Domain architecture of a Caenorhabditis elegans AKAP suggests a novel AKAP function

A-kinase anchoring proteins (AKAPs) are adapter proteins that are involved in directing cAMP-dependent protein kinase and some other signaling enzymes to certain intracellular locations. In this study, we investigate the domain architecture of an AKAP from Caenorhabditis elegans (AKAP(CE)). We show that AKAP(CE) shares two domains with the Smad anchor for receptor activation, a FYVE-finger and a transforming growth factor beta (TGFbeta) receptor binding domain, suggesting that AKAP(CE) may interact with a receptor belonging to the TGFbeta receptor family. This predicted novel AKAP function supports the recent view of AKAPs as adapter proteins that can be involved in various signaling pathways.     

Role of an electrostatic network of residues in the enzymatic action of the Rhizomucor miehei lipase family

We have used continuum electrostatic methods to investigate the role of electrostatic interactions in the structure, function, and pH-dependent stability of the fungal Rhizomucor miehei lipase (RmL) family. We identify a functionally important electrostatic network which includes residues S144, D203, H257, Y260, H143, Y28, R80, and D91 (residue numbering is from RmL). This network consists of residues belonging to the catalytic triad (S144, D203, H257), residues located in proximity to the active site (Y260), residues stabilizing the geometry of the active site (Y28, H143), and residues located in the lid (D91) or close to the first hinge (R80). The lid and the first hinge are associated with the interfacial activation of lipases, where an alpha-helical lid opens up by rotating around two hinge regions. All network residues are well conserved in a set of 12 lipase homologues, and 6 of the network residues are located in sequence motifs. We observe that the effects of modeled mutations R86L, D91N, and H257F on the pH-dependent electrostatic free energies differ significantly in the closed and open conformations of RmL. Mutation R86L is especially interesting since it stabilizes the closed conformation but destabilizes the open one. Site-site electrostatic interaction energies reveal that interactions between R86 and D61, D113, and E117 stabilize the open conformation.     

Kinetics of association of anti-lysozyme monoclonal antibody D44.1 and hen-egg lysozyme

Association rate constants for antigen/antibody associations have been computed by Brownian Dynamics simulations of D. L. Ermak and J. A. McCammon, J. Chem. Phys. 69:1352-1360, 1978. The model of monoclonal antibody (mAb) D44.1 is based on crystallographic data (B. C. Braden et al., J. Mol. Biol. 243:767-781, 1994). Electrostatic forces that steer the antigen to the antibody-combining site are computed by solving the linearized Poisson-Boltzmann equation. D44. 1-HEL complex displays very similar association motifs to a related anti-lysozyme antibody, HyHEL-5-HEL system. The computed association rate constants are comparable in the two systems, although the experimental affinity constants differ by three orders of magnitude (D. Tello et al., Biochem. Soc. Trans. 21:943-946, 1993; K. A. Hibbits et al., Biochemistry. 33:3584-3590, 1994). Simulations suggest that the origin of the differences in the affinity come from dissociation rate constants. We have also carried out simulation experiments on a number of mutant antibody fragment-HEL associations to address the role of electrostatics and, to a limited extent, the orientational aspects of association.     

A novel microarray strategy for detecting genes and pathways in microbes with unsequenced genomes

Expression profile analysis of genes provides valuable information concerning the genetic response of cells to stimuli. We describe an adaptation of this technology that can be used to probe for the expression of specific families of genes in microbial species. In our method a combination of sets of oligonucleotide probes representing fingerprint sequences specific to protein families is used to identify the presence and expression levels of family homologs in a microbial cell. We demonstrate computationally, using exemplars, that when the cDNA complement from an organism is sequentially screened against a set of specific motif oligonucleotides, statistically significant information can be obtained concerning the expression of the corresponding genes. This method can be used to identify specific genes and pathways simultaneously in several organisms of interest even in the absence of sequence information from the organisms.     

Trans cooperativity by a split DNA recombinase: the central and catalytic domains of bacteriophage lambda integrase cooperate in cleaving DNA substrates when the two domains are not covalently linked

Site-specific recombinases of the lambda-integrase family recognize and cleave their cognate DNA sites through cooperative binding to opposite sides of the DNA substrate by a C-terminal catalytic domain and a flexibly linked "core-binding" domain; regulation of this cleavage is achieved via the formation of higher-order complexes. We report that the core-binding domain of lambda-integrase is able to stimulate the activity of the catalytic domain even when the two domains are not linked. This trans stimulation is accomplished without significantly increasing the affinity of the catalytic domain for its DNA substrate. Moreover, we show that mutations in the DNA substrate can abrogate this effect while retaining specificity determinants for cleavage. Since the domains do not significantly interact directly, this finding implies that trans activation is achieved via the DNA substrate in a manner that may be mechanistically important in this and similar DNA binding and cleaving enzymes.     

Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential

Background

We have previously shown that prostate cancer LNCaP cells are resistant to TRAIL, and downregulation of PI-3K/Akt pathway by molecular and pharmacological means sensitizes cells to undergo apoptosis by TRAIL and curcumin. The purpose of this study was to examine the molecular mechanisms by which resveratrol sensitized TRAIL-resistant LNCaP cells.

Results

Resveratrol inhibited growth and induced apoptosis in androgen-dependent LNCaP cells, but had no effect on normal human prostate epithelial cells. Resveratrol upregulated the expression of Bax, Bak, PUMA, Noxa, Bim, TRAIL-R1/DR4 and TRAIL-R2/DR5, and downregulated the expression of Bcl-2, Bcl-X_L, survivin and XIAP. Treatment of LNCaP cells with resveratrol resulted in generation of reactive oxygen species, translocation of Bax and p53 to mitochondria, subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, AIF, Smac/DIABLO and Omi/HtrA2), activation of caspase-3 and caspase-9 and induction of apoptosis. The ability of resveratrol to sensitize TRAIL-resistant LNCaP cells was inhibited by dominant negative FADD, caspase-8 siRNA or N-acetyl cysteine. Smac siRNA inhibited resveratrol-induced apoptosis, whereas Smac N7 peptide induced apoptosis and enhanced the effectiveness of resveratrol.

Conclusion

Resveratrol either alone or in combination with TRAIL or Smac can be used for the prevention and/or treatment of human prostate cancer.     

Curcumin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells: molecular mechanisms of apoptosis, migration and angiogenesis

Background

We have recently shown that curcumin (a diferuloylmethane) inhibits growth and induces apoptosis, and also demonstrated that TRAIL induces apoptosis by binding to specific cell surface death receptors in prostate cancer cells. The objectives of this paper were to investigate the molecular mechanisms by which curcumin enhanced the apoptosis-inducing potential of TRAIL in prostate cancer cells.

Results

Curcumin enhanced the apoptosis-inducing potential of TRAIL in androgen-unresponsive PC-3 cells and sensitized androgen-responsive TRAIL-resistant LNCaP cells. Curcumin inhibited the expressions of Bcl-2, Bcl-X_L, survivin and XIAP, and induced the expressions Bax, Bak, PUMA, Bim, and Noxa and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) in both cell lines. Overexpression of dominant negative FADD inhibited the interactive effects of curcumin and TRAIL on apoptosis. Treatment of these cells with curcumin resulted in activation of caspase-3, and caspase-9, and drop in mitochondrial membrane potential, and these events were further enhanced when combined with TRAIL. Curcumin inhibited capillary tube formation and migration of HUVEC cells and these effects were further enhanced in the presence of MEK1/2 inhibitor PD98059.

Conclusion

The ability of curcumin to inhibit capillary tube formation and cell migration, and enhance the therapeutic potential of TRAIL suggests that curcumin alone or in combination with TRAIL can be used for prostate cancer prevention and/or therapy.     

A novel role for connexin hemichannel in oxidative stress and smoking-induced cell injury

Oxidative stress is linked to many pathological conditions, including ischemia, atherosclerosis and neurodegenerative disorders. The molecular mechanisms of oxidative stress induced pathophysiology and cell death are currently poorly understood. Our present work demonstrates that oxidative stress induced by reactive oxygen species and cigarette smoke extract depolarize the cell membrane and open connexin hemichannels. Under oxidative stress, connexin expression and connexin silencing resulted in increased and reduced cell deaths, respectively. Morphological and live/dead assays indicate that cell death is likely through apoptosis. Our studies provide new insights into the mechanistic role of hemichannels in oxidative stress induced cell injury.     

A review of some issues and identification of some barriers in the implementation of FMS

Global competition, advancements in technology and ever changing customers’ demand have made the manufacturing companies to realize the importance of flexible manufacturing systems (FMS). These organizations are looking at FMS as a viable alternative to enhance their competitive edge. But, implementation of this universally accepted and challenging technology is not an easy task. A large number of articles have been reviewed and it is found that the existing literature lacks in providing a clear picture about the implementation of FMS. In this paper, work of various researchers has been studied and it is found that it is really a very difficult task for any organization to transform into FMS on the basis of existing research results. A wide gap exists between the proposed approaches/algorithms for the design of different components of FMS and the real-life complexities. Besides describing the gap in various issues related to FMS, some barriers, which inhibit the adaptation and implementation of FMS, have also been identified in this paper.     

TFAP2C- and p63-Dependent Networks Sequentially Rearrange Chromatin Landscapes to Drive Human Epidermal Lineage Commitment

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