Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG)

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2^+/+ and RSK2^-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.     

Crystal structure of the apoptosis-inducing human granzyme A dimer

Granzyme A (GzmA) belongs to a family of trypsin-like serine proteases localized in cytoplasmic granules of activated lymphocytes and natural killer (NK) cells. In contrast to the related granzyme B (GzmB), GzmA forms a stable disulfide-linked homodimer and triggers target-cell death in a caspase-independent way. Limited proteolysis of a high-molecular-mass complex containing SET (also named putative HLA-associated protein II or PHAPII), PHAPI (pp32, leucine-rich acidic nuclear protein) and HMG2 by GzmA liberates NM23-H1, a Mg2+-dependent DNase that causes single-stranded breaks in nuclear DNA. By analyzing the dimeric GzmA structure at a resolution of 2.5 A, we determined the substrate-binding constraints and selective advantages of the two domains arranged as a unique functional tandem. The active sites of the two subunits point in opposite directions and the nearby noncatalytic surfaces can function as exosites, presenting substrates to the active site region of the adjacent partner in a manner analogous to staphylokinase or streptokinase, which present plasminogen to the cofactor-plasmin and cofactor-plasminogen complexes.     

Über Zusammenhänge zwischen CO2-Assimilation und Photoperiodismus bei Kalanchoe Blossfeldiana

Zusammenfassung Assimilation und Atmung vonKalanchoe Blossfeldiana aus verschieden langem Tag wurden mit der manometrischen Methode geprüft. Pflanzen, die von der Keimung an im Kurztag gestanden hatten, und auch solche, die erst nach längerer Zeit in Kurztagsbehandlung kamen, wiesen zur Zeit der Blüte erheblich gesteigerte Photosynthese und Atmung auf. Im Knospenstadium waren die Verhältnisse noch extremer. Bei Säckchenpflanzen war auch assimilatorisch eine Aufteilung der Pflanze in eine Kurztags- und eine Langtagsseite festzustellen. Nach der Fruchtanlage war die photosynthetische Tätigkeit wieder bedeutend geringer. Durch Kurztagsbehandlung war beiKalanchoe eine Vermehrung des Farbstoffgehaltes hervorgerufen.     

TraG Encoded by the pIP501 Type IV Secretion System Is a Two-Domain Peptidoglycan-Degrading Enzyme Essential for Conjugative Transfer

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.     

Effects of Inorganic Turbidity on the Phytoplankton of an Amazonian Lake Impacted by Bauxite Tailings

The dumping of bauxite tailings into Batata Lake, an Amazonian clear-water lake, generated high levels of turbidity and caused a serious decrease in phytoplankton densities, which could possibly be the result of a photosynthetic limitation due to light attenuation together with an increase in algal sinking due to the adhesion of clay particles. This study aimed to investigate the sinking process through the addition of different suspended clay concentrations in columns containing Batata lake water. Since no effect of the suspended clays on Batata Lake phytoplankton sinking was observed, it was then evaluated, under laboratory conditions, whether the low conductivity of the Batata Lake water could interfere with the algae-clay aggregation process. Cultures of two algal species known to be capable to aggregate to Batata Lake suspended clays in algal culture medium: Staurodesmus convergens and Phormidium amoenum, were added to both the low conductivity Batata Lake water (14 μS cm^?1) and the high conductivity algal culture media (WC – 300 μS cm^?1 and Z8 – 560 μS cm^?1) together with Batata lake suspended clays. In both algal culture media and Batata lake water the two species had their sinking accelerated due to clay adhesion. It is thus suggested that the decrease in phytoplankton densities recorded in Batata Lake may not be related to an increase in phytoplankton loss rates due to algal-clay aggregation, but rather are a consequence of decreasing growth rates because of light attenuation.     

Serum apolipoprotein AI synthesis in rat hepatocytes and its secretion as proform

Rat hepatocytes in monolayer or suspension culture synthesize serum lipoprotein AI. It is secreted into the serum-free culture medium. Synthesis and secretion processes were studied in the presence of radiolabelled amino acids. The synthesis product of the hepatocytes and the secretion product from the medium were isolated by immunoprecipitation with a mono-specific rabbit antiserum against rat apolipoprotein AI. The intracellular and secreted products were homogeneous and identical in polyacrylamide gel electrophoresis but had reduced electrophoretic mobility as compared to native apolipoprotein AI. They were submitted to automated Edman degradation. They were present in their proform, the N-terminus of which is extended by a hexapeptide. In the presence of rat serum the proform is proteolytically transformed into the mature form of apolipoprotein AI.     

Distinguishing social from nonsocial navigation in moving animal groups

Many animals, such as migrating shoals of fish, navigate in groups. Knowing the mechanisms involved in animal navigation is important when it comes to explaining navigation accuracy, dispersal patterns, population and evolutionary dynamics, and consequently, the design of conservation strategies. When navigating toward a common target, animals could interact socially by sharing available information directly or indirectly, or each individual could navigate by itself and aggregations may not disperse because all animals are moving toward the same target. Here we present an analysis technique that uses individual movement trajectories to determine the extent to which individuals in navigating groups interact socially, given knowledge of their target. The basic idea of our approach is that the movement directions of individuals arise from a combination of responses to the environment and to other individuals. We estimate the relative importance of these responses, distinguishing between social and nonsocial interactions. We develop and test our method, using simulated groups, and we demonstrate its applicability to empirical data in a case study on groups of guppies moving toward shelter in a tank. Our approach is generic and can be extended to different scenarios of animal group movement.     

A cometabolic kinetics model incoroporating enzyme inhibition, inactivation, and recovery: II. Trichloroethylene degradaation experiments

A Cometabolism enzyme kinetics model has been presented which takes into account changes in bacterial activity associated with enzyme inhibitiion, inactivation, inactivation of enzyme resulting from product toxicty, and respondent synthesis of new enzyme. Although this process is inherently unsteady-state, the model assumes that cometabolic degradation of a compound exhibiting product toxicity can be modeled as pseudo-steady-staate under certain conditions. In its simplified from, the model also assumes that enzyme inactivation is directly propoertional to nongrawth substrate oxidation, and that recovery is directly proportionla to growth substrate oxidation. In part 1, model drivation, simplification, and analyses were described. In this articles, model assuptiions are tested by analyzing data from experiments exmining trichloroethylene (TCE) degradation by the ammoniaoxidizing baceterium Nitrosomonas europaea in a quasisteady-state bioreactor. Model solution results showed steady-state bioreactor. Model solution results showed TCE to be a competitive inhibitoer of ammonia oxidation, with TCE affinity for ammonia monooxygenase (AMO) being about four times greater than that of ammonia for the enzyme. Inhibition was independent fo TCE oxidation and occurred essentially instantly upon exposure to TCE. In contrast, inactivation of AMO occurred more gradually and was proportional to the rate and amount of TCE oxidized. Evaluation of other O(2)-dependent enzymes and electron transport proteins suggested that TCE-related damage was predominantly confined to AMO. In response to inhibition and/or inactivation, bacterial recovery was initiated, even in the presence of TCE, implying that membranes adn protein synthesis systems were functioning. Analysis of data and comparison of model results showed the inhibition/inactivation/recovery concept to provide a reasonable basis for understandign the effects fo TCE on AMO function and bacterial response. The model assumptions were verified except tht questions remain regarding the factores controlling recovery and its role in the long term. (c) 1995 John Wiley & Sons, Inc.     

Solution structure of a synthetic peptide corresponding to a receptor binding region of FSH (hFSH-beta 33-53).

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH- chain amino acid sequences 33–53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990),Biochemistry 29, 1194–1200], 81–95 [Santa-Coloma, T. A., and Reichert, L. E., Jr. (1990),J. Biol. Chem. 265, 5037–5042], and the combined sequence (33–53)–(81–95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991),Mol. Cell. Endocrinol. 78, 197–204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH--(33–53), H₂N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel -pleated sheet, turns including a -turn, other structures, and a small amount ofa-helix. The major characteristics of the structure were found to be relatively stable at acidicpH and the predominant effect of increased solvent polarity was a small increase ina-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution atpH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH--(33–53). These features included an antiparallel -sheet (residues 38–51 and 46–48), turns within residues 41–46, and 50–52 (a -turn) and a small N-terminal helical region comprised of amino acids 34–36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to thetrans conformation, whereas proline-42 favored thetrans conformer (70%) over thecis (30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containingtrans-42 andtrans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptide's structure. The hFSH--(33–53) structure has a highly polar surface composed of six cationic amino acid (arginie-35, lysine-40, arginine-44, lysine-46, glutamine-48, and lysine-49) and two anionic residues (aspartate-36 and aspartic acid-41). A hydrophobic region in the structure is composed of residues in the antiparallel -sheet and -turn which fold to produce a distorted hairpin. The structure of this domain, together with the protruding and positively charged region in the vicinity of residues 42–45, may mimic the surface of hFSH that binds to the receptor.Abreviations used: hFSH, human follicle-stimulating hormone; PB, 25 mM Na₂KPO₄, 25 mM KH₂PO₄, and 5 mM Mg Cl₂; CD, circular dichroism spectrapolarimetry; NMR, nuclear magnetic resonance spectrometry; COSY, homonuclear correlated spectroscopy; NOESY, 2D nuclear Overhauser effect spectroscopy; HOHAHA, homonuclear Hartman-Han coherence transfer; HMQCHY, reverse-detected heteronuclear multiple shift correlation, one bond; HMBC, reverse-detected heteronuclear multiple bond correlation; S/N, signal to noise ratio; TFE, trifluoroethanol.Dr. Santa-Coloma is on leave of absence from the National Research Council of Argentina (CONICET).