Myb-binding protein 1a augments AhR-dependent gene expression

We have studied the mechanism by which an acidic domain (amino acids 515-583) of the aromatic hydrocarbon receptor (AhR) transactivates a target gene. Studies with glutathione S-transferase fusion proteins demonstrate that the wild-type acidic domain associates in vitro with Myb-binding protein la, whereas a mutant domain (F542A, 1569A) does not. AhR-defective cells reconstituted with an AhR containing the wild-type acidic domain exhibit normal AhR function; however, cells reconstituted with an AhR containing the mutant acidic domain do not function normally. Transient transfection of Myb-binding protein la into mouse hepatoma cells is associated with augmentation of AhR-dependent gene expression. Such augmentation does not occur when Myb-binding protein la is transfected into AhR-defective cells that have been reconstituted with an AhR that lacks the acidic domain. We infer that 1) Myb-binding protein la associates with AhR, thereby enhancing transactivation, and 2) the presence of AhR's acidic domain is both necessary and sufficient for Myb-binding protein la to increase AhR-dependent gene expression.

Myb-binding Protein 1a is a Nucleocytoplasmic Shuttling Protein that Utilizes CRM1-dependent and Independent Nuclear Export Pathways

Myb-binding protein 1a (Mybbp1a) is a novel nuclear protein localized predominantly, but not exclusively, in nucleoli. Although initially isolated as a c-Myb interacting protein, Mybbp1a is expressed ubiquitously, associates with a number of different transcription factors, and may play a role in both RNA polymerase I- and II-mediated transcriptional regulation. However, its precise function remains unclear. In this study we show that Mybbp1a is a nucleocytoplasmic shuttling protein and investigate the mechanisms responsible for both nuclear import and export. The carboxyl terminus of Mybbp1a, which contains seven short basic amino acid repeat sequences, is responsible for both nuclear and nucleolar localization, and this activity can be transferred to a heterologous protein. Deletion mapping demonstrated that these repeat sequences appear to act incrementally, with successive deletions resulting in a corresponding increase in the proportion of protein localized in the cytoplasm. Glutathione S-transferase pulldown experiments showed that the nuclear receptor importin-alpha/beta mediates Mybbp1a nuclear import. Interspecies heterokaryons were used to demonstrate that Mybbp1a was capable of shuttling between the nucleus and the cytoplasm. Deletion analysis and in vivo export studies using a heterologous assay system identified several nuclear export sequences which facilitate Mybbp1a nuclear export of Mybbp1a by CRM1-dependent and -independent pathways. (C) 2003 Elsevier Science (USA). All rights reserved.

Theoretical and numerical analyses of convective instability in porous media with temperature-dependent viscosity

Exact analytical solutions of the critical Rayleigh numbers have been obtained for a hydrothermal system consisting of a horizontal porous layer with temperature-dependent viscosity. The boundary conditions considered are constant temperature and zero vertical Darcy velocity at both the top and bottom of the layer. Not only can the derived analytical solutions be readily used to examine the effect of the temperature-dependent viscosity on the temperature-gradient driven convective flow, but also they can be used to validate the numerical methods such as the finite-element method and finite-difference method for dealing with the same kind of problem. The related analytical and numerical results demonstrated that the temperature-dependent viscosity destabilizes the temperature-gradient driven convective flow and therefore, may affect the ore body formation and mineralization in the upper crust of the Earth. Copyright (C) 2003 John Wiley Sons, Ltd.

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted.We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mM, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe(6), and the second involving a charge-charge interaction with the Asp(25) residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine - A mechanism for the slow acetylator phenotype and substrate-dependent down-regulation

Many drugs and chemicals found in the environment are either detoxified by N-acetyltransferase 1 (NAT1, EC 2.3.1.5) and eliminated from the body or bioactivated to metabolites that have the potential to cause toxicity and/or cancer. NAT1 activity in the body is regulated by genetic polymorphisms as well as environmental factors such as substrate-dependent down-regulation and oxidative stress. Here we report the molecular mechanism for the low protein expression from mutant NAT1 alleles that gives rise to the slow acetylator phenotype and show that a similar process accounts for enzyme down-regulation by NAT1 substrates. NAT1 allozymes NAT1 14, NAT1 15, NAT1 17, and NAT1 22 are devoid of enzyme activity and have short intracellular half-lives (similar to4 h) compared with wild-type NAT1 4 and the active allozyme NAT1 24. The inactive allozymes are unable to be acetylated by cofactor, resulting in ubiquitination and rapid degradation by the 26 S proteasome. This was confirmed by site-directed mutagenesis of the active site cysteine 68. The NAT1 substrate p-aminobenzoic acid induced ubiquitination of the usually stable NAT1 4, leading to its rapid degradation. From this study, we conclude that NAT1 exists in the cell in either a stable acetylated state or an unstable non-acetylated state and that mutations in the NAT1 gene that prevent protein acetylation produce a slow acetylator phenotype.

Therapeutic administration of KM+ lectin protects mice against Paracoccidioides brasiliensis infection via interleukin-12 production in a Toll-like receptor 2-dependent mechanism

KM+ is a mannose-binding lectin from Artocarpus integrifolia that induces interleukin (IL)-12 production by macrophages and protective T helper I immune response against Leishmania major infection. in this study, we performed experiments to evaluate the therapeutic activity of jackfruit KM+ (jfKM(+)) and its recombinant counterpart (rKM(+)) in experimental paracoccidioidomycosis. To this end, jfKM(+) or rKM(+) was administered to BALB/c mice 10 days after infection with Paracoccidiodes brasiliensis. Thirty days postinfection, lungs from the KM+-treated mice contained significantly fewer colony-forming units and little to no organized granulomas compared to the controls. In addition, lung homogenates from the KM+-treated mice presented higher levels of nitric oxide, IL-12, interferon-gamma, and tumor necrosis factor-a, whereas higher levels of IL-4 and IL-10 were detected in the control group. With mice deficient in IL-12, Toll-like receptor (TLR) 2, TLR4, or TLR adaptor molecule MyD88, we demonstrated that KM+ led to protection against P. brasiliensis infection through IL-12 production, which was dependent on TLR2. These results demonstrated a beneficial effect of KM+ on the severity of P. brasiliensis infection and may expand its potential use as a novel immunotherapeutic molecule.

Interpretation of negative values of the interaction parameter in the adsorption equation through the effects of surface layer heterogeneity

The problem of the negative values of the interaction parameter in the equation of Frumkin has been analyzed with respect to the adsorption of nonionic molecules on energetically homogeneous surface. For this purpose, the adsorption states of a homologue series of ethoxylated nonionic surfactants on air/water interface have been determined using four different models and literature data (surface tension isotherms). The results obtained with the Frumkin adsorption isotherm imply repulsion between the adsorbed species (corresponding to negative values of the interaction parameter), while the classical lattice theory for energetically homogeneous surface (e.g., water/air) admits attraction alone. It appears that this serious contradiction can be overcome by assuming heterogeneity in the adsorption layer, that is, effects of partial condensation (formation of aggregates) on the surface. Such a phenomenon is suggested in the Fainerman-Lucassen-Reynders-Miller (FLM) 'Aggregation model'. Despite the limitations of the latter model (e.g., monodispersity of the aggregates), we have been able to estimate the sign and the order of magnitude of Frumkin's interaction parameter and the range of the aggregation numbers of the surface species. (C) 2004 Elsevier B.V All rights reserved.

Non-diagonal solutions of the reflection equation for the trigonometric A((1)) (n-1) vertex model

We obtain a class of non-diagonal solutions of the reflection equation for the trigonometric A(n-1)((1)) vertex model. The solutions can be expressed in terms of intertwinner matrix and its inverse, which intertwine two trigonometric R-matrices. In addition to a discrete (positive integer) parameter l, 1 less than or equal to l less than or equal to n, the solution contains n + 2 continuous boundary parameters.

Characterization of temperature dependent and substrate-binding cleft movements in Bacillus circulans family 11 xylanase: A molecular dynamics investigation

Background: Xylanases (EC 3.2.1.8) hydrolyze xylan, one of the most abundant plant polysaccharides found in nature, and have many potential applications in biotechnology. Methods: Molecular dynamics simulations were used to investigate the effects of temperature between 298 to 338 K and xylobiose binding on residues located in the substrate-binding cleft of the family 11 xylanase from Bacillus circulans (BcX). Results: In the absence of xylobiose the BcX exhibits temperature dependent movement of the thumb region which adopts an open conformation exposing the active site at the optimum catalytic temperature (328 K). In the presence of substrate, the thumb region restricts access to the active site at all temperatures, and this conformation is maintained by substrate/protein hydrogen bonds involving active site residues, including hydrogen bonds between Tyr69 and the 2` hydroxyl group of the substrate. Substrate access to the active site is regulated by temperature dependent motions that are restricted to the thumb region, and the BcX/substrate complex is stabilized by extensive intermolecular hydrogen bonding with residues in the active site. General significance: These results call for a revision of both the ""hinge-bending"" model for the activity of group 11 xylanases, and the role of Tyr69 in the catalytic mechanism. (C) 2009 Elsevier B.V. All rights reserved.

Root Secretion of Defense-related Proteins Is Development-dependent and Correlated with Flowering Time

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.