bluuebunny/crossref_metadata_2025 huggingface dataset:
Epstein–Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells
Authors: Maruo Seiji , Wu Yi , Ishikawa Satoko , Kanda Teru , Iwakiri Dai , Takada Kenzo
Published: December 2006

Epstein–Barr virus (EBV) infection converts primary human B cells into continuously proliferating lymphoblastoid cell lines (LCLs). To examine the role of EBV nuclear antigen (EBNA) 3C in the proliferation of LCLs, we established LCLs infected with an EBV recombinant that expresses EBNA3C with a C-terminal fusion to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor, E3C–HT. In the presence of 4HT, LCLs expressed the E3C–HT protein and grew like WT LCLs. When E3C–HT EBV-infected LCLs were transferred to medium without 4HT, E3C–HT protein slowly disappeared, and the LCLs gradually ceased growing. WT EBNA3C expression from an oriP plasmid transfected into E3C–HT LCLs protected the LCLs from growth arrest in medium without 4HT, whereas expression of EBNA3A or EBNA3B did not. The expression of other EBNA proteins and of LMP1, CD21, CD23, and c-myc was unaffected by EBNA3C inactivation. However, EBNA3C inactivation resulted in the accumulation of p16
INK4A
, a decrease in the hyperphosphorylated form of the retinoblastoma protein, and a decrease in the proportion of cells in S or G
2
/M phase. These results indicate that EBNA3C has an essential role in cell cycle progression and the growth maintenance of LCLs.
Presenilin mutations linked to familial Alzheimer's disease cause an imbalance in phosphatidylinositol 4,5-bisphosphate metabolism
Authors: Landman Natalie , Jeong Soon Youn , Shin Sun Young , Voronov Sergey V. , Serban Geo , Kang Min Suk , Park Myung Kyu , Di Paolo Gilbert , Chung Sungkwon , Kim Tae-Wan
Published: December 2006

Phosphatidylinositol 4,5-bisphosphate (PIP
2
) is an important cellular effector whose functions include the regulation of ion channels and membrane trafficking. Aberrant PIP
2
metabolism has also been implicated in a variety of human disease states, e.g., cancer and diabetes. Here we report that familial Alzheimer's disease (FAD)-associated presenilin mutations cause an imbalance in PIP
2
metabolism. We find that the transient receptor potential melastatin 7 (TRPM7)-associated Mg
2+
-inhibited cation (MIC) channel underlies ion channel dysfunction in presenilin FAD mutant cells, and the observed channel deficits are restored by the addition of PIP
2
, a known regulator of the MIC/TRPM7 channel. Lipid analyses show that PIP
2
turnover is selectively affected in FAD mutant presenilin cells. We also find that modulation of cellular PIP
2
closely correlates with 42-residue amyloid β-peptide (Aβ42) levels. Our data suggest that PIP
2
imbalance may contribute to Alzheimer's disease pathogenesis by affecting multiple cellular pathways, such as the generation of toxic Aβ42 as well as the activity of the MIC/TRPM7 channel, which has been linked to other neurodegenerative conditions. Thus, our study suggests that brain-specific modulation of PIP
2
may offer a therapeutic approach in Alzheimer's disease.
Flux control and excess capacity in the enzymes of glycolysis and their relationship to flight metabolism inDrosophila melanogaster
Authors: Eanes Walter F. , Merritt Thomas J. S. , Flowers Jonathan M. , Kumagai Seiji , Sezgin Efe , Zhu Chen-Tseh
Published: December 2006

An important question in evolutionary and physiological genetics is how the control of flux-base phenotypes is distributed across the enzymes in a pathway. This control is often related to enzyme-specific levels of activity that are reported to be in excess of that required for demand. In glycolysis, metabolic control is frequently considered vested in classical regulatory enzymes, each strongly displaced from equilibrium. Yet the contribution of individual steps to control is unclear. To assess enzyme-specific control in the glycolytic pathway, we usedP-element excision-derived mutagenesis inDrosophila melanogasterto generate full and partial knockouts of seven metabolic genes and to measure tethered flight performance. For most enzymes, we find that reduction to half of the normal activity has no measurable impact on wing beat frequency. The enzymes catalyzing near-equilibrium reactions, phosphoglucose isomerase, phosphoglucomutase, and triosephosphate isomerase fail to show any decline in flight performance even when activity levels are reduced to 17% or less. At reduced activities, the classic regulatory enzymes, hexokinase and glycogen phosphorylase, show significant drops in flight performance and are nearer to saturation. Our results show that flight performance is canalized or robust to the activity variation found in natural populations. Furthermore, enzymes catalyzing near-equilibrium reactions show strong genetic dominance down to low levels of activity. This implies considerable excess enzyme capacity for these enzymes.
Discrete Radon transform has an exact, fast inverse and generalizes to operations other than sums along lines
Authors: Press William H.
Published: December 2006

Götz, Druckmüller, and, independently, Brady have defined a discrete Radon transform (DRT) that sums an image's pixel values along a set of aptly chosen discrete lines, complete in slope and intercept. The transform is fast,
O
(
N
2
log
N
) for an
N
×
N
image; it uses only addition, not multiplication or interpolation, and it admits a fast, exact algorithm for the adjoint operation, namely backprojection. This paper shows that the transform additionally has a fast, exact (although iterative) inverse. The inverse reproduces to machine accuracy the pixel-by-pixel values of the original image from its DRT, without artifacts or a finite point-spread function. Fourier or fast Fourier transform methods are not used. The inverse can also be calculated from sampled sinograms and is well conditioned in the presence of noise. Also introduced are generalizations of the DRT that combine pixel values along lines by operations other than addition. For example, there is a fast transform that calculates median values along all discrete lines and is able to detect linear features at low signal-to-noise ratios in the presence of pointlike clutter features of arbitrarily large amplitude.
Epstein–Barr nuclear antigen leader protein coactivates transcription through interaction with histone deacetylase 4
Authors: Portal D. , Rosendorff A. , Kieff E.
Published: December 2006

Epstein–Barr nuclear antigen (EBNA) leader protein (EBNALP) coactivates promoters with EBNA2 and is important for Epstein–Barr virus immortalization of B cells. Investigation of the role of histone deacetylases (HDACs) in EBNALP and EBNA2 promoter regulation has now identified EBNALP and EBNA2 to be associated with HDAC4 in a lymphoblastoid cell line. Furthermore, a transcription-deficient EBNALP point mutant did not associate with HDAC4. HDAC4 and 5 overexpression repressed EBNA2 activation and EBNALP coactivation, whereas other HDACs had little effect. Moreover, EBNALP expression decreased nuclear HDAC4. Expression of 14-3-3 anchors HDAC4 in the cytoplasm, increased EBNALP effects, and reversed HDAC4 or 5 repression. HDAC4 reversal depended on the HDAC4 nuclear export sequence. Consistent with EBNALP coactivation being mediated by nuclear HDAC4 depletion, HDAC4 overexpression increased nuclear HDAC4 and specifically repressed EBNA2-dependent activation as well as EBNALP-dependent coactivation. Also, EBNALP, HDAC4, and 14-3-3 could be immunoprecipitated in a single complex. Thus, these data strongly support a model in which EBNALP coactivates transcription by relocalizing HDAC4 and 5 from EBNA2 activated promoters to the cytoplasm. The observed EBNALP effects are likely also in part through HDAC5, which is highly homologous to HDAC4.
Polyoma and SV40 proteins differentially regulate PP2A to activate distinct cellular signaling pathways involved in growth control
Authors: Rodriguez-Viciana Pablo , Collins Crista , Fried Mike
Published: December 2006

Binding of Src family kinases to membrane-associated polyoma virus middle T-antigen (PyMT) can result in the phosphorylation of PyMT tyrosine 250, which serves as a docking site for the binding of Shc and subsequent activation of the Raf-MEK-ERK (MAP) kinase cascade. In a screen for PyMT variants that could not activate the ARF tumor suppressor, we isolated a cytoplasmic nontransforming mutant (MTA) that encoded a C-terminal truncated form of the PyMT protein. Surprisingly, MTA was able to strongly activate the MAP kinase pathway in the absence of Src family kinase and Shc binding. Interestingly, the polyoma small T-antigen (PyST), which shares with MTA both partial amino acid sequence homology and cellular location, also activates the MAP kinase cascade. Activation of the MAP kinase cascade by both MTA and PyST has been demonstrated to be PP2A-dependent. Neither MTA nor PyST activate the phosphorylation of AKT. The SV40 small T-antigen, which is similar to PyST in containing a J domain and in binding to the PP2A AC dimer, does not activate the MAP kinase cascade, but does stimulate phosphorylation of AKT in a PP2A-dependent manner. These findings highlight a novel role of PP2A in stimulating the MAP kinase cascade and indicate that the similar polyoma and SV40 small T-antigens influence PP2A to activate discrete cellular signaling pathways involved in growth control.
Aboveground sink strength in forests controls the allocation of carbon below ground and its [CO2]-induced enhancement
Authors: Palmroth Sari , Oren Ram , McCarthy Heather R. , Johnsen Kurt H. , Finzi Adrien C. , Butnor John R. , Ryan Michael G. , Schlesinger William H.
Published: December 2006

The partitioning among carbon (C) pools of the extra C captured under elevated atmospheric CO2concentration ([CO2]) determines the enhancement in C sequestration, yet no clear partitioning rules exist. Here, we used first principles and published data from four free-air CO2enrichment (FACE) experiments on forest tree species to conceptualize the total allocation of C to below ground (TBCA) under current [CO2] and to predict the likely effect of elevated [CO2]. We show that at a FACE site where leaf area index (L) ofPinus taedaL. was altered through nitrogen fertilization, ice-storm damage, and droughts, changes inL, reflecting the aboveground sink for net primary productivity, were accompanied by opposite changes in TBCA. A similar pattern emerged when data were combined from the four FACE experiments, using leaf area duration (LD) to account for differences in growing-season length. Moreover, elevated [CO2]-induced enhancement of TBCA in the combined data decreased from ≈50% (700 g C m−2y−1) at the lowestLDto ≈30% (200 g C m−2y−1) at the highestLD. The consistency of the trend in TBCA withLand its response to [CO2] across the sites provides a norm for predictions of ecosystem C cycling, and is particularly useful for models that useLto estimate components of the terrestrial C balance.
Conservation of the metabolomic response to starvation across two divergent microbes
Authors: Brauer Matthew J. , Yuan Jie , Bennett Bryson D. , Lu Wenyun , Kimball Elizabeth , Botstein David , Rabinowitz Joshua D.
Published: December 2006

We followed 68 cellular metabolites after carbon or nitrogen starvation of
Escherichia coli
and
Saccharomyces cerevisiae
, using a filter-culture methodology that allows exponential growth, nondisruptive nutrient removal, and fast quenching of metabolism. Dynamic concentration changes were measured by liquid chromatography–tandem mass spectrometry and viewed in clustered heat-map format. The major metabolic responses anticipated from metabolite-specific experiments in the literature were observed as well as a number of novel responses. When the data were analyzed by singular value decomposition, two dominant characteristic vectors were found, one corresponding to a generic starvation response and another to a nutrient-specific starvation response that is similar in both organisms. Together these captured a remarkable 72% of the metabolite concentration changes in the full data set. The responses described by the generic starvation response vector (42%) included, for example, depletion of most biosynthetic intermediates. The nutrient-specific vector (30%) included key responses such as increased phosphoenolpyruvate signaling glucose deprivation and increased α-ketoglutarate signaling ammonia deprivation. Metabolic similarity across organisms extends from the covalent reaction network of metabolism to include many elements of metabolome response to nutrient deprivation as well.
Estrogen receptor β expression in the embryonic brain regulates development of calretinin-immunoreactive GABAergic interneurons
Authors: Fan Xiaotang , Warner Margaret , Gustafsson Jan-Åke
Published: December 2006

Our previous studies with estrogen receptor β knockout (ERβ
−/−
) mice demonstrated that ERβ is necessary for embryonic development of the brain as early as embryonic day 14.5 (E14.5) and is involved in neuronal migration. Such early effects of ER were unexpected because estradiol synthesis and action in the brain occur at E18.5. In the present study, we examined the distribution of ERβ in the developing brain and identified a population of ERβ-regulated interneurons. ERβ appears in the brain at E12.5, mainly localized in the wall of the midbrain, neuromere, hypothalamus, thalamus, and basal plate of pons. At E15.5 and E16.5, ERβ expression increased in the hypothalamus, thalamus, and midbrain and appeared in the limbic forebrain. At E18.5, ERβ expression was strongly expressed throughout the brain, including the cerebellum and striatum, whereas there were very few positive cells in the ventricular region. In the paraventricular thalamic nucleus and parafascicular nucleus, most of the calretinin-immunopositive interneurons expressed ERβ. In ERβ
−/−
mice, calretinin expression was markedly lower than in WT mice in the hippocampus, thalamus, and amygdala both at E16.5 and at E18.5. Epidermal growth factor receptor expression was lower in the cortex of ERβ
−/−
than in WT mice at E15.5 and, unlike WT mice, was absent from the superficial marginal zone. These findings suggest that ERβ in the embryonic brain is necessary for the development of calretinin-immunoreactive GABAergic interneurons and for neuronal migration in the cortex through modulating epidermal growth factor receptor expression at middle and later embryonic stages.
Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages
Authors: Perera Rushika M. , Zoncu Roberto , Lucast Louise , De Camilli Pietro , Toomre Derek
Published: December 2006

Phosphoinositides are thought to play an important role in clathrin-coated pit (CCP) dynamics. Biochemical and structural studies have shown a direct interaction of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P
2
] with endocytic clathrin adaptors, whereas functional studies using cell-free systems or intact cells have demonstrated the importance of PI(4,5)P
2
synthesis and dephosphorylation in clathrin coating and uncoating, respectively. Furthermore, genetic manipulations of kinases and phosphatases involved in PI(4,5)P
2
metabolism result in major defects in synaptic vesicle recycling and other forms of clathrin-dependent endocytosis. However, live imaging studies of these enzymes at CCPs have not been conducted. We have used multicolor total internal reflection fluorescence microscopy (TIRFM) to visualize the spatial-temporal recruitment of synaptojanin 1 (SJ1), a polyphosphoinositide phosphatase, and its binding partner endophilin to CCPs. Strikingly, we observed differential temporal recruitment of the two major SJ1 splice variants to CCPs. The 145-kDa isoform, the predominant isoform expressed in the brain, was rapidly recruited as a “burst,” together with endophilin, at a late stage of CCP formation. In contrast, the nonneuronal ubiquitously expressed 170-kDa isoform of SJ1 was present at all stages of CCP formation. These results raise the possibility that dynamic phosphoinositide metabolism may occur throughout the lifetime of a CCP.
Rhamm−/− fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair
Authors: Tolg Cornelia , Hamilton Sara R. , Nakrieko Kerry-Ann , Kooshesh Fatemeh , Walton Paul , McCarthy James B. , Bissell Mina J. , Turley Eva A.
Published: December 2006

Rhamm (receptor for hyaluronan-mediated motility) is an hyaluronan binding protein with limited expression in normal tissues and high expression in advanced cancers. To understand its physiological functions and identify the molecular mechanisms underlying these functions, we created mice with a genetic deletion of Rhamm. We show that Rhamm−/− fibroblasts fail to resurface scratch wounds >3 mm or invade hyaluronan-supplemented collagen gels in culture. We identify a requirement for Rhamm in the localization of CD44 to the cell surface, formation of CD44–ERK1,2 (extracellular-regulated kinase 1,2) complexes, and activation/subcellular targeting of ERK1,2 to the cell nucleus. We also show that cell surface Rhamm, restricted to the extracellular compartment by linking recombinant protein to beads, and expression of mutant active mitogen-activated kinase kinase 1 (Mek1) are sufficient to rescue aberrant signaling through CD44–ERK1,2 complexes in Rh−/− fibroblasts. ERK1,2 activation and fibroblast migration/differentiation is also defective during repair of Rh−/− excisional skin wounds and results in aberrant granulation tissue in vivo. These results identify Rhamm as an essential regulator of CD44–ERK1,2 fibroblast motogenic signaling required for wound repair.
Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism
Authors: Pelicano Hélène , Xu Rui-hua , Du Min , Feng Li , Sasaki Ryohei , Carew Jennifer S. , Hu Yumin , Ramdas Latha , Hu Limei , Keating Michael J. , Zhang Wei , Plunkett William , Huang Peng
Published: December 2006

Cancer cells exhibit increased glycolysis for ATP production due, in part, to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration, how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (ρ-) harboring mitochondrial DNA deletion exhibit dependency on glycolysis, increased NADH, and activation of Akt, leading to drug resistance and survival advantage in hypoxia. Similarly, chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism, leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents.
β4 integrin activates a Shp2–Src signaling pathway that sustains HGF-induced anchorage-independent growth
Authors: Bertotti Andrea , Comoglio Paolo M. , Trusolino Livio
Published: December 2006

Despite being a cell–matrix adhesion molecule, β4 integrin can prompt the multiplication of neoplastic cells dislodged from their substrates (anchorage-independent growth). However, the molecular events underlying this atypical behavior remain partly unexplored. We found that activation of the Met receptor for hepatocyte growth factor results in the tyrosine phosphorylation of β4, which is instrumental for integrin-mediated recruitment of the tyrosine phosphatase Shp2. Shp2 binding to β4 enhances the activation of Src, which, in turn, phosphorylates the multiadaptor Gab1 predominantly on consensus sites for Grb2 association, leading to privileged stimulation of the Ras–extracellular signal-regulated kinase (ERK) cascade. This signaling axis can be inhibited by small interfering RNA–mediated β4 depletion, by a β4 mutant unable to bind Shp2, and by pharmacological and genetic inhibition of Shp2 or Src. Preservation of the β4 docking sites for Shp2 as well as the integrity of Shp2, Src, or ERK activity are required for the β4-mediated induction of anchorage-independent growth. These results unravel a novel pathway whereby β4 directs tyrosine kinase–based signals toward adhesion-unrelated outcomes.
Metabolic regulation of Akt: roles reversed
Authors: Coloff Jonathan L. , Rathmell Jeffrey C.
Published: December 2006

The respiration-deficient, highly glycolytic metabolic phenotype of cancer cells known as the “Warburg effect” has been appreciated for many years. A new study (see Pelicano et al. on p. 913 of this issue) demonstrates that respiration deficiency caused by mitochondrial mutation or hypoxia may directly promote the enormous survival advantage observed in cancer cells by activation of the phosphatidylinositol 3-kinase–Akt survival pathway. We discuss these and other recent findings that show how metabolic changes associated with cancer can play a significant role in tumor biology.
Bcl-xl does not have to bind Bax to protect T cells from death
Authors: Liu Xinqi , Zhu Yanan , Dai Shaodong , White Janice , Peyerl Fred , Kappler John W. , Marrack Philippa
Published: December 2006

Activated T cells die when antigen disappears from animals. This death is caused by proteins related to Bcl-2. Two hypotheses have been suggested to explain the actions of the different types of Bcl-2 proteins. One hypothesis suggests that, when T cells prepare to die, Bak and Bax, the proteins that actually kill activated T cells, are released from antiapoptotic proteins such as Bcl-2 and Bcl-xl. Another hypothesis suggests that Bak and Bax are normally free and are triggered to kill cells by release of messenger proteins, such as Bim, from Bcl-2 and Bcl-xl. Here, a form of Bcl-xl, which lacks a long unstructured loop, is used to show that the first hypothesis is not correct. Bcl-xl without its loop protects activated T cells from death, yet Bcl-xl without its loop cannot bind any form of Bak and Bax. Thus, binding of Bcl-xl to Bak or Bax is not involved in T cell life or death. The loop of Bcl-xl is also somewhat involved in Bcl-xl's binding of Bim because Bcl-xl without its loop binds Bim less well than wild-type Bcl-xl. Moreover, the loop may have additional, as yet unknown, functions because it changes its shape when Bcl-xl binds Bim.
Neutrophil histamine contributes to inflammation in mycoplasma pneumonia
Authors: Xu Xiang , Zhang Dongji , Zhang Hong , Wolters Paul J. , Killeen Nigel P. , Sullivan Brandon M. , Locksley Richard M. , Lowell Clifford A. , Caughey George H.
Published: December 2006

Mycoplasmas cause chronic inflammation and are implicated in asthma. Mast cells defend against mycoplasma infection and worsen allergic inflammation, which is mediated partly by histamine. To address the hypothesis that mycoplasma provokes histamine release, we exposed mice to Mycoplasma pulmonis, comparing responses in wild-type and mast cell–deficient KitW-sh/KitW-sh (W-sh) mice. Low histamine levels in uninfected W-sh mice confirmed the conventional wisdom that mast cells are principal sources of airway and serum histamine. Although mycoplasma did not release histamine acutely in wild-type airways, levels rose up to 50-fold above baseline 1 week after infection in mice heavily burdened with neutrophils. Surprisingly, histamine levels also rose profoundly in infected W-sh lungs, increasing in parallel with neutrophils and declining with neutrophil depletion. Furthermore, neutrophils from infected airway were highly enriched in histamine compared with naive neutrophils. In vitro, mycoplasma directly stimulated histamine production by naive neutrophils and strongly upregulated mRNA encoding histidine decarboxylase, the rate-limiting enzyme in histamine synthesis. In vivo, treatment with antihistamines pyrilamine or cimetidine decreased lung weight and severity of pneumonia and tracheobronchitis in infected W-sh mice. These findings suggest that neutrophils, provoked by mycoplasma, greatly expand their capacity to synthesize histamine, thereby contributing to lung and airway inflammation.
Regulation of ATM/p53-dependent suppression of myc-induced lymphomas by Wip1 phosphatase
Authors: Shreeram Sathyavageeswaran , Hee Weng Kee , Demidov Oleg N. , Kek Calvina , Yamaguchi Hiroshi , Fornace Albert J. , Anderson Carl W. , Appella Ettore , Bulavin Dmitry V.
Published: December 2006

The ataxia telangiectasia mutated (ATM) kinase is a key tumor suppressor that regulates numerous cell cycle checkpoints as well as apoptosis. Here, we report that ATM is a critical player in the regulation of apoptosis and lymphomagenesis in the presence of c-myc. In turn, deletion of the inhibitory ATM phosphatase, Wip1, results in ATM up-regulation and suppression of Eμ-myc–induced B cell lymphomas. Using mouse genetic crosses, we show that the onset of myc-induced lymphomas is dramatically delayed in Wip1-null mice in an ATM- and p53-, but not p38 MAPK– or Arf-, dependent manner. We propose that Wip1 phosphatase is critical for regulating the ATM-mediated tumor surveillance network.
Muscle Chloride Channel Dysfunction in Two Mouse Models of Myotonic Dystrophy
Authors: Lueck John D. , Mankodi Ami , Swanson Maurice S. , Thornton Charles A. , Dirksen Robert T.
Published: December 2006

Muscle degeneration and myotonia are clinical hallmarks of myotonic dystrophy type 1 (DM1), a multisystemic disorder caused by a CTG repeat expansion in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Transgenic mice engineered to express mRNA with expanded (CUG)250 repeats (HSALR mice) exhibit prominent myotonia and altered splicing of muscle chloride channel gene (Clcn1) transcripts. We used whole-cell patch clamp recordings and nonstationary noise analysis to compare and biophysically characterize the magnitude, kinetics, voltage dependence, and single channel properties of the skeletal muscle chloride channel (ClC-1) in individual flexor digitorum brevis (FDB) muscle fibers isolated from 1–3-wk-old wild-type and HSALR mice. The results indicate that peak ClC-1 current density at −140 mV is reduced >70% (−48.5 ± 3.6 and −14.0 ± 1.6 pA/pF, respectively) and the kinetics of channel deactivation increased in FDB fibers obtained from 18–20- d-old HSALR mice. Nonstationary noise analysis revealed that the reduction in ClC-1 current density in HSALR FDB fibers results from a large reduction in ClC-1 channel density (170 ± 21 and 58 ± 11 channels/pF in control and HSALR fibers, respectively) and a modest decrease in maximal channel open probability(0.91 ± 0.01 and 0.75 ± 0.03, respectively). Qualitatively similar results were observed for ClC-1 channel activity in knockout mice for muscleblind-like 1 (Mbnl1ΔE3/ΔE3), a second murine model of DM1 that exhibits prominent myotonia and altered Clcn1 splicing (Kanadia et al., 2003). These results support a molecular mechanism for myotonia in DM1 in which a reduction in both the number of functional sarcolemmal ClC-1 and maximal channel open probability, as well as an acceleration in the kinetics of channel deactivation, results from CUG repeat–containing mRNA molecules sequestering Mbnl1 proteins required for proper CLCN1 pre-mRNA splicing and chloride channel function.
Comments on the size effect on the microcantilever quality factor in free air space
Authors: Lee Jiunn-Horng , Lee Sheng-Ta , Yao Chih-Min , Fang Weileun
Published: December 2006

This study presents a numerical approach to investigate the size effect on the quality factor associated with the first mode of microcantilever vibration in 1 atm air. The numerical simulation results are verified by experiments and compared with the approximate analytical solutions. Bulk micromachined cantilever arrays are employed as the test vehicles. Based on the experimental and numerical results, this paper proposes a modification to the existing approximate models for air damping analysis by taking into account the geometry effects of the microcantilevers. The arrived semi-empirical equation suggests that the quality factors of the microcantilevers are approximately proportional toL−1.62andb0.62at a low kinetic Reynolds number. Thus, the quality factor of the microcantilever resulting from the free space air can be precisely predicted for design purposes.
Causality and pathway search in microarray time series experiment
Authors: Mukhopadhyay Nitai D. , Chatterjee Snigdhansu
Published: December 2006

Motivation: Interaction among time series can be explored in many ways. All the approach has the usual problem of low power and high dimensional model. Here we attempted to build a causality network among a set of time series. The causality has been established by Granger causality, and then constructing the pathway has been implemented by finding the Minimal Spanning Tree within each connected component of the inferred network. False discovery rate measurement has been used to identify the most significant causalities.

Results: Simulation shows good convergence and accuracy of the algorithm. Robustness of the procedure has been demonstrated by applying the algorithm in a non-stationary time series setup. Application of the algorithm in a real dataset identified many causalities, with some overlap with previously known ones. Assembled network of the genes reveals features of the network that are common wisdom about naturally occurring networks.

Contact:  nitai@lilly.com; chatterjee@stat.umn.edu
Assembling millions of short DNA sequences using SSAKE
Authors: Warren René L. , Sutton Granger G. , Jones Steven J. M. , Holt Robert A.
Published: December 2006

Summary: Novel DNA sequencing technologies with the potential for up to three orders magnitude more sequence throughput than conventional Sanger sequencing are emerging. The instrument now available from Solexa Ltd, produces millions of short DNA sequences of 25 nt each. Due to ubiquitous repeats in large genomes and the inability of short sequences to uniquely and unambiguously characterize them, the short read length limits applicability for de novo sequencing. However, given the sequencing depth and the throughput of this instrument, stringent assembly of highly identical sequences can be achieved. We describe SSAKE, a tool for aggressively assembling millions of short nucleotide sequences by progressively searching through a prefix tree for the longest possible overlap between any two sequences. SSAKE is designed to help leverage the information from short sequence reads by stringently assembling them into contiguous sequences that can be used to characterize novel sequencing targets.

Availability:  

Contact:  rwarren@bcgsc.ca
Comparing Incomparable Survey Responses: Evaluating and Selecting Anchoring Vignettes
Authors: King Gary , Wand Jonathan
Published: December 2006

When respondents use the ordinal response categories of standard survey questions in different ways, the validity of analyses based on the resulting data can be biased. Anchoring vignettes is a survey design technique, introduced by King et al. (2004, Enhancing the validity and cross-cultural comparability of measurement in survey research.American Political Science Review94 [February]: 191–205), intended to correct for some of these problems. We develop new methods both for evaluating and choosing anchoring vignettes and for analyzing the resulting data. With surveys on a diverse range of topics in a range of countries, we illustrate how our proposed methods can improve the ability of anchoring vignettes to extract information from survey data, as well as saving in survey administration costs.
Genetic organization of pre-CTX and CTX prophages in the genome of an environmental Vibrio cholerae non-O1, non-O139 strain
Authors: Maiti Diganta , Das Bhabatosh , Saha Arjun , Nandy Ranjan K. , Nair G. Balakrish , Bhadra Rupak K.
Published: December 2006

The cholera toxin (CT) is a critical determinant of the virulence of epidemic Vibrio cholerae strains. The ctxAB operon encoding CT is part of the genome of a filamentous bacteriophage CTXΦ, which may integrate as a single copy or as multiple copies in the genome of V. cholerae. The CTXΦ genome is composed of RS2 (2.4 kb) and core (4.5 kb) regions. In the present study extensive genetic mapping analyses indicated that two copies of tandemly arrayed CTX prophages are integrated in the small chromosome of an environmental V. cholerae strain, VCE232, belonging to serogroup O4. Further mapping revealed that the integration of prophages has occurred in the same genetic locus of the small chromosome of VCE232 as that of V. cholerae O1 biotype El Tor strains. Interestingly, a new type of RS2-like element 3.5 kb in size was found in the CTX prophage genome in the small chromosome of VCE232. Cloning followed by sequencing of the new RS2-like element of VCE232 revealed the presence of three ORFs, which probably encode highly divergent types of phage regulatory proteins. Furthermore, the strain VCE232 also harbours two copies of a tandemly arranged CTX prophage devoid of the ctxAB genes, called pre-CTX prophage, in its large chromosome. The presence of multiple copies of diverse CTX prophages in both the chromosomes of VCE232 suggests that toxigenic environmental V. cholerae non-O1, non-O139 strains could play a role in the emergence of new epidemic clones.
Characterization of DNA-binding specificity and analysis of binding sites of the Pseudomonas aeruginosa global regulator, Vfr, a homologue of the Escherichia coli cAMP receptor protein
Authors: Kanack Kristen J. , Runyen-Janecky Laura J. , Ferrell Evan P. , Suh Sang-Jin , West Susan E. H.
Published: December 2006

Vfr, a global regulator ofPseudomonas aeruginosavirulence factors, is a homologue of theEscherichia colicAMP receptor protein, CRP. Vfr is 91 % similar to CRP and maintains many residues important for CRP to bind cAMP, bind DNA, and interact with RNA polymerase at target promoters. Whilevfrcan complement anE. coli crpmutant inβ-galactosidase production, tryptophanase production and catabolite repression,crpcan only complement a subset of Vfr-dependent phenotypes inP. aeruginosa. Using specific CRP binding site mutations, it is shown that Vfr requires the same nucleotides as CRP for optimal transcriptional activity from theE. coli lacpromoter. In contrast, CRP did not bind Vfr target sequences in the promoters of thetoxAandregAgenes. Footprinting analysis revealed Vfr protected sequences upstream oftoxA,regA, and the quorum sensing regulatorlasR, that are similar to but significantly divergent from the CRP consensus binding sequence, and Vfr causes similar DNA bending to CRP in bound target sequences. Using a preliminary Vfr consensus binding sequence deduced from the Vfr-protected sites, Vfr target sequences were identified upstream of the virulence-associated genesplcN,plcHR,pbpG,prpLandalgD, and in thevfr/orfX,argH/fimS,pilM/ponAintergenic regions. From these sequences the Vfr consensus binding sequence, 5′-ANWWTGNGAWNY : AGWTCACAT-3′, was formulated. This study suggests that Vfr shares many of the same functions as CRP, but has specialized functions, at least in terms of DNA target sequence binding, required for regulation of a subset of genes in its regulon.
Repellents have functionally replaced hydrophobins in mediating attachment to a hydrophobic surface and in formation of hydrophobic aerial hyphae in Ustilago maydis
Authors: Teertstra Wieke R. , Deelstra Heine J. , Vranes Miroslav , Bohlmann Ralph , Kahmann Regine , Kämper Jörg , Wösten Han A. B.
Published: December 2006

Ustilago maydis contains one repellent and two class I hydrophobin genes in its genome. The repellent gene rep1 has been described previously. It encodes 11 secreted repellent peptides that result from the cleavage of a precursor protein at KEX2 recognition sites. The hydrophobin gene hum2 encodes a typical class I hydrophobin of 117 aa, while hum3 encodes a hydrophobin that is preceded by 17 repeat sequences. These repeats are separated, like the repellent peptides, by KEX2 recognition sites. Gene hum2, but not hum3, was shown to be expressed in a cross of two compatible wild-type strains, suggesting a role of the former hydrophobin gene in aerial hyphae formation. Indeed, aerial hyphae formation was reduced in a Δhum2 cross. However, the reduction in aerial hyphae formation was much more dramatic in the Δrep1 cross. Moreover, colonies of the Δrep1 cross were completely wettable, while surface hydrophobicity was unaffected and only slightly reduced in the Δhum2 and the Δhum2Δhum3 cross, respectively. It was also shown that the repellents and not the hydrophobins are involved in attachment of hyphae to hydrophobic Teflon. Deleting either or both hydrophobin genes in the Δrep1 strains did not further affect aerial hyphae formation, surface hydrophobicity and attachment. From these data it is concluded that hydrophobins of U. maydis have been functionally replaced, at least partially, by repellents.
Plasmid pBP136 from Bordetella pertussis represents an ancestral form of IncP-1β plasmids without accessory mobile elements
Authors: Kamachi Kazunari , Sota Masahiro , Tamai Yuji , Nagata Noriyo , Konda Toshifumi , Inoue Toshiro , Top Eva M. , Arakawa Yoshichika
Published: December 2006

The complete 41 268 bp nucleotide sequence of the IncP-1βplasmid pBP136 from the human pathogenBordetella pertussis, the primary aetiological agent of whooping cough, was determined and analysed. This plasmid carried a total of 46 ORFs: 44 ORFs corresponding to the genes in the conserved IncP-1βbackbone, and 2 ORFs similar to theXF1596andXF1597genes with unknown function of the plant pathogenXylella fastidiosa. Interestingly, pBP136 had no accessory genes carrying genetic traits such as antibiotic or mercury resistance and/or xenobiotic degradation. Moreover, pBP136 had only two of theklegenes (kleAE) that have been reported to be important for the stability of IncP-1 plasmid inPseudomonas aeruginosa. Phylogenetic analysis of the Kle proteins revealed that the KleA and KleE of pBP136 were phylogenetically distant from those of the present IncP-1 plasmids. In contrast, IncC1 and KorC, encoded upstream and downstream of theklegenes respectively, and the replication-initiation protein, TrfA, were closely related to those of the IncP-1β‘R751 group’. These results suggest that (i) pBP136 without any apparent accessory genes diverged early from an ancestor of the present IncP-1βplasmids, especially those of the R751 group, and (ii) theklegenes might be incorporated independently into the backbone region of the IncP-1 plasmids for their stable maintenance in various host cells.
C terminus of NisI provides specificity to nisin
Authors: Takala Timo M. , Saris Per E. J.
Published: December 2006

Nisin-producing Lactococcus lactis protects its own cell membrane against the bacteriocin with the ABC transporter NisFEG, and the immunity lipoprotein NisI. In this study, in order to localize a site for specific nisin interaction in NisI, a C-terminal deletion series of NisI was constructed, and the C-terminally truncated NisI proteins were expressed in L. lactis. The shortest deletion (5 aa) decreased the nisin immunity capacity considerably in the nisin-negative strain MG1614, resulting in approximately 78 % loss of immunity function compared with native NisI. A deletion of 21 aa decreased the immunity level even more, but longer deletions, up to 74 aa, provided the same level of nisin immunity as the 21 aa deletion, i.e. approximately 14 % of the immunity provided by native NisI. Similar to native NisI, all the C-terminally truncated NisI proteins provided higher immunity to nisin in the NisFEG-expressing strain NZ9840 than in MG1614, i.e. approximately 40–50 % of the immunity capacity of native NisI. Then, it was determined whether the NisI C-terminal 21 aa fragment could protect cells against nisin. To target the 21 aa fragment to its natural location, 21 C-terminal amino acids from the subtilin-specific immunity lipoprotein SpaI were replaced by 21 C-terminal amino acids from NisI. The expression of the SpaI′–′NisI fusion in L. lactis strains significantly increased their nisin immunity. This is the first time the immunity function of a lantibiotic immunity protein has been transferred to another protein. However, unlike native NisI, and the C-terminally truncated NisI fragments, the increase in nisin immunity conferred by the SpaI′–′NisI fusion was the same in both the NisFEG strain NZ9840 and MG1614. In conclusion, the SpaI′–′NisI fusion could not enhance nisin immunity by interacting with NisFEG, whereas the C-terminally truncated NisI fragments and native NisI were able to enhance nisin immunity, probably by co-operation with NisFEG. The results made it evident that the C terminus of NisI is involved in specific interaction with nisin, and that it confers specificity for the NisI immunity lipoprotein.
Two membrane proteins from Bifidobacterium breve UCC2003 constitute an ABC-type multidrug transporter
Authors: Margolles Abelardo , Flórez Ana Belén , Moreno José Antonio , van Sinderen Douwe , de los Reyes-Gavilán Clara G.
Published: December 2006

Intrinsic resistance to drugs is one of the main determining factors in bacterial survival in the intestinal ecosystem. This is mediated by, among others, multidrug resistance (MDR) transporters, membrane proteins which extrude noxious compounds with very different chemical structures and cellular targets. Two genes from Bifidobacterium breve encoding hypothetical membrane proteins with a high homology with members of the ATP-binding cassette (ABC) family of multidrug efflux transporters, were expressed separately and jointly in Lactococcus lactis. Cells co-expressing both proteins exhibited enhanced resistance levels to the antimicrobials nisin and polymyxin B. Furthermore, the drug extrusion activity in membrane vesicles was increased when both proteins were co-expressed, compared to membranes in which the proteins were produced independently. Both proteins were co-purified from the membrane as a stable complex in a 1 : 1 ratio. This is believed to be the first study of a functional ABC-type multidrug transporter in Bifidobacterium and contributes to our understanding of the molecular mechanisms underlying the capacity of intestinal bacteria to tolerate cytotoxic compounds.
tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I
Authors: Wang Zhijun , Yuan Zhenghong , Xiang Li , Shao Junjie , Węgrzyn Grzegorz
Published: December 2006

Effects of tRNAAla(UGC) and its derivative devoid of the 3′-ACCA motif [tRNAAla(UGC)ΔACCA] on the cleavage of the ColE1-like plasmid-derived RNA I were analysedin vivoandin vitro. In an amino-acid-starvedrelAmutant, in which uncharged tRNAs occur in large amounts, three products of specific cleavage of RNA I were observed, in contrast to an otherwise isogenicrelA+host. Overexpression of tRNAAla(UGC), which under such conditions occurs inEscherichia colimostly in an uncharged form, induced RNA I cleavage and resulted in an increase in ColE1-like plasmid DNA copy number. Such effects were not observed during overexpression of the 3′-ACCA-truncated tRNAAla(UGC). Moreover, tRNAAla(UGC), but not tRNAAla(UGC)ΔACCA, caused RNA I cleavagein vitroin the presence of MgCl2. These results strongly suggest that tRNA-dependent RNA I cleavage occurs in ColE1-like plasmid-bearingE. coli, and demonstrate that tRNAAla(UGC) participates in specific degradation of RNA Iin vivoandin vitro. This reaction is dependent on the presence of the 3′-ACCA motif of tRNAAla(UGC).
A conserved G protein (Drg1p) plays a role in regulation of invasive filamentation in Candida albicans
Authors: Chen Xi , Kumamoto Carol A.
Published: December 2006

During infection, the opportunistic fungal pathogenCandida albicansgrows invasively into the tissues of its host, forming filaments that penetrate the host tissue. To search for genes that are important for invasive filamentation, a screen for mutants that were defective in invasion of agar medium was conducted. A mutant carrying an insertion mutation in the locus of a gene, termed hereDRG1, was identified.DRG1encodes a highly conserved cytoplasmic G protein, with orthologues in the genomes of organisms from humans to yeast and archaea.C. albicansstrains lacking Drg1p were defective in producing filaments that penetrated agar media, but produced filaments normally under other conditions, such as during liquid growth. When inoculated intravenously into mice, thedrg1null mutant caused delayed lethality accompanied by delayed invasive growth in the kidneys of the host, in comparison with those of the wild-type strain. These results implicate Drg1p in the control of invasive filamentation in the laboratory, and in the progression of invasive disease in the host.
Cloning and characterization of the chromosomal arsenic resistance genes from Acidithiobacillus caldus and enhanced arsenic resistance on conjugal transfer of ars genes located on transposon TnAtcArs
Authors: Kotze Andre A. , Tuffin I. Marla , Deane Shelly M. , Rawlings Douglas E.
Published: December 2006

All strains of the moderately thermophilic, acidophilic, sulphur-oxidizing bacteriumAcidithiobacillus caldusthat have been tested contain a set of chromosomal arsenic resistance genes. Highly arsenic-resistant strains isolated from commercial arsenopyrite bio-oxidation tanks contain additional transposon-located (TnAtcArs) arsenic resistance genes. The chromosomalAt. caldus arsgenes were cloned and found to consist ofarsRandarsCgenes transcribed in one direction, andarsBin the opposite direction. ThearsRCgenes were co-transcribed with ORF1, andarsBwith ORF5 in bothAt. caldusandEscherichia coli, although deletion of ORFs 1 and 5 did not appear to affect resistance to arsenate or arsenite inE. coli. ORFs 1 and 5 have not previously been reported as part of thearsoperons, and had high amino acid identity to hypothetical proteins fromPolaromonas naphthalenivorus(76 %) andLegionella pneumophila(60 %), respectively. Reporter-gene studies showed that the arsenic operon of transposon origin (TnAtcArs) was expressed at a higher level, and was less tightly regulated inE. colithan were theAt. caldus arsgenes of chromosomal origin. Plasmid pSa-mediated conjugal transfer of TnAtcArsfromE. colitoAt. caldusstrains lacking the transposon was successful, and resulted in greatly increased levels of resistance to arsenite.
Study of the population structure of Haemophilus parasuis by multilocus sequence typing
Authors: Olvera Alex , Cerdà-Cuéllar Marta , Aragon Virginia
Published: December 2006

Haemophilus parasuisis the aetiological agent of Glässer's disease in swine. In addition, this bacterium causes other clinical outcomes and can also be isolated from the upper respiratory tract of healthy pigs. Isolates ofH. parasuisdiffer in phenotypic features (e.g. protein profiles, colony morphology or capsule production) and pathogenic capacity. Differences among strains have also been demonstrated at the genetic level. Several typing methods have been used to classifyH. parasuisfield strains, but they had resolution or implementation problems. To overcome these limitations, a multilocus sequence typing (MLST) system, using partial sequences of the house-keeping genesmdh,6pgd,atpD,g3pd,frdB,infBandrpoB, was developed. Eleven reference strains and 120 field strains were included in this study. The number of alleles per locus ranged from 14 to 41,6pgdbeing the locus with the highest diversity. The high genetic heterogeneity of this bacterium was confirmed with MLST, since the strains were divided into 109 sequence types, and only 13 small clonal complexes were detected by the Burst algorithm. Further analysis by unweighted-pair group method with arithmetic mean (UPGMA) identified six clusters. When the clinical background of the isolates was examined, one cluster was statistically associated with nasal isolation (putative non-virulent), while another cluster showed a significant association with isolation from clinical lesions (putative virulent). The remaining clusters did not show a statistical association with the clinical background of the isolates. Finally, although recombination amongH. parasuisstrains was detected, two divergent branches were found when a neighbour-joining tree was constructed with the concatenated sequences. Interestingly, one branch included almost all isolates of the putative virulent UPGMA cluster.