A HIF-1 target, ATIA, protects cells from apoptosis by modulating the mitochondrial thioredoxin, TRX2

The regulation of apoptosis is critical for controlling tissue homeostasis and preventing tumor formation and growth. Reactive oxygen species (ROS) generation plays a key role in such regulation. Here, we describe a HIF-1 target, Vasn/ATIA (anti-TNFα-induced apoptosis), which protects cells against TNFα- and hypoxia-induced apoptosis. Through the generation of ATIA knockout mice, we show that ATIA protects cells from apoptosis through regulating the function of the mitochondrial antioxidant, thioredoxin-2, and ROS generation. ATIA is highly expressed in human glioblastoma, and ATIA knockdown in glioblastoma cells renders them sensitive to hypoxia-induced apoptosis. Therefore, ATIA is not only a HIF-1 target that regulates mitochondrial redox pathways but also a potentially diagnostic marker and therapeutic target in human glioblastoma.     

Arsenate arrests flagellar rotation in cytoplasm-free envelopes of bacteria.

The effect of arsenate on flagellar rotation in cytoplasm-free flagellated envelopes of Escherichia coli and Salmonella typhimurium was investigated. Flagellar rotation ceased as soon as the envelopes were exposed to arsenate. Inclusion of phosphate intracellularly (but not extracellular) prevented the inhibition by arsenate. In a parallel experiment, the rotation was not affected by inclusion of an ATP trap (hexokinase and glucose) within the envelopes. It is concluded that arsenate affects the motor in a way other than reversible deenergization. This may be an irreversible damage to the cell or direct inhibition of the motor by arsenate. The latter possibility suggests that a process of phosphorylation or phosphate binding is involved in the motor function.     

CGHAnalyzer: a stand-alone software package for cancer genome analysis using array-based DNA copy number data

SUMMARY: This synopsis provides an overview of array-based comparative genomic hybridization data display, abstraction and analysis using CGHAnalyzer, a software suite, designed specifically for this purpose. CGHAnalyzer can be used to simultaneously load copy number data from multiple platforms, query and describe large, heterogeneous datasets and export results. Additionally, CGHAnalyzer employs a host of algorithms for microarray analysis that include hierarchical clustering and class differentiation. AVAILABILITY: CGHAnalyzer, the accompanying manual, documentation and sample data are available for download at http://acgh.afcri.upenn.edu. This is a Java-based application built in the framework of the TIGR MeV that can run on Microsoft Windows, Macintosh OSX and a variety of Unix-based platforms. It requires the installation of the free Java Runtime Environment 1.4.1 (or more recent) (http://www.java.sun.com).     

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.     

Sizing DNA using a nanometer-diameter pore

Each species from bacteria to human has a distinct genetic fingerprint. Therefore, a mechanism that detects a single molecule of DNA represents the ultimate analytical tool. As a first step in the development of such a tool, we have explored using a nanometer-diameter pore, sputtered in a nanometer-thick inorganic membrane with a tightly focused electron beam, as a transducer that detects single molecules of DNA and produces an electrical signature of the structure. When an electric field is applied across the membrane, a DNA molecule immersed in electrolyte is attracted to the pore, blocks the current through it, and eventually translocates across the membrane as verified unequivocally by gel electrophoresis. The relationship between DNA translocation and blocking current has been established through molecular dynamics simulations. By measuring the duration and magnitude of the blocking current transient, we can discriminate single-stranded from double-stranded DNA and resolve the length of the polymer.     

A Comprehensive Model of the Spectrin Divalent Tetramer Binding Region Deduced Using Homology Modeling and Chemical Cross-linking of a Mini-spectrin

Spectrin dimer-tetramer interconversion is a critical contributor to red cell membrane stability, but some properties of spectrin tetramer formation cannot be studied effectively using monomeric recombinant domains. To address these limitations, a fused αβ mini-spectrin was produced that forms wild-type divalent tetramer complexes. Using this mini-spectrin, a medium-resolution structure of a seven-repeat bivalent tetramer was produced using homology modeling coupled with chemical cross-linking. Inter- and intramolecular cross-links provided critical distance constraints for evaluating and optimizing the best conformational model and appropriate docking interfaces. The two strands twist around each other to form a super-coiled, rope-like structure with the AB helix face of one strand associating with the opposing AC helix face. Interestingly, two tetramer site hereditary anemia mutations that exhibit wild-type binding in univalent head-to-head assays are located in the interstrand region. This suggests that perturbations of the interstrand region can destabilize spectrin tetramers and the membrane skeleton. The α subunit N-terminal cross-links to multiple sites on both strands, demonstrating that this non-homologous tail remains flexible and forms heterogeneous structures in the tetramer complex. Although no cross-links were observed involving the β subunit non-homologous C-terminal tail, several cross-links were observed only when this domain was present, suggesting it induces subtle conformational changes to the tetramer site region. This medium-resolution model provides a basis for further studies of the bivalent spectrin tetramer site, including analysis of functional consequences of interstrand interactions and mutations located at substantial molecular distances from the tetramer site.     

Changes in the Min Oscillation Pattern before and after Cell Birth

The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.Rod-shaped bacteria, such as Escherichia coli, divide by binary fission and thus assemble their cell division apparatus (the divisome) at the cell midpoint. Tubulin-like FtsZ is the major cytoskeletal protein of the divisome (17) and assembles into a polymeric ring on the inner surface of the cytoplasmic membrane (the Z ring). Assembly and eventual contraction of the Z ring are crucial for divisome function, and thus it is not surprising that many regulatory factors control FtsZ assembly (25). Notably, two negatively acting spatial regulatory systems, the Min system and nucleoid occlusion, ensure that the Z ring is located properly at the cell midpoint (18). Whereas a major component of the nucleoid occlusion system can be deleted with no major effects on cell division (2), inactivation of the Min system causes cells to divide either at midcell or aberrantly at cell poles (27). The result of polar cell division is the formation of chromosome-free minicells.The Min system consists of three proteins, MinC, MinD, and MinE (7). MinC has two separate domains, each of which binds to FtsZ and promotes disassembly of FtsZ polymers and polymer bundles (6, 29, 30). MinC also binds to MinD, an ATPase with a carboxy-terminal amphipathic helix that binds to the membrane only when the protein is bound to ATP (11, 12). MinD also forms polymers (31). Finally, MinE is a small protein that binds to MinD and stimulates hydrolysis of its bound ATP in the presence of membranes. By doing so, MinE helps to dislodge MinD from the membrane, although MinE itself can bind to the membrane (10). The result is that MinD and MinE form zones that oscillate from one cell pole to the other, with an oscillation period of seconds to minutes, depending on a number of factors, including temperature (9, 23, 24, 34). In typical cells, MinD spends most of its time bound to the membrane at a cell pole, forming a U-shaped zone, and its transit to the opposite pole is rapid compared to its dwell time (23). MinE typically forms a ring at the edge of the MinD zone (22, 24). The direction of the oscillation is determined strongly by cell geometry (5, 35). Other factors, such as membrane phospholipid composition, also influence MinD oscillation; MinD-ATP preferentially binds anionic phospholipids, such as cardiolipin, which is enriched at cell poles (15, 21, 32).Because MinC binds to MinD, MinC oscillates in concert with MinD and therefore is present at the cell poles for longer times than anywhere else in the cell (13, 22). This sets up a gradient of MinC, with the average smallest amount of MinC at midcell at any one time. The current model is that Z rings are most likely to assemble at the trough of the MinC gradient and are discouraged from assembling at cell poles at the peak of the gradient (14). This is supported by the observation that nonring FtsZ itself oscillates from pole to pole, presumably being chased back and forth by the alternating zones of high MinC concentration (33).However, recent work in Bacillus subtilis has shed new light on the possible function of MinC on the Z ring and the divisome. B. subtilis lacks MinE and thus relies on a static MinC gradient. This is set up by the recruitment of MinC and MinD (MinCD) to the Z ring during formation of the division septum (19, 20). This seems paradoxical, as the presence of MinCD at the Z ring is predicted to destabilize it. However, in B. subtilis, Z rings containing MinCD remain functional. Therefore, MinCD seems to have an important role in preventing the immediate reassembly of Z rings at developing cell poles next to a recently used ring (4, 8).This recruitment of MinCD to the Z ring of B. subtilis prompted us to examine in more detail Min oscillations in E. coli cells undergoing septation. We hypothesized that MinCD might bind to the Z ring at later stages of septation, perhaps helping the Z ring to function by stimulation of FtsZ disassembly. Previous results with green fluorescent protein (GFP)-MinC suggested that MinC could transiently localize to the Z ring during septation (13). Consequently, we tested if MinD, the driving force of the oscillation, could also localize to the Z ring and if this localization was dependent on MinC. We also hypothesized that a more central localization of MinCD during the time of septum formation might explain how Min proteins are partitioned equitably to both daughter cells.     

Diverse antiapoptotic signaling pathways activated by vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase in prostate cancer cells converge on BAD

It has been demonstrated that vasoactive intestinal polypeptide, epidermal growth factor, and chronic activation of phosphatidylinositol 3-kinase can protect prostate cancer cells from apoptosis; however, the signaling pathways that they use and molecules that they target are unknown. We report that vasoactive intestinal polypeptide, epidermal growth factor, and phosphatidylinositol 3-kinase activate independent signaling pathways that phosphorylate the proapoptotic protein BAD. Vasoactive intestinal polypeptide operated via protein kinase A, epidermal growth factor required Ras activity, and effects of phosphatidylinositol 3-kinase were predominantly mediated by Akt. BAD phosphorylation was critical for the antiapoptotic effects of each signaling pathway. None of these survival signals was able to rescue cells that express BAD with mutations in phosphorylation sites, whereas knockdown of BAD expression with small hairpin RNA rendered cells insensitive to apoptosis. Taken together, these results identify BAD as a convergence point of several antiapoptotic signaling pathways in prostate cells.