Distinct structural and adhesive roles of Ca2+ in membrane binding of blood coagulation factors

The GLA domain, a common membrane-anchoring domain of several serine protease coagulation factors, is a key element in membrane association and activation of these factors in a highly Ca2+-dependent manner. However, the critical role of Ca2+ ions in binding is only poorly understood. Here, we present the atomic model of a membrane-bound GLA domain by using MD simulations of the GLA domain of human factor VIIa and an anionic lipid bilayer. The binding is furnished through a complete insertion of the omega-loop into the membrane and through direct interactions of structurally bound Ca2+ ions and protein side chains with negative lipids. The model suggests that Ca2+ ions play two distinct roles in the process: the four inner Ca2+ ions are primarily responsible for optimal folding of the GLA domain for membrane insertion, whereas the outer Ca2+ ions anchor the protein to the membrane through direct contacts with lipids.     

DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G₁-like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc ^scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc ^scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc ^scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc ^scid . Applying the same dose of IR to Prdkc ^?/? and Ku ^?/? mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc ^?/? cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku ^?/? MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.     

Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization.

Cleavage of Bid has been shown to promote apoptosis by inducing mitochondrial membrane permeabilization with the resultant release of apoptosis-inducing proteins from the intermembrane space into the cytosol. However, direct visualization of the Bid-induced release of various proteins from the highly compartmentalized intermembrane space and the changes in the mitochondrial metabolic machinery remain elusive. Using green fluorescent protein fusion proteins and immunostaining in individual permeabilized HepG2 cells, first we demonstrated that truncated Bid (15.5-kDa C-terminal fragment, tBid) evoked a rapid and essentially complete release of cytochrome c and Smac/DIABLO from every mitochondrion. To establish at a resolution of seconds the kinetics of tBid-induced cytochrome c and Smac/DIABLO release and depolarization, we monitored the mitochondrial membrane potential (DeltaPsi(m)) fluorimetrically in permeabilized cells and applied a rapid filtration method to obtain cytosolic fractions for Western blotting. We found that subnanomolar doses of tBid were sufficient to evoke cytochrome c release and mitochondrial depolarization, whereas full-length Bid was 100-fold less effective. Bcl-x(L) prevented tBid-induced cytochrome c release and depolarization. In response to 2.5 nm tBid, cytochrome c release started after a 10 s delay, displayed rapid progression, and was complete at 50-70 s. Release of Smac/DIABLO was synchronized with cytochrome c release, whereas the loss of DeltaPsi(m) lagged slightly behind cytochrome c release. Furthermore, tBid-induced cytochrome c release was insensitive to changes in substrate composition, but tBid-induced depolarization did not occur in the presence of extramitochondrial ATP supply. Thus, tBid-induced permeabilization of the outer membrane permits rapid release of cytochrome c and Smac/DIABLO from all domains of the intermembrane space. The tBid-induced loss of DeltaPsi(m) occurs after cytochrome c release and reflects impairment of oxidative metabolism.     

New 1,2,3-triazole linked ciprofloxacin-chalcones induce DNA damage by inhibiting human topoisomerase I& II and tubulin polymerization

A series of novel 1,2,3-triazole-linked ciprofloxacin-chalcones 4a-j were synthesised as potential anticancer agents. Hybrids 4a-j exhibited remarkable anti-proliferative activity against colon cancer cells. Compounds 4a-j displayed IC₅₀s ranged from 2.53-8.67 µM, 8.67–62.47 µM, and 4.19–24.37 µM for HCT116, HT29, and Caco-2 cells; respectively, whereas the doxorubicin, showed IC₅₀ values of 1.22, 0.88, and 4.15 µM. Compounds 4a, 4b, 4e, 4i, and 4j were the most potent against HCT116 with IC₅₀ values of 3.57, 4.81, 4.32, 4.87, and 2.53 µM, respectively, compared to doxorubicin (IC₅₀ = 1.22 µM). Also, hybrids 4a, 4b, 4e, 4i, and 4j exhibited remarkable inhibitory activities against topoisomerase I, II, and tubulin polymerisation. They increased the protein expression level of γH2AX, indicating DNA damage, and arrested HCT116 in G2/M phase, possibly through the ATR/CHK1/Cdc25C pathway. Thus, the novel ciprofloxacin hybrids could be exploited as potential leads for further investigation as novel anticancer medicines to fight colorectal carcinoma.     

Genetic aspects of inflammation and cancer

Chronic inflammation is involved in the pathogenesis of most common cancers. The aetiology of the inflammation is varied and includes microbial, chemical and physical agents. The chronically inflamed milieu is awash with pro-inflammatory cytokines and is characterized by the activation of signalling pathways that cross-talk between inflammation and carcinogenesis. Many of the factors involved in chronic inflammation play a dual role in the process, promoting neoplastic progression but also facilitating cancer prevention. A comprehensive understanding of the molecular and cellular inflammatory mechanisms involved is vital for developing preventive and therapeutic strategies against cancer. The purpose of the present review is to evaluate the mechanistic pathways that underlie chronic inflammation and cancer with particular emphasis on the role of host genetic factors that increase the risk of carcinogenesis.     

Conformational coupling of the nucleotide-binding and the transmembrane domains in ABC transporters

Basic architecture of ABC transporters includes two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Although the transport process takes place in the TMDs, which provide the substrate translocation pathway across the cell membrane and control its accessibility between the two sides of the membrane, the energy required for the process is provided by conformational changes induced in the NBDs by binding and hydrolysis of ATP. Nucleotide-dependent conformational changes in the NBDs, therefore, need to be coupled to structural changes in the TMDs. Using molecular dynamics simulations, we have investigated the structural elements involved in the conformational coupling between the NBDs and the TMDs in the Escherichia coli maltose transporter, an ABC importer for which an intact structure is available both in inward-facing and outward-facing conformations. The prevailing model of coupling is primarily based on a single structural motif, known as the coupling helices, as the main structural element for the NBD-TMD coupling. Surprisingly, we find that in the absence of the NBDs the coupling helices can be conformationally decoupled from the rest of the TMDs, despite their covalent connection. That is, the structural integrity of the coupling helices and their tight coupling to the core of the TMDs rely on the contacts provided by the NBDs. Based on the conformational and dynamical analysis of the simulation trajectories, we propose that the core coupling elements in the maltose transporter involve contributions from several structural motifs located at the NBD-TMD interface, namely, the EAA loops from the TMDs, and the Q-loop and the ENI motifs from the NBDs. These three structural motifs in small ABC importers show a high degree of correlation in motion and mediate the necessary conformational coupling between the core of TMDs and the helical subdomains of NBDs. A comprehensive analysis of the structurally known ABC transporters shows a high degree of conservation of the identified 3-motif coupling elements only in the subfamily of small ABC importers, suggesting a distinct mode of NBD-TMD coupling from the other two major ABC transporter folds, namely large ABC importers and ABC exporters.     

Nitric oxide conduction by the brain aquaporin AQP4

Involvement of aquaporins in gas conduction across the membrane and the physiological significance of this process have attracted marked attention from both experimental and theoretical studies. Previous work demonstrated that AQP1 is permeable to both CO₂ and O₂. Here we employ various simulation techniques to examine the permeability of the brain aquaporin AQP4 to NO and O₂ and to describe energetics and pathways associated with these phenomena. The energy barrier to NO and O₂ permeation through AQP4 central pore is found to be only ～3 kcal mol^?1. The results suggest that the central pore of AQP4, similar to that of AQP1, can indeed conduct gas molecules. Interestingly, despite a longer and narrower central pore, AQP4 appears to provide an energetically more favorable permeation pathway for gas molecules than AQP1, mainly due to the different orientation of its charged residues near the pore entrance. Although the low barrier against gas permeation through AQP4 indicates that it can participate in gas conduction across the cellular membrane, physiological relevance of the phenomenon remains to be established experimentally, particularly since pure lipid bilayers appear to present a more favorable pathway for gas conduction across the membrane. With an energy well of ?1.8 kcal mol^?1, the central pore of AQP4 may also act as a reservoir for NO molecules to accumulate in the membrane. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Exploring the cardioprotective effects of canagliflozin against cisplatin-induced cardiotoxicity: Role of iNOS/NF-κB,Nrf2, and Bax/cytochrome C/Bcl-2 signals

Cardiotoxicity is a severe considerable side effect of cisplatin (CDDP) that requires much medical attention. The current study investigates the cardioprotective effects of canagliflozin (CA) against CDDP-induced heart toxicity. Rats were allocated to the control group; the CA group was administered CA 10 mg/kg/day orally for 10 days; the CDDP group was injected with 7 mg/kg, intraperitoneal as a single dose on the 5th day, and the CDDP + CA group. Compared to the CDDP-treated group, CA effectively attenuated CDDP-induced heart injury as evidenced by a decrease of serum aspartate aminotransferase, alkaline phosphatase, creatine kinase-MB, and lactate dehydrogenase enzymes and supported by the alleviation of histopathological changes in cardiac tissues. Biochemically, CA attenuated cardiac oxidative injury through upregulation of the nuclear factor-erythroid 2 related factor 2 (Nrf2) signal. CA suppressed inflammation by decreasing cardiac NO₂⁻, MPO, iNOS, nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha, and interleukin 1-beta levels. Besides, CA significantly upregulated cardiac levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and p-AKT proteins. Moreover, CA remarkably mitigated CDDP-induced apoptosis via modulation of Bax, cytochrome C, and Bcl-2 protein levels. Together, the present study revealed that CA could be a good candidate for preventing CDDP-induced cardiac injury by modulating iNOS/NF-κB, Nrf2, PI3K/AKT, and Bax/cytochrome C/Bcl-2 signals.     

Long-Term Treatment with Glucocorticoids Increases Synthesis and Stability of Junctional Acetylcholine Receptors on Innervated Cultured Human Muscle

Abstract: We have studied the effect of long-term treatment with hydrocortisone on the expression of acetylcholine receptors (AChRs) at the neuromuscular junctions of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord motoneurons. Hydrocortisone increased accumulation of junctional AChRs in a dose-and time-dependent fashion. This increase was due to both decreased degradation and increased synthesis of AChRs. Other glucocorticoids, dexamethasone and prednisolone, exerted similar effects. Our study demonstrates a novel action of glucocorticoids on human junctional AChRs.