exosomes. Exosomal microRNAs (miRNAs) released by MSCs are promising therapeutic hotspot target for MI. This could be attributed to the role of miRNA in cardiac biology, including cardiac regeneration, stem cell differentiation, apoptosis, neovascularization, cardiac contractility and cardiac remodeling. Furthermore, gene‐modified MSCs could be a recent promising therapy for MI to enhance the paracrine effects of MSCs, including better homing and effective cell targeted tissue regeneration. Although MSC therapy has achieved considerable attention and progress, there are critical challenges that remains to be overcome to achieve the most effective successful cell‐based therapy in MI.     

Cellulolytic activities ofChaetomium globosum on different cellulosic substrates

The effect of cellulosic substrates on the production of extra-cellular cellulases and their cellulolytic activity inChaetomium globosum has been studied in shake flask cultures. Production of endoglucanase, exoglucanase and filter paper cellulase was highest with pure cellulose whereas -glucosidase was maximally induced by wheat straw. A suitable pretreatment for wheat straw was peracetic acid followed by NaOH and that of bagasse with NaOH for saccharification.

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Résumé On a étudié l'effet de substrats cellulosiques sur la production de cellulases extracellulaires et leur activité cellulolytique chezChaetomium globosum en culture en flacons agités. La production d'endoglucanase, d'exoglucanase et de cellulase type papier-filtre est maximum avec la cellulose pure tandis que la -glucosidase est induite de manière maximum par la paille de froment. La paille de froment est traitée de manière adéquate pour la saccharification par l'acide peracétique suivi de NaOH, la bagasse, par NaOH.

     

Cepharanthine triggers apoptosis in a human hepatocellular carcinoma cell line (HuH-7) through the activation of JNK1/2 and the downregulation of Akt

Cepharanthine (CEP), a biscoclaurine alkaloid, has been reported to induce cell death, however, the molecular mechanism of this phenomenon remains unclear. We herein report that CEP induced apoptosis in HuH-7 cells through nuclear fragmentation, DNA ladder formation, cytochrome c release, caspase-3 activation and poly-(ADP-ribose)-polymerase cleavage. CEP triggered the generation of reactive oxygen intermediates, the activation of mitogen activated protein kinase (MAPK) p38, JNK1/2 and p44/42, and the downregulation of protein kinase B/Akt. Antioxidants and SP600125, an inhibitor of JNK1/2, but not inhibitors of p38 MAPK and MEK1/2, significantly prevented cell death, thus implying that reactive oxygen species and JNK1/2 play crucial roles in the CEP-induced apoptosis of HuH-7 cells.