Green tea epigallocatechin gallate binds to and inhibits respiratory complexes in swelling but not normal rat hepatic mitochondria

Epigallocatechin gallate (EGCG), the major flavonoid in green tea, is consumed via tea products and dietary supplements, and has been tested in clinical trials. However, EGCG can cause hepatotoxicity in humans and animals by unknown mechanisms. Here EGCG effects on rat liver mitochondria were examined. EGCG showed negligible effects on oxidative phosphorylation at 7.5–100 μM in normal mitochondria. However, respiratory chain complexes (RCCs) were profoundly inhibited by EGCG in mitochondria undergoing Ca²⁺ overload-induced mitochondrial permeability transition (MPT). As RCCs are located in mitochondrial inner membranes (IM) and matrix, it was reasoned that EGCG could not readily pass through IM to affect RCCs in normal mitochondria but may do so when IM integrity is compromised. This speculation was substantiated in three ways. (1) Purified EGCG-bound proteins were barely detectable in normal mitochondria and contained no RCCs as determined by Western blotting, but swelling mitochondria contained about 1.5-fold more EGCG-bound proteins which included four RCC subunits together with cyclophilin D that locates in mitochondrial matrix. (2) Swelling mitochondria consumed more EGCG than normal ones. (3) The MPT blocker cyclosporine A diminished the above-mentioned difference. Among four subunits of RCC II, only SDHA and SDHB which locate in mitochondrial matrix, but not SDHC or SDHD which insert into the IM, were found to be EGCG targets. Interestingly, EGCG promoted Ca²⁺ overload-induced MPT only when moderate MPT already commenced. This study identified hepatic RCCs as targets for EGCG in swelling but not normal mitochondria, suggesting EGCG may trigger hepatotoxicity by worsening pre-existing mitochondria abnormalities.     

Tug-of-war model for the two-bandit problem: Nonlocally-correlated parallel exploration via resource conservation

We propose a model - the “tug-of-war (TOW) model” - to conduct unique parallel searches using many nonlocally-correlated search agents. The model is based on the property of a single-celled amoeba, the true slime mold Physarum, which maintains a constant intracellular resource volume while collecting environmental information by concurrently expanding and shrinking its branches. The conservation law entails a “nonlocal correlation” among the branches, i.e., volume increment in one branch is immediately compensated by volume decrement(s) in the other branch(es). This nonlocal correlation was shown to be useful for decision making in the case of a dilemma. The multi-armed bandit problem is to determine the optimal strategy for maximizing the total reward sum with incompatible demands, by either exploiting the rewards obtained using the already collected information or exploring new information for acquiring higher payoffs involving risks. Our model can efficiently manage the “exploration-exploitation dilemma” and exhibits good performances. The average accuracy rate of our model is higher than those of well-known algorithms such as the modified ?-greedy algorithm and modified softmax algorithm, especially, for solving relatively difficult problems. Moreover, our model flexibly adapts to changing environments, a property essential for living organisms surviving in uncertain environments.     

Determination of aconitine,hypaconitine and mesaconitine in urine using hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography

Hollow fiber liquid-phase microextraction (HF-LPME) coupled with high-performance liquid chromatography was used to simultaneously determine three Aconitum alkaloids, including aconitine (AC), hypaconitine (HA) and mesaconitine (MA) in human urine sample. Analytes were extracted from 5 mL urine sample containing 1.0 mmol/L NaOH into 1-octanol membrane phase impregnated in the pores of hollow fiber wall, and then back extracted into acidified aqueous solution in the lumen of the hollow fiber. After extraction, 10 μL of the acceptor phase was analyzed directly by HPLC. In this method, some important extraction parameters, such as organic solvent, extraction time, stirring rate, pH of donor phase and acceptor phase, temperature, and the volume of acceptor phase were optimized. This method provided 98- to 288-fold enrichment factors within 60 min of extraction and good repeatability with RSDs of 0.99–7.22%. The calibration curves were linear over the ranges of 16.0–128.0 μg/L for AC, 11.0–88.0 μg/L for HA and 8.1–64.8 μg/L for MA in human urine sample, with correlation coefficients of 0.9949, 0.9969 and 0.9904, respectively. Limits of detection were from 0.7 to 1.5 μg/L, and recoveries from spiked urine sample varied from 84.4% to 106.2% for AC, 77.3% to 85.6% for HA and 90.1% to 100.8% for MA.     

The N-terminal region of the starch-branching enzyme from Phaseolus vulgaris L. is essential for optimal catalysis and structural stability

Starch-branching enzymes (SBEs) play a pivotal role in determining the fine structure of starch by catalyzing the syntheses of alpha-1,6-branch points. They are the members of the alpha-amylase family and have four conserved regions in a central (beta/alpha)8 barrel, including the catalytic sites. Although the role of the catalytic barrel domain of an SBE is known, that of its N- and C-terminal regions remain unclear. We have previously shown that the C-terminal regions of the two SBE isozymes (designated as PvSBE1 and PvSBE2) from kidney bean (Phaseolus vulgaris L.) have different roles in branching enzyme activity. To understand the contribution of the N-terminal region to catalysis, six chimeric enzymes were constructed between PvSBE1 and PvSBE2. Only one enzyme (1Na/2Nb)-II, in which a portion of the N-terminal region of PvSBE2 was substituted by the corresponding region of PvSBE1, retained 6% of the PvSBE2 activity. The N-terminal truncated form (DeltaN46-PvSBE2), lacking 46 N-terminal residues of PvSBE2, lost enzyme activity and stability to proteolysis. To investigate the possible function of this region, three residues (Asp-15, His-24, and Arg-28) among these 46 residues were subjected to site-directed mutagenesis. The purified mutant enzymes showed nearly the same K(m) values as PvSBE2 but had lower V(max) values and heat stabilities than PvSBE2. These results suggest that the N-terminal region of the kidney bean SBE is essential for maximum enzyme activity and thermostability.     

Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.     

A P system and a constructive membrane-inspired DNA algorithm for solving the Maximum Clique Problem

We present a P system with replicated rewriting to solve the Maximum Clique Problem for a graph. Strings representing cliques are built gradually. This involves the use of inhibitors that control the space of all generated solutions to the problem. Calculating the maximum clique for a graph is a highly relevant issue not only on purely computational grounds, but also because of its relationship to fundamental problems in genomics. We propose to implement the designed P system by means of a DNA algorithm. This algorithm is then compared with two standard papers that addressed the same problem and its DNA implementation in the past. This comparison is carried out on the basis of a series of computational and physical parameters. Our solution features a significantly lower cost in terms of time, the number and size of strands, as well as the simplicity of the biological implementation.     

Human gliosarcoma-associated ganglioside composition is complex and distinctive as evidenced by high-performance mass spectrometric determination and structural characterization

Gangliosides (GGs), involved in malignant alteration and tumor progression/invasiveness, are considered as tumor biomarkers or therapeutic targets. Here, we describe the first systematic GG composition characterization in human gliosarcoma versus normal brain tissue using our recently developed mass spectrometry (MS) methods, based on nano-electrospray (nano-ESI), Fourier-transform ion cyclotron resonance (FT-ICR), and chip nano-ESI quadrupole time-of-flight (QTOF), complemented by thin-layer chromatographic (TLC) analysis and quantification. Combined MS enabled detection and structural assignment of 73 distinct GG species: many more than reported so far for investigated gliomas. Apart from the 7.4-times lower total GG content, gliosarcoma contained all major brain-associated species, however, in very altered proportions, exhibiting a highly distinctive pattern: GD3 (48.9%)>GD1a/nLD1>GD2/GT3>GM3>GT1b>GM2>GM1a/GM1b/nLM1>LM1>GD1b>GQ1b. MS also revealed abundant O-Ac-GD3; its sequencing provided structural evidence to postulate a novel O-Ac-GD3 isomer O-acetylated at the inner Neu5Ac-residue, previously not structurally confirmed. The high sensitivity and mass accuracy permitted the assignment of unusual minor species: GM4, Hex-HexNAc-nLM1, Gal-GD1, Fuc-GT1, GalNAc-GT1, O-Ac-GM3, di- O-Ac-GD3O-Ac-GD3, and O-Ac-GT3, not previously reported as glioma-associated. The gliosarcoma-expressed GA2 might represent a marker distinguishing astrocytic from oligodendroglial tumors. This is, to our knowledge, so far the most complete GG composition characterization of certain glioma, which demonstrates that our MS-based approach could provide essential structural information relevant to glycosphingolipid role(s) in brain tumor biology, differential diagnosis/prognosis and novel treatment concepts.     

Malleable applications for scalable high performance computing

Iterative applications are known to run as slow as their slowest computational component. This paper introduces malleability, a new dynamic reconfiguration strategy to overcome this limitation. Malleability is the ability to dynamically change the data size and number of computational entities in an application. Malleability can be used by middleware to autonomously reconfigure an application in response to dynamic changes in resource availability in an architecture-aware manner, allowing applications to optimize the use of multiple processors and diverse memory hierarchies in heterogeneous environments. The modular Internet Operating System (IOS) was extended to reconfigure applications autonomously using malleability. Two different iterative applications were made malleable. The first is used in astronomical modeling, and representative of maximum-likelihood applications was made malleable in the SALSA programming language. The second models the diffusion of heat over a two dimensional object, and is representative of applications such as partial differential equations and some types of distributed simulations. Versions of the heat application were made malleable both in SALSA and MPI. Algorithms for concurrent data redistribution are given for each type of application. Results show that using malleability for reconfiguration is 10 to 100 times faster on the tested environments. The algorithms are also shown to be highly scalable with respect to the quantity of data involved. While previous work has shown the utility of dynamically reconfigurable applications using only computational component migration, malleability is shown to provide up to a 15% speedup over component migration alone on a dynamic cluster environment. This work is part of an ongoing research effort to enable applications to be highly reconfigurable and autonomously modifiable by middleware in order to efficiently utilize distributed environments. Grid computing environments are becoming increasingly heterogeneous and dynamic, placing new demands on applications’ adaptive behavior. This work shows that malleability is a key aspect in enabling effective dynamic reconfiguration of iterative applications in these environments.

Plumbagin and juglone induce caspase-3-dependent apoptosis involving the mitochondria through ROS generation in human peripheral blood lymphocytes

The phytochemicals plumbagin and juglone have recently been gaining importance because of their various pharmacological activities. In this study, these compounds are shown to induce concentration- and time-dependent toxicity in human peripheral blood lymphocytes via the apoptotic pathway. Flow cytometry data revealed the occurrence of about 28% early apoptotic cells after 6 h exposure to 10 μM plumbagin and 35% late apoptotic cells and about 43% sub-G1 population after 24 h. The cytotoxic effect of plumbagin was at least twofold higher than that of juglone as evidenced by the IC₅₀ value for cytotoxicity. Characteristic apoptotic features such as chromatin condensation and apoptotic body formation were observed through TEM, and membrane blebbing and cell surface smoothening were seen in SEM studies. Generation of ROS was evidenced through the HPLC analysis of superoxide-specific 2-OH-E+ formation. In addition, a decrease in GSH levels parallel to ROS production was observed. Reversal of apoptosis in both NAC- and Tempol-pretreated cells indicates the involvement of both ROS generation and GSH depletion in plumbagin- and juglone-induced apoptosis. The mechanistic pathway involves a decrease in MMP; alterations in the levels of Bcl-2, Bax, and cytosolic cytochrome c; and PARP-1 cleavage subsequent to caspase-3 activation.     

Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO₂, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.