Beta-amyrin from Ardisia elliptica Thunb. is more potent than aspirin in inhibiting collagen-induced platelet aggregation

Ardisia elliptica Thunberg (Myrsinaceae) is a medicinal plant traditionally used for alleviating chest pains, treatment of fever, diarrhoea, liver poisoning and parturition complications. The objectives of the study were to investigate the effect of A. elliptica on collagen induced platelet aggregation and to isolate and purify potential antiplatelet components. Fresh A. elliptica leaves were extracted using methanol (70% v/v) by Soxhlet extraction and the extract was analysed for its inhibition of collagen-induced platelet aggregation. Inhibition of platelet aggregation was assessed by incubating the extracts with rabbit blood and collagen in a whole blood aggregometer and measuring the impedance. The leaf extract was found to inhibit platelet aggregation with an IC50 value of 167 microg/ml. Using bioassay guided fractionation, beta-amyrin was isolated and purified. The IC50 value of beta-amyrin was found to be 4.5 microg/ml (10.5 microM) while that of aspirin was found to be 11 microg/ml (62.7 microM), indicating that beta-amyrin was six times as active as aspirin in inhibiting platelet aggregation. This paper is the first report that beta-amyrin isolated from A. elliptica is more potent than aspirin in inhibiting collagen-induced platelet aggregation. In conclusion, A. elliptica leaves were found to inhibit collagen-induced platelet aggregation and one of the bioactive components responsible for the observed effect was determined to be beta-amyrin.     

Roles of RubisCO and the RubisCO-Like Protein in 5-Methylthioadenosine Metabolism in the Nonsulfur Purple Bacterium Rhodospirillum rubrum

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the assimilation of atmospheric CO₂ into organic matter and is thus central to the existence of life on earth. The beginning of the 2000s was marked by the discovery of a new family of proteins, the RubisCO-like proteins (RLPs), which are structural homologs of RubisCO. RLPs are unable to catalyze CO₂ fixation. The RLPs from Chlorobaculum tepidum, Bacillus subtilis, Geobacillus kaustophilus, and Microcystis aeruginosa have been shown to participate in sulfur metabolism. Whereas the precise function of C. tepidum RLP is unknown, the B. subtilis, G. kaustophilus, and M. aeruginosa RLPs function as tautomerases/enolases in a methionine salvage pathway (MSP). Here, we show that the form II RubisCO enzyme from the nonsulfur purple bacterium Rhodospirillum rubrum is also able to function as an enolase in vivo as part of an MSP, but only under anaerobic conditions. However, unlike B. subtilis RLP, R. rubrum RLP does not catalyze the enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate. Instead, under aerobic growth conditions, R. rubrum RLP employs another intermediate of the MSP, 5-methylthioribulose-1-phosphate, as a substrate, resulting in the formation of different products. To further determine the interrelationship between RubisCOs and RLPs (and the potential integration of cellular carbon and sulfur metabolism), the functional roles of both RubisCO and RLP have been examined in vivo via the use of specific knockout strains and complementation studies of R. rubrum. The presence of functional, yet separate, MSPs in R. rubrum under both aerobic (chemoheterotrophic) and anaerobic (photoheterotrophic) growth conditions has not been observed previously in any organism. Moreover, the aerobic and anaerobic sulfur salvage pathways appear to be differentially controlled, with novel and previously undescribed steps apparent for sulfur salvage in this organism.Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) is the key enzyme of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway. This enzyme catalyzes the primary CO₂ fixation reaction and is found in diverse organisms, including plants, most photosynthetic and chemoautotrophic microorganisms, and many archaea (25). On the basis of amino acid sequence similarities, the RubisCO family of proteins has been classified into four groups, i.e., form I, form II, form III, and form IV (Fig. ​(Fig.1).1). The enzymes classified under forms I, II, and III are all able to catalyze the RubisCO reaction, i.e., carboxylation/oxygenation of RuBP. The most recently discovered group of enzymes in the RubisCO family are the form IV or RubisCO-like proteins (RLPs). These proteins have thus far been identified in proteobacteria, cyanobacteria, archaea, and algae (2, 4, 8, 11, 12, 21, 25, 26). RLPs have been further divided into six different subgroups based on sequence similarities within the group: IV-Photo, IV-Nonphoto, IV-YkrW, IV-DeepYrkW, IV-GOS (Global Ocean Sequencing), and IV-AMC (Acid Mine Consortium) (25, 26). Despite sharing a level of sequence similarity with the bonafide RubisCOs, the RLPs are unable to carry out CO₂/O₂ fixation because their sequences contain dissimilar residues at positions analogous to RubisCO''s active-site residues (25). The structures of the Geobacillus kaustophilus and Chlorobaculum tepidum RLPs have now been solved, and there are indeed differences between the tertiary structures of these two proteins and the bonafide RubisCO enzymes (14, 17, 25). Moreover, distinct patterns of active-site residue identities among the different clades of the RLP lineage suggest that these subgroups of RLPs are likely to utilize different substrates and perform dissimilar reactions (23, 25, 26).Open in a separate windowFIG. 1.Summary of the different classes of RubisCO found in nature so far (25). Forms I, II, and III catalyze bonafide CO₂/O₂ fixation reactions by using RuBP as the substrate. Form IV RubisCO (RLP) does not catalyze RuBP-dependent CO₂/O₂ fixation and is divided into six known clades (25), with only representatives of the type IV-YkrW and IV-DeepYkrW subgroups shown to catalyze defined, yet distinct, reactions (Fig. ​(Fig.22).Previous studies performed with the Chlorobaculum tepidum RLP (of the IV-Photo group) gave the first indication that the RLPs may be involved in some aspect of sulfur metabolism (12, 13). This was later substantiated when the precise function was established for the Bacillus subtilis (2), Microcystis aeruginosa (4), and Geobacillus kaustophilus (14) RLPs of the IV-YkrW group. All three proteins catalyze a tautomerase/enolase reaction of a methionine salvage pathway (MSP) in which the substrate 2,3-diketo-5-methylthiopentanyl-1-phosphate (DK-MTP 1P) is converted to 2-hydroxy-3-keto-5-thiomethylpent-1-ene 1-phosphate (HK-MTP 1P) (Fig. ​(Fig.2).2). This reaction is very reminiscent of the enolization of RuBP catalyzed by RubisCO. Moreover, form II RubisCO from Rhodospirillum rubrum was shown to complement an RLP mutant strain of B. subtilis, with the ability to catalyze the identical tautomerase/enolase reaction (2). Interestingly, in addition to the presence of a form II RubisCO gene (cbbM), the genome of R. rubrum also encodes an RLP that clusters with the IV-DeepYkrW group (25). The function of this protein was recently determined, and it was shown to catalyze a distinct reaction that uses 5-methylthioribulose-1-phosphate as the substrate (15). Via an unprecedented 1,3-proton transfer, with two successive 1,2-proton transfers from its substrate, R. rubrum RLP catalyzes the formation of two products, i.e., 1-thiomethyl-d-xylulose-5-phosphate and 1-thiomethyl-d-ribulose-5-phosphate, at a 3:1 ratio (15) (Fig. ​(Fig.2).2). The novel reaction catalyzed by this RLP suggests that R. rubrum likely uses a different pathway to salvage sulfur compounds.Open in a separate windowFIG. 2.Distinct reactions catalyzed by type IV-YkrW (A) and type IV-DeepYkrW (B) classes of form IV RubisCO/RLP, exemplified by the proteins from B. subtilis and R. rubrum, respectively.The presence of an RLP-encoding gene triggered the search for additional genes in the R. rubrum genome that might be homologs of known enzymes that participate in a conventional MSP. Several genes were indeed identified to encode homologs of MSP enzymes. However, to this point there is no experimental evidence for the existence of a functional MSP (21) in R. rubrum. Thus, in this study, we sought to determine the role of the RLP and RubisCO protein in sulfur salvage since each protein catalyzes different reactions and RubisCO is known to be synthesized only under anaerobic conditions (6, 7). Moreover, it is well appreciated that R. rubrum possesses a versatile metabolic capacity and is able to grow under both anaerobic and aerobic conditions, using a variety of carbon sources. The involvement of RLP and RubisCO in sulfur salvage was thus determined and found to be associated with aerobic and anaerobic metabolism, respectively.     

LDLR Expression and Localization Are Altered in Mouse and Human Cell Culture Models of Alzheimer's Disease

Background

Alzheimer''s disease (AD) is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the ε-4 allele of apolipoprotein E (apoE), the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR) has the highest affinity for apoE and plays an important role in brain cholesterol metabolism.

Methodology/Principal Findings

Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Aβ-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of γ- and α-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network.

Conclusions/Significance

These data suggest that increased APP expression and Aβ exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.     

Reconstruction of the chemotaxis receptor-kinase assembly

In bacterial chemotaxis, an assembly of transmembrane receptors, the CheA histidine kinase and the adaptor protein CheW processes environmental stimuli to regulate motility. The structure of a Thermotoga maritima receptor cytoplasmic domain defines CheA interaction regions and metal ion-coordinating charge centers that undergo chemical modification to tune receptor response. Dimeric CheA-CheW, defined by crystallography and pulsed ESR, positions two CheWs to form a cleft that is lined with residues important for receptor interactions and sized to clamp one receptor dimer. CheW residues involved in kinase activation map to interfaces that orient the CheW clamps. CheA regulatory domains associate in crystals through conserved hydrophobic surfaces. Such CheA self-contacts align the CheW receptor clamps for binding receptor tips. Linking layers of ternary complexes with close-packed receptors generates a lattice with reasonable component ratios, cooperative interactions among receptors and accessible sites for modification enzymes.     

Inhibition of beta(S)-chain dependent polymerization by synergistic complementation of contact site perturbations of alpha-chain: application of semisynthetic chimeric alpha-chains

Mouse alpha(1-30)-horse alpha(31-141) chimeric alpha-chain, a semisynthetic super-inhibitory alpha-chain, inhibits beta(S)-chain dependent polymerization better than both parent alpha-chains. Although contact site sequence differences are absent in the alpha(1-30) region of the chimeric chain, the four sequence differences of the region alpha(17-22) could induce perturbations of the side chains at alpha(16), alpha(20) and alpha(23), the three contact sites of the region. A synergistic complementation of such contact site perturbation with that of horse alpha(31-141) probably results in the super-inhibitory activity of the chimeric alpha-chain. The inhibitory contact site sequence differences, by themselves, could also exhibit similar synergistic complementation. Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin (LM) mutation [His20(alpha)-->Gln], a contact site sequence difference, engineered into human-horse chimeric alpha-chain has been investigated to map such a synergistic complementation. Gln20(alpha) has little effect on the O(2) affinity of HbS, but in human-horse chimeric alpha-chain it reduces the O(2) affinity slightly. In the chimeric alpha-chain, Gln20(alpha) increased sensitivity of the betabeta cleft for the DPG influence, reflecting a cross-talk between the alpha(1)beta(1) interface and betabeta cleft in this semisynthetic chimeric HbS. In the human alpha-chain frame, the polymerization inhibitory activity of Gln20(alpha) is higher compared with horse alpha(1-30), but lower than mouse alpha(1-30). Gln20(alpha) synergistically complements the inhibitory propensity of horse alpha(31-141). However, the inhibitory activity of LM-horse chimeric alpha-chain is still lower than that of mouse-horse chimeric alpha-chain. Therefore, perturbation of multiple contact sites in the alpha(1-30) region of the mouse-horse chimeric alpha-chain and its linkage with the inhibitory propensity of horse alpha(31-141) has been now invoked to explain the super-inhibitory activity of the chimeric alpha-chain. The 'linkage-map' of contact sites can serve as a blueprint for designing synergistic complementation of multiple contact sites into alpha-chains as a strategy for generating super-inhibitory antisickling hemoglobins for gene therapy of sickle cell disease.     

Two-stage designs for gene-disease association studies with sample size constraints

Gene-disease association studies based on case-control designs may often be used to identify candidate polymorphisms (markers) conferring disease risk. If a large number of markers are studied, genotyping all markers on all samples is inefficient in resource utilization. Here, we propose an alternative two-stage method to identify disease-susceptibility markers. In the first stage all markers are evaluated on a fraction of the available subjects. The most promising markers are then evaluated on the remaining individuals in Stage 2. This approach can be cost effective since markers unlikely to be associated with the disease can be eliminated in the first stage. Using simulations we show that, when the markers are independent and when they are correlated, the two-stage approach provides a substantial reduction in the total number of marker evaluations for a minimal loss of power. The power of the two-stage approach is evaluated when a single marker is associated with the disease, and in the presence of multiple disease-susceptibility markers. As a general guideline, the simulations over a wide range of parametric configurations indicate that evaluating all the markers on 50% of the individuals in Stage 1 and evaluating the most promising 10% of the markers on the remaining individuals in Stage 2 provides near-optimal power while resulting in a 45% decrease in the total number of marker evaluations.     

Side-chain anchoring strategy for solid-phase synthesis of peptide acids with C-terminal cysteine

Many naturally occurring peptide acids, e.g., somatostatins, conotoxins, and defensins, contain a cysteine residue at the C-terminus. Furthermore, installation of C-terminal cysteine onto epitopic peptide sequences as a preliminary to conjugating such structures to carrier proteins is a valuable tactic for antibody preparation. Anchoring of N(alpha)-Fmoc, S-protected C-terminal cysteine as an ester onto the support for solid-phase peptide synthesis is known to sometimes occur in low yields, has attendant risks of racemization, and may also result in conversion to a C-terminal 3-(1-piperidinyl)alanine residue as the peptide chain grows by Fmoc chemistry. These problems are documented for several current strategies, but can be circumvented by the title anchoring strategy, which features the following: (a). conversion of the eventual C-terminal cysteine residue, with Fmoc for N(alpha)-amino protection and tert-butyl for C(alpha)-carboxyl protection, to a corresponding S-xanthenyl ((2)XAL(4)) preformed handle derivative; and (b). attachment of the resultant preformed handle to amino-containing supports. This approach uses key intermediates that are similar to previously reported Fmoc-XAL handles, and builds on earlier experience with Xan and related protection for cysteine. Implementation of this strategy is documented here with syntheses of three small model peptides, as well as the tetradecapeptide somatostatin. Anchoring occurs without racemization, and the absence of 3-(1-piperidinyl)alanine formation is inferred by retention of chains on the support throughout the cycles of Fmoc chemistry. Fully deprotected peptides, including free sulfhydryl peptides, are released from the support in excellent yield by using cocktails containing a high concentration (i.e., 80-90%) of TFA plus appropriate thiols or silanes as scavengers. High-yield release of partially protected peptides is achieved by treatment with cocktails containing a low concentration (i.e., 1-5%) of TFA. In peptides with two cysteine residues, the corresponding intramolecular disulfide-bridged peptide is obtained by either (a). oxidation, in solution, of the dithiol product released by acid; (b). simultaneous acidolytic cleavage and disulfide formation, achieved by addition of the mild oxidant DMSO to the cleavage cocktail; or (c). concomitant cleavage/cooxidation (involving a downstream S-Xan protected cysteine), using reagents such as iodine or thallium tris(trifluoroacetate) in acetic acid.     

Site specific integration of FLP recombinase in BHK-21 cell line

A binary system for gene activation and site specific integration based on conditional recombination of transfected sequences mediated by FLP recombinase from yeast was implemented in mammalian cells. In several cell lines, FLP rapidly and precisely recombined copies of its specific target sequences to activate an otherwise silent beta-galactosidase reporter gene. Clones of marked cells were generated by excisional recombination within a chromosomally integrated copy of the silent reporters. These clones exhibited intense blue colour with X-Gal staining solution.     

Chiral synthesis of (2S,3S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol

Resolution of (2RS,3RS)-2-[alpha-(2-methoxymethoxyphenoxy)phenylmethyl]morpholine, 11, with (+) mandelic acid led to the formation of (+)-(2S,3S)-2-[alpha-(2-methoxymethoxyphenoxy)phenyl methyl] morpholine (11a). Compound 11 was synthesized in seven steps from (2RS,3RS)-cinnamyl alcohol-2,3-epoxide (4), with an overall yield of 17%. Cleavage of the methoxymethyl group of the Fmoc derivative 12 with catalytic amounts of p-toluenesulfonic acid in methanol afforded (+)-(2S,3S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol 2. The synthetic utility as well as the configuration of compound 2 has been demonstrated by converting (S,S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol 2 to (2S,3S)-2-[alpha-(2-ethoxyphenoxy)phenylmethyl]morpholine (1) and (2S,3S)-2-(2-methoxyphenoxy) benzyl)morpholine (16), two potential norepinephrine reuptake inhibitors under clinical evaluation.