LC-MS/MS-based metabolites of Eurycoma longifolia (Tongkat Ali) in Malaysia (Perak and Pahang)

A number of three LC-MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small metabolites (m/z 100-1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triterpene and biphenylneolignans from the aqueous extracts of Eurycoma longifolia. The metabolite profiles of small molecules showed four significant clusters in the principle component analysis for the aqueous extracts of E. longifolia, which had been collected from different geographical terrains (Perak and Pahang) and processed at different extraction temperatures (35°C and 100°C). A small peptide of leucine (m/z 679) and a new hydroxyl methyl β-carboline propionic acid have been identified to differentiate E. longifolia extracts that prepared at 35°C and 100°C, respectively. From the targeted metabolites identification, it was found that 3,4?-dihydroeurycomanone (quassinoids) and eurylene (squalene-type triterpene) could only be detected in the Pahang extract, whereas canthin-6-one-3N-oxide could only be detected in the Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone and its derivatives compared to the other groups of metabolites. However, the concentration of canthin-6-one and β-carboline alkaloids was significantly increased when the roots of the plant samples were extracted at 100°C.     

Chemically inhibited ATP synthesis promoted detachment of different-age biofilms from membrane surface

This study investigated the response of different-age biofilms developed on membrane surface to a chemical uncoupler 3, 3', 4', 5-tetrachlorosalicylanilide (TCS). Results showed that adenosine triphosphate (ATP) dissipation caused by TCS would promote different-age biofilms detachment, whereas chemically inhibited cellular ATP synthesis subsequently suppressed autoinducer-2 (AI-2) and extracellular polymeric substances (EPS) production. The extent of biofilm detachment was found to be closely related to AI-2-regulated EPS contents of bacteria. It was revealed that energy dissipation induced biofilm detachability was controlled by AI-2 regulated cellular communication via AI-2-mediated EPS secretion. This study would lead to a new cleaning strategy of biologically fouled membrane.     

Expression of vascular cell adhesion molecule-1 indicates the differentiation potential of human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.     

Importance of SARS-CoV spike protein Trp-rich region in viral infectivity

SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalanine substitutions. Our results show that individually substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10-25% infectivity comparing to the wild-type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity.     

Parasites in food webs: the ultimate missing links

Parasitism is the most common consumer strategy among organisms, yet only recently has there been a call for the inclusion of infectious disease agents in food webs. The value of this effort hinges on whether parasites affect food‐web properties. Increasing evidence suggests that parasites have the potential to uniquely alter food‐web topology in terms of chain length, connectance and robustness. In addition, parasites might affect food‐web stability, interaction strength and energy flow. Food‐web structure also affects infectious disease dynamics because parasites depend on the ecological networks in which they live. Empirically, incorporating parasites into food webs is straightforward. We may start with existing food webs and add parasites as nodes, or we may try to build food webs around systems for which we already have a good understanding of infectious processes. In the future, perhaps researchers will add parasites while they construct food webs. Less clear is how food‐web theory can accommodate parasites. This is a deep and central problem in theoretical biology and applied mathematics. For instance, is representing parasites with complex life cycles as a single node equivalent to representing other species with ontogenetic niche shifts as a single node? Can parasitism fit into fundamental frameworks such as the niche model? Can we integrate infectious disease models into the emerging field of dynamic food‐web modelling? Future progress will benefit from interdisciplinary collaborations between ecologists and infectious disease biologists.     

H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes

The present study was aimed to investigate the regulatory effect of protein kinase C (PKC) on intracellular Ca(2+) handling in hydrogen sulfide (H(2)S)-preconditioned cardiomyocytes and its consequent effects on ischemia challenge. Immunoblot analysis was used to assess PKC isoform translocation in the rat cardiomyocytes 20 h after NaHS (an H(2)S donor, 10(-4) M) preconditioning (SP, 30 min). Intracellular Ca(2+) was measured with a spectrofluorometric method using fura-2 ratio as an indicator. Cell length was compared before and after ischemia-reperfusion insults to indicate the extent of hypercontracture. SP motivated translocation of PKCalpha, PKCepsilon, and PKCdelta to membrane fraction but only translocation of PKCepsilon and PKCdelta was abolished by an ATP-sensitive potassium channel blocker glibenclamide. It was also found that SP significantly accelerated the decay of both electrically and caffeine-induced intracellular [Ca(2+)] transients, which were reversed by a selective PKC inhibitor chelerythrine. These data suggest that SP facilitated Ca(2+) removal via both accelerating uptake of Ca(2+) into sarcoplasmic reticulum and enhancing Ca(2+) extrusion through Na(+)/Ca(2+) exchanger in a PKC-dependent manner. Furthermore, blockade of PKC also attenuated the protective effects of SP against Ca(2+) overload during ischemia and against myocyte hypercontracture at the onset of reperfusion. We demonstrate for the first time that SP activates PKCalpha, PKCepsilon, and PKCdelta in cardiomyocytes via different signaling mechanisms. Such PKC activation, in turn, protects the heart against ischemia-reperfusion insults at least partly by ameliorating intracellular Ca(2+) handling.     

Differential molecular assemblies underlie the dual function of Axin in modulating the WNT and JNK pathways.

Axin is a multidomain scaffold protein that exerts a dual function in the Wnt signaling and MEKK1/JNK pathways. This raises a critical question as to whether Axin-based differential molecular assemblies exist and how these may act to coordinate the two separate pathways. Here we show that both wild-type glycogen synthase kinase-3 beta (GSK-3 beta) and kinase-dead GSK-3 beta-Y216F (capable of binding to Axin), but not GSK-3 beta-K85M (incapable of binding to Axin in mammalian cells), prevented MEKK1 binding to the Axin complex, thereby inhibiting JNK activation. We further show that casein kinase I epsilon also inhibited Axin-mediated JNK activation by competing against MEKK1 binding. In contrast, beta-catenin and adenomatous polyposis coli binding did not affect MEKK1 binding to the same Axin complex. This suggests that even when Axin is "switched" to activate the JNK pathway, it is still capable of sequestering free beta-catenin, which is a critical aspect for cellular homeostasis. Our results clearly demonstrate that differential molecular assemblies underlie the duality of Axin functions in the negative regulation of Wnt signaling and activation of the JNK MAPK pathway.     

Axin forms a complex with MEKK1 and activates c-Jun NH(2)-terminal kinase/stress-activated protein kinase through domains distinct from Wnt signaling

Axin negatively regulates the Wnt pathway during axis formation and plays a central role in cell growth control and tumorigenesis. We found that Axin also serves as a scaffold protein for mitogen-activated protein kinase activation and further determined the structural requirement for this activation. Overexpression of Axin in 293T cells leads to differential activation of mitogen-activated protein kinases, with robust induction for c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase, moderate induction for p38, and negligible induction for extracellular signal-regulated kinase. Axin forms a complex with MEKK1 through a novel domain that we term MEKK1-interacting domain. MKK4 and MKK7, which act downstream of MEKK1, are also involved in Axin-mediated JNK activation. Domains essential in Wnt signaling, i. e. binding sites for adenomatous polyposis coli, glycogen synthase kinase-3beta, and beta-catenin, are not required for JNK activation, suggesting distinct domain utilization between the Wnt pathway and JNK signal transduction. Dimerization/oligomerization of Axin through its C terminus is required for JNK activation, although MEKK1 is capable of binding C terminus-deleted monomeric Axin. Furthermore, Axin without the MEKK1-interacting domain has a dominant-negative effect on JNK activation by wild-type Axin. Our results suggest that Axin, in addition to its function in the Wnt pathway, may play a dual role in cells through its activation of JNK/stress-activated protein kinase signaling cascade.     

Dimerization choices control the ability of axin and dishevelled to activate c-Jun N-terminal kinase/stress-activated protein kinase

Axin and Dishevelled are two downstream components of the Wnt signaling pathway. Dishevelled is a positive regulator and is placed genetically between Frizzled and glycogen synthase kinase-3beta, whereas Axin is a negative regulator that acts downstream of glycogen synthase kinase-3beta. It is intriguing that they each can activate the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) when expressed in the cell. We set out to address if Axin and Dishevelled are functionally cooperative, antagonistic, or entirely independent, in terms of the JNK activation event. We found that in contrast to Axin, Dvl2 activation of JNK does not require MEKK1, and complex formation between Dvl2 and Axin is independent of Axin-MEKK1 binding. Furthermore, Dvl2-DIX and Dvl2-DeltaDEP proteins deficient for JNK activation can attenuate Axin-activated JNK activity by disrupting Axin dimerization. However, Axin-DeltaMID, Axin-DeltaC, and Axin-CT proteins deficient for JNK activation cannot interfere with Dvl2-activated JNK activity. These results indicate that unlike the strict requirement of homodimerization for Axin function, Dvl2 can activate JNK either as a monomer or homodimer/heterodimer. We suggest that there may be a switch mechanism based on dimerization combinations, that commands cells to activate Wnt signaling or JNK activation, and to turn on specific activators of JNK in response to various environmental cues.