Sodium binding site of factor Xa: role of sodium in the prothrombinase complex

The nature of residue 225 on a consensus loop in serine proteases determines whether a protease can bind Na(+). Serine proteases with a Pro at this position are unable to bind Na(+), but those with a Tyr or Phe can bind Na(+). Factor Xa (FXa), the serine protease of the prothrombinase complex, contains a Tyr at this position. Na(+) is also known to stimulate the amidolytic activity of FXa toward cleavage of small synthetic substrates, but the role of Na(+) in the prothrombinase complex has not been investigated. In this study, we engineered a Gla-domainless form of FX (GDFX) in which residue Tyr(225) was replaced with a Pro. We found that Na(+) stimulated the cleavage rate of chromogenic substrates by FXa or GDFXa approximately 8-24-fold with apparent dissociation constants [K(d(app))] of 37 and 182 mM in the presence and absence of Ca(2+), respectively. In contrast, Na(+) minimally affected the cleavage rate of these substrates by the mutant, and no K(d(app)) for Na(+) binding to the mutant could be estimated. Unlike the wild-type enzyme, the reactivity of the mutant with antithrombin was independent of Na(+) and impaired approximately 32-fold. Ca(2+) improved the reactivity of the mutant with antithrombin approximately 5-fold. Affinity of the mutant for binding to factor Va was weakened and its ability to activate prothrombin was severely impaired. Further studies with the wild-type prothrombinase complex revealed that FXa binds to factor Va with a similar K(d(app)) of 1. 1-1.8 nM in the presence of Na(+), K(+), Li(+), Ch(+), and Tris(+) and that the catalytic efficiency of prothrombinase is enhanced less than 1.5-fold by the specific effect of Na(+) in the reaction buffer. These results suggest that (1) the loop including residue 225 (225-loop) is a Na(+) binding site in FXa, (2) the Na(+)- and Ca(2+)-binding loops of FXa are allosterically linked, and (3) the Tyr conformer of the 225-loop is critical for factor Xa function; however, both Na(+)-bound and Na(+)-free forms of factor Xa in the prothrombinase complex can efficiently activate prothrombin.     

atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. Here, we show that ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch.     

Mechanochemical regulation of oscillatory follicle cell dynamics in the developing Drosophila egg chamber

During tissue elongation from stage 9 to stage 10 in Drosophila oogenesis, the egg chamber increases in length by ∼1.7-fold while increasing in volume by eightfold. During these stages, spontaneous oscillations in the contraction of cell basal surfaces develop in a subset of follicle cells. This patterned activity is required for elongation of the egg chamber; however, the mechanisms generating the spatiotemporal pattern have been unclear. Here we use a combination of quantitative modeling and experimental perturbation to show that mechanochemical interactions are sufficient to generate oscillations of myosin contractile activity in the observed spatiotemporal pattern. We propose that follicle cells in the epithelial layer contract against pressure in the expanding egg chamber. As tension in the epithelial layer increases, Rho kinase signaling activates myosin assembly and contraction. The activation process is cooperative, leading to a limit cycle in the myosin dynamics. Our model produces asynchronous oscillations in follicle cell area and myosin content, consistent with experimental observations. In addition, we test the prediction that removal of the basal lamina will increase the average oscillation period. The model demonstrates that in principle, mechanochemical interactions are sufficient to drive patterning and morphogenesis, independent of patterned gene expression.     

Killing cancer with platycodin D through multiple mechanisms

Cancer is a multi‐faceted disease comprised of a combination of genetic, epigenetic, metabolic and signalling aberrations which severely disrupt the normal homoeostasis of cell growth and death. Rational developments of highly selective drugs which specifically block only one of the signalling pathways have been associated with limited therapeutic success. Multi‐targeted prevention of cancer has emerged as a new paradigm for effective anti‐cancer treatment. Platycodin D, a triterpenoid saponin, is one the major active components of the roots of Platycodon grandiflorum and possesses multiple biological and pharmacological properties including, anti‐nociceptive, anti‐atherosclerosis, antiviral, anti‐inflammatory, anti‐obesity, immunoregulatory, hepatoprotective and anti‐tumour activities. Recently, the anti‐cancer activity of platycodin D has been extensively studied. The purpose of this review was to give our perspectives on the current status of platycodin D and discuss its anti‐cancer activity and molecular mechanisms which may help the further design and conduct of pre‐clinical and clinical trials to develop it successfully into a potential lead drug for oncological therapy. Platycodin D has been shown to fight cancer by inducing apoptosis, cell cycle arrest, and autophagy and inhibiting angiogenesis, invasion and metastasis by targeting multiple signalling pathways which are frequently deregulated in cancers suggesting that this multi‐target activity rather than a single effect may play an important role in developing platycodin D into potential anti‐cancer drug.     

Emotional Noun Processing: An ERP Study with Rapid Serial Visual Presentation

Reading is an important part of our daily life, and rapid responses to emotional words have received a great deal of research interest. Our study employed rapid serial visual presentation to detect the time course of emotional noun processing using event-related potentials. We performed a dual-task experiment, where subjects were required to judge whether a given number was odd or even, and the category into which each emotional noun fit. In terms of P1, we found that there was no negativity bias for emotional nouns. However, emotional nouns elicited larger amplitudes in the N170 component in the left hemisphere than did neutral nouns. This finding indicated that in later processing stages, emotional words can be discriminated from neutral words. Furthermore, positive, negative, and neutral words were different from each other in the late positive complex, indicating that in the third stage, even different emotions can be discerned. Thus, our results indicate that in a three-stage model the latter two stages are more stable and universal.     

Effects of hypo- and hypersalinity on photosynthetic performance of Sargassum fusiforme (Fucales,Heterokontophyta)

Photoprotection mechanisms protect photosynthetic organisms, especially under stress conditions, against photodamage that may inhibit photosynthesis. We investigated the effects of short-term immersion in hypo- and hypersalinity sea water on the photosynthesis and xanthophyll cycle in Sargassum fusiforme (Harvey) Setchell. The results indicated that under moderate light [110 μmol(photon) m^?2 s^?1], the effective quantum yield of PSII was not reduced in S. fusiforme fronds after 1 h in hyposalinity conditions, even in fresh water, but it was significantly affected by extreme hypersalinity treatment (90‰ sea water). Under high light [HL, 800 μmol(photon) m^?2 s^?1], photoprotective mechanisms operated efficiently in fronds immersed in fresh water as indicated by high reversible nonphotochemical quenching of chlorophyll fluorescence (NPQ) and de-epoxidation state; the quantum yield of PSII recovered during the subsequent relaxation period. In contrast, fronds immersed in 90‰ sea water did not withstand HL, barely developed reversible NPQ, and accumulated little antheraxanthin and zeaxanthin during HL, while recovery of the quantum yield of PSII was severely inhibited during the subsequent relaxation period. The data provided concrete evidence supporting the short-term tolerance of S. fusiforme to immersion in fresh water compared to hypersalinity conditions. The potential practical implications of these results were also discussed.