bluuebunny/crossref_metadata_2025 huggingface dataset:
Implementation of a ray‐tracing operator for ground‐based GPS Slant Delay observation modeling
Authors: Eresmaa Reima , Healy Sean , Järvinen Heikki , Salonen Kirsti
Published: June 2008

Implementation of an observation‐modeling algorithm is necessary in order to make use of Slant Delay (SD) observations, processed from ground‐based Global Positioning System (GPS) measurements, in Numerical Weather Prediction (NWP). This article introduces an algorithm for SD observation modeling. The algorithm is an extension of radio‐occultation bending‐angle observation modeling to ground‐based GPS meteorology, and it is based on ray‐tracing in a two‐dimensional plane. The new algorithm is implemented in the framework of the High Resolution Limited Area Model (HIRLAM) in order to allow comparison with a reference model, that has been published earlier. Only insignificant differences are found between observation minus model background (OmB) statistics of the two implementations. In terms of computational efficiency, the new approach is found to be cheaper to apply in the NWP systems currently in use. However, the computational efficiency of the new implementation is shown to rapidly increase with increasing NWP model resolution.
Coupling and evaluating gas/particle mass transfer treatments for aerosol simulation and forecast
Authors: Hu Xiao‐Ming , Zhang Yang , Jacobson Mark Z. , Chan Chak K.
Published: June 2008

Simulating gas/particle mass transfer in three‐dimensional (3‐D) air quality models (AQMs) represents one of the major challenges for both hindcasting and forecasting. The lack of an efficient yet accurate gas/particle mass transfer treatment for aerosol simulation and forecast in 3‐D AQMs warrants its development, improvement, and evaluation. In this paper, several condensation/evaporation schemes (e.g., the Bott, Trajectory‐Grid (T‐G), Walcek, and analytical predictor of condensation (APC)) are first tested in a condensation‐only case. The APC and Walcek schemes are shown to be more accurate than the Bott and T‐G schemes. The Walcek and the APC schemes are then incorporated into the Model of Aerosol Dynamics, Reaction, Ionization, and Dissolution (MADRID) to solve the gas/particle mass transfer process explicitly. The test simulations with MADRID are initialized with measurements available from three sites with representative meteorological and emission characteristics. The results are evaluated using benchmark based on the kinetic approach with 500‐section for all cases and available measurements from two sites. The box MADRID tests have shown that the bulk equilibrium approach fails to predict the distribution of semivolatile species (e.g., ammonium, chloride, and nitrate) because of the equilibrium and internal mixture assumptions. The hybrid approach exhibits the same problem for some cases as the bulk equilibrium approach since it assumes bulk equilibrium for fine particles. The kinetic approaches (including the APC and Walcek schemes for the condensation/evaporation equations) predict the most accurate solutions. Among all approaches tested, the bulk equilibrium approach is the most computationally efficient, and the kinetic/Walcek scheme provides an accurate solution but is the slowest due to its requirement for a small time step. Overall, the kinetic/APC and hybrid/APC schemes are attractive for 3‐D applications in terms of both accuracy and computational efficiency.
Lateral organic carbon supply to the deep Canada Basin
Authors: Hwang Jeomshik , Eglinton Timothy I. , Krishfield Richard A. , Manganini Steven J. , Honjo Susumu
Published: June 2008

Understanding the processes driving the carbon cycle in the Arctic Ocean is important for assessing the impacts of the predicted rapid and amplified climate change in this region. We analyzed settling particle samples intercepted by a time‐series sediment trap deployed in the abyssal Canada Basin (at 3067 m) in order to examine carbon export to the deep Arctic Ocean. Strikingly old radiocarbon ages (apparent mean 14C age = ∼1900 years) of the organic carbon, abundant lithogenic material (∼80%), and mass flux variations temporally decoupled from the cycle of primary productivity in overlying surface waters together suggest that, unlike other ocean basins, the majority of the particulate organic carbon entering the deep Canada Basin is supplied from the surrounding margins.
Cerebral Blood Flow Response in Adenosine 2a Receptor Knockout Mice during Transient Hypoxic Hypoxia
Authors: Miekisiak Greg , Kulik Tobias , Kusano Yoshikazu , Kung David , Chen Jiang-Fan , Winn H Richard
Published: June 2008

We evaluated cerebral blood flow by laser Doppler during 30 secs of hypoxia (0.10 FiO2) in anesthetized, ventilated adenosine 2a receptor knockout (A2aR KO) and wild-type (WT) mice to test the hypothesis that cerebral hypoxic hyperemia in KO mice would be attenuated. We also studied the effects of selective and nonselective A2aR antagonists. During 30 secs of hypoxia, PaO2decreased significantly ( P < 0.05) and to a similar degree in both types of mice, whereas PaCO2remained relatively stable. However, mean arterial blood pressure (MABP) decreased to a greater extent ( P < 0.05) during hypoxia in KO mice (58.6 ± 1.5 mm Hg) than in WT animals (76.1 ± 3.2 mm Hg). Consequently, in a separate group of mice, we stabilized and matched MABP during hypoxia. Hypoxic hyperemia was attenuated by 38% ( P < 0.05) in KO animals whose MABP was uncontrolled, and by 81% ( P < 0.05) in KO animals whose MABP changes were matched to the MABP in the hypoxic WT mice. In animals treated with adenosine antagonists, hypoxic hyperemia was decreased by 44% to 48% ( P < 0.05) in WT mice, but was without effect in KO mice. We conclude that adenosine via A2aR is responsible for a significant proportion of the hyperemia during hypoxia.
Web 2.0 and the ever elusive balance between information explosion and data mining
Authors: Krishna Abhay
Published: June 2008

In a fascinating tussle of perspectives in Volume 6 and issue no 3 of april 2008 Frontiers in Ecology and Evolution, Martin A Nuñez and Gregory M Crustinger enlist ways to brace up with the recent literature. They advocate that PhD students should choose a well studied system to have better chances of success. On the other side of this fascinating tussle Daniel Simberloff and Nathan J Sanders agree with there enthusiastic case and approaches for bracing up with the recent literature. However, they disagree with the play safe strategy of start-ups in ecology and evolution.For me this tussle of perspectives is really fascinating. It underlines the story of our times. In the times of information explosion, should we concentrate on mining this information for knowledge or should we keep adding to this information explosion. This dilemma fits well in the context of the tussle by a quote from Nassim Nicholas Taleb “ It is almost impossible these days to finish PhD without excessive intellectual curiosity and it is impossible to get a faculty position without narrowly specializing in a chosen field ”.Here in this letter, I want to harmonize the two perspectives by arguing that coexistence of information explosion and data mining is possible. We should strive for that ever elusive balance and I discuss below how Web 2.0 will enable it. Web 2.0, still in its infancy already shows promising results and I discuss below these usages.
Biosynthesis of fluorescent allophycocyanin α‐subunits by autocatalysis in Escherichia coli
Authors: Zhang Weijie , Guan Xiangyu , Yang Yu , Ge Baosheng , Chen Huaxin , Li Fuchao , Qin Song
Published: June 2008

APC (allophycocyanin) is widely used for fluorescence tagging and may be a promising antioxidant agent for use within the food and pharmaceutical industries. Chromophore attachment to apo‐ApcA (apo‐APC α‐subunit without chromophore) can be autocatalysed both in vitro and in vivo. In the present study, a plasmid containing genes of apo‐ApcA and chromophore synthetases [HO1 (ferredoxin‐dependent haem oxygenase) and PcyA (phycocyanobilin:ferredoxin oxidoreductase)] was constructed and expressed in Escherichia coli. The results show that holo‐ApcA (APC α‐subunit with chromophore) can be synthesized by autocatalysis in E. coli. Recombinant holo‐ApcA showed the same spectral and fluorescent properties as PC (phycocyanin) and could serve as a good substitute for native PC for fluorescent tagging. Moreover, recombinant ApcA can inhibit hydroxyl and peroxyl radicals more strongly than holo‐ApcA and native APC. The EC50 values were 296.4±22.4 μg/ml against hydroxyl radicals and 38.5±2.6 μg/ml against peroxyl radicals.
Production of the monoterpene limonene and modulation of apoptosis‐related proteins in embryonic‐mouse NIH 3T3 fibroblast cells by introduction of the limonene synthase gene isolated from Japanese catnip (Schizonepeta tenuifolia)
Authors: Satomi Yoshiko , Ohara Kazuaki , Yazaki Kazufumi , Ito Michiho , Honda Gisho , Nishino Hoyoku
Published: June 2008

The monoterpene D‐limonene shows cancer preventative and cancer therapeutic activities in vitro and in vivo. Unlike plants, animals are unable to synthesize limonene de novo and obtain limonene through dietary sources. In the present study we established transgenic mouse embryonic NIH 3T3 fibroblast cells that produce limonene by introducing the D‐limonene synthase gene obtained from Japanese catnip (Schizonepeta tenuifolia). Apoptosis was not observed in the limonene‐producing cells. A concomitant increase in the level of apoptosis‐related protein Bcl‐2 (B‐cell lymphoma protein 2) and decreases in the levels of Bad (Bcl‐2 antagonist of cell death) and phosphorylated JNK (c‐Jun N‐terminal kinase) were observed in limonene‐producing cells. Limonene‐producing cells may provide a useful new system to investigate the in vivo function of this monoterpene.
The 5′–3′ exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered
Authors: Grima Dominic P. , Sullivan Melanie , Zabolotskaya Maria V. , Browne Cathy , Seago Julian , Wan Kay Chong , Okada Yoshio , Newbury Sarah F.
Published: June 2008

Background information. Ribonucleases have been well studied in yeast and bacteria, but their biological significance to developmental processes in multicellular organisms is not well understood. However, there is increasing evidence that specific timed transcript degradation is critical for regulation of many cellular processes, including translational repression, nonsense‐mediated decay and RNA interference. The Drosophila gene pacman is highly homologous to the major yeast exoribonuclease XRN1 and is the only known cytoplasmic 5′–3′ exoribonuclease in eukaryotes. To determine the effects of this exoribonuclease in development we have constructed a number of mutations in pacman by P‐element excision and characterized the resulting phenotypes.

Results. Mutations in pacman resulted in flies with a number of specific phenotypes, such as low viability, dull wings, crooked legs, failure of correct dorsal/thorax closure and defects in wound healing. The epithelial sheet movement involved in dorsal/thorax closure is a conserved morphogenetic process which is similar to that of hind‐brain closure in vertebrates and wound healing in humans. As the JNK (c‐Jun N‐terminal kinase) signalling pathway is known to be involved in dorsal/thorax closure and wound healing, we tested whether pacman affects JNK signalling. Our experiments demonstrate that pacman genetically interacts with puckered, a phosphatase that negatively regulates the JNK signalling pathway.

Conclusions. These results reveal that the 5′–3′ exoribonuclease pacman is required for a critical aspect of epithelial sheet sealing in Drosophila. Since these mutations result in specific phenotypes, our data suggest that the exoribonuclease Pacman targets a specific subset of mRNAs involved in this process. One of these targets could be a member of the JNK signalling pathway, although it is possible that a parallel pathway may instead be affected. The exoribonuclease pacman is highly conserved in all eukaryotes, therefore it is likely that it is involved in similar morphological processes, such as wound healing in human cells.
Regulation of Nedd4-2 self-ubiquitination and stability by a PY motif located within its HECT-domain
Authors: Bruce M. Christine , Kanelis Voula , Fouladkou Fatemeh , Debonneville Anne , Staub Olivier , Rotin Daniela
Published: June 2008

Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 domain, four WW domains (protein–protein interaction domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) domain. In the present paper we show that the WW domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT domain and inhibit auto-ubiquitination. Pulse–chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-domain–HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.
The secretion inhibitor Exo2 perturbs trafficking of Shiga toxin between endosomes and the trans-Golgi network
Authors: Spooner Robert A. , Watson Peter , Smith Daniel C. , Boal Frédéric , Amessou Mohammed , Johannes Ludger , Clarkson Guy J. , Lord J. Michael , Stephens David J. , Roberts Lynne M.
Published: June 2008

The small-molecule inhibitor Exo2 {4-hydroxy-3-methoxy-(5,6,7,8-tetrahydrol[1]benzothieno[2,3-d]pyrimidin-4-yl)hydraz-one benzaldehyde} has been reported to disrupt the Golgi apparatus completely and to stimulate Golgi–ER (endoplasmic reticulum) fusion in mammalian cells, akin to the well-characterized fungal toxin BFA (brefeldin A). It has also been reported that Exo2 does not affect the integrity of the TGN (trans-Golgi network), or the direct retrograde trafficking of the glycolipid-binding cholera toxin from the TGN to the ER lumen. We have examined the effects of BFA and Exo2, and found that both compounds are indistinguishable in their inhibition of anterograde transport and that both reagents significantly disrupt the morphology of the TGN in HeLa and in BS-C-1 cells. However, Exo2, unlike BFA, does not induce tubulation and merging of the TGN and endosomal compartments. Furthermore, and in contrast with its effects on cholera toxin, Exo2 significantly perturbs the delivery of Shiga toxin to the ER. Together, these results suggest that the likely target(s) of Exo2 operate at the level of the TGN, the Golgi and a subset of early endosomes, and thus Exo2 provides a more selective tool than BFA for examining membrane trafficking in mammalian cells.
Down-regulation of hepatic nuclear factor 4α on expression of human hepatic stimulator substance via its action on the proximal promoter in HepG2 cells
Authors: Guo Duo , Dong Ling-yue , Wu Yuan , Yang Lin , An Wei
Published: June 2008

hHSS (human hepatic stimulator substance) stimulates hepatocyte growth. To understand the mechanism controlling hHSS expression, we analysed the proximal promoter activity and identified two regulatory regions (−212/−192 and −152/−132) that were important for transcription in HepG2 cells. Using the luciferase reporter assay, gel-shift experiments and ChIP (chromatin immunoprecipitation), we found that the transcription factors HNF4α (hepatocyte nuclear factor 4α) and Sp1 (stimulating protein-1) were essential for hHSS promoter activity and could directly bind to regions −209/−204 and −152/−145 respectively. We also confirmed that activation and repression of hHSS transcription induced by Sp1 and HNF4α resulted from binding of these factors to these two cis-elements respectively. Overexpression of HNF4α led to a dramatic repression of the promoter activity and, in contrast, the activity was markedly elevated by overexpression of Sp1. Furthermore, overexpression of HNF4α1, one of the HNF4α isoforms, resulted in a dramatic suppression of the promoter activity. Moreover, repression of HNF4α expression by siRNA (small interfering RNA) remarkably enhanced the hHSS mRNA level. It has been reported previously that expression of HNF4α is functionally regulated by dexamethasone. To further confirm the transcriptional control of HNF4α on hHSS, we tested the effect of dexamethasone on hHSS transcription in HepG2 cells. In the present study we have demonstrated that the expression of the hHSS gene was down-regulated at the transcriptional level by dexamethasone in HepG2 cells. A deletion and decoy assay revealed that binding of HNF4α to nucleotides −209/−204 was responsible for the suppression of hHSS promoter activity by dexamethasone. Increases in the HNF4α-binding activity and expression were simultaneously observed in an electrophoretic mobility-shift assay and Western blot analysis. These results suggested that Sp1 activates hHSS basal expression, but HNF4α inhibits hHSS gene expression.
Molecular basis of the substrate specificity and the catalytic mechanism of citramalate synthase from Leptospira interrogans
Authors: Ma Jun , Zhang Peng , Zhang Zilong , Zha Manwu , Xu Hai , Zhao Guoping , Ding Jianping
Published: June 2008

Leptospira interrogans is the causative agent for leptospirosis, a zoonotic disease of global importance. In contrast with most other micro-organisms, L. interrogans employs a pyruvate pathway to synthesize isoleucine and LiCMS (L. interrogans citramalate synthase) catalyses the first reaction of the pathway which converts pyruvate and acetyl-CoA into citramalate, thus making it an attractive target for the development of antibacterial agents. We report here the crystal structures of the catalytic domain of LiCMS and its complexes with substrates, and kinetic and mutagenesis studies of LiCMS, which together reveal the molecular basis of the high substrate specificity and the catalytic mechanism of LiCMS. The catalytic domain consists of a TIM barrel flanked by an extended C-terminal region. It forms a homodimer in the crystal structure, and the active site is located at the centre of the TIM barrel near the C-terminal ends of the β-strands and is composed of conserved residues of the β-strands of one subunit and the C-terminal region of the other. The substrate specificity of LiCMS towards pyruvate against other α-oxo acids is dictated primarily by residues Leu81, Leu104 and Tyr144, which form a hydrophobic pocket to accommodate the C2-methyl group of pyruvate. The catalysis follows the typical aldol condensation reaction, in which Glu146 functions as a catalytic base to activate the methyl group of acetyl-CoA to form an enolated acetyl-CoA intermediate and Arg16 as a general acid to stabilize the intermediate.
In vivo modular control analysis of energy metabolism in contracting skeletal muscle
Authors: Arsac Laurent M. , Beuste Christophe , Miraux Sylvain , Deschodt-Arsac Véronique , Thiaudiere Eric , Franconi Jean-Michel , Diolez Philippe H.
Published: June 2008

We used 31P MRS (magnetic resonance spectroscopy) measurements of energetic intermediates [ATP, Pi and PCr (phosphocreatine)] in combination with the analytical tools of metabolic control analysis to study in vivo energy metabolism in the contracting skeletal muscle of anaesthetized rats over a broad range of workload. According to our recent MoCA (modular control analysis) used to describe regulatory mechanisms in beating heart, we defined the energetic system of muscle contraction as two modules (PCr-Producer and PCr-Consumer) connected by the energetic intermediates. Hypoxia and electrical stimulation were used in this in vivo study as reasonably selective modulations of Producer and Consumer respectively. As quantified by elasticity coefficients, the sensitivities of each module to PCr determine the control of steady-state contractile activity and metabolite concentrations. The magnitude of the elasticity of the producer was high (4.3±0.6) at low workloads and decreased 5-fold (to 0.9±0.2) at high workloads. By contrast, the elasticity of the consumer remained low (0.5–1.2) over the range of metabolic rates studied. The control exerted by each module over contraction was calculated from these elasticities. The control of contraction was found on the consumer at low workloads and then swung to the producer, due to the workload-dependent decrease in the elasticity of producer. The workload-dependent elasticity and control pattern of energy production in muscle is a major difference from heart. Since module rate and elasticity depend on the concentrations of substrates and products, the absence of homoeostasis of the energetic intermediates in muscle, by contrast with heart, is probably the origin of the workload-dependent elasticity of the producer module.
Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor
Authors: Kong Huihui , Jones Peter P. , Koop Andrea , Zhang Lin , Duff Henry J. , Chen S. R. Wayne
Published: June 2008

Caffeine has long been used as a pharmacological probe for studying RyR (ryanodine receptor)-mediated Ca2+ release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. In the present study, we investigated the effects of caffeine on spontaneous Ca2+ release and on the response of single RyR2 (cardiac RyR) channels to luminal or cytosolic Ca2+. We found that HEK-293 cells (human embryonic kidney cells) expressing RyR2 displayed partial or ‘quantal’ Ca2+ release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca2+ release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca2+ revealed that caffeine reduced the luminal Ca2+ threshold at which spontaneous Ca2+ release occurs. Interestingly, spontaneous Ca2+ release in the form of Ca2+ oscillations persisted in the presence of 10 mM caffeine, and was diminished by ryanodine, demonstrating that unlike ryanodine, caffeine, even at high concentrations, does not hold the channel open. At the single-channel level, caffeine markedly reduced the threshold for luminal Ca2+ activation, but had little effect on the threshold for cytosolic Ca2+ activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca2+ activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca2+ activation of RyR2, and increased the propensity for spontaneous Ca2+ release, mimicking the effects of disease-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca2+ release by reducing the threshold for luminal Ca2+ activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.
Localization and characterization of the novel protein encoded by C20orf3
Authors: Ilhan Aysegul , Gartner Wolfgang , Nabokikh Anastasiya , Daneva Teodora , Majdic Otto , Cohen Gerald , Böhmig Georg A. , Base Wolfgang , Hörl Walter H. , Wagner Ludwig
Published: June 2008

In the present study, we characterized the gene product of open reading frame 3 encoded at human chromosome 20 (C20orf3), which represents a member of the lactonohydrolase super family. Multiple-tissue Northern blot analysis showed ubiquitous expression of the 2.4 kb transcript coding for 416 amino acids, with highest levels in human liver, placenta and kidney. After recombinant production of protein variants in Escherichia coli and insect cells, antibodies directed against different epitopes within the C20orf3 gene product were generated. Using these immunoreagents, protein expression was demonstrated in the liver, and glomerular and tubular structures of the kidney, as well as in endothelial cells and arterial wall. Positive staining was also observed at the pancreatic islets of Langerhans. Using immunoblotting, we identified three size variants. In line with the results of in silico analysis demonstrating a single transmembrane sequence (amino acids 40–61) at the N-terminus of the full-length protein, FACS cell-surface staining confirmed a mainly extracellular localization of the full-length protein. Sucrose density gradient cell fractionation revealed membrane association of the dominant 50 kDa variant in HepG2 and Rin-5F cells. The finding of a strong arylesterase activity with β-naphthyl acetate and phenyl acetate of the C20orf3 protein-containing fractions suggests potential involvement of this protein in enzymatic processes. C20orf3 promoter-driven reporter assays, which were verified by gene-specific RT-qPCR (real-time quantitative PCR) showed a strong inhibitory effect of human serum on transcription using the HEK-293 human embryonic kidney cell line. In conclusion, we characterized the structure and expression pattern of the C20orf3 gene product. According to a series of analogies with PON (paraoxonase) family members, we speculate that the C20orf3 gene product represents a new member of this important protein family present at the cellular level.
Probing structure–activity relationships in bactericidal peptide βpep-25
Authors: Dings Ruud P. M. , Haseman Judith R. , Mayo Kevin H.
Published: June 2008

Cationic peptides, known to disrupt bacterial membranes, are being developed as promising agents for therapeutic intervention against infectious disease. In the present study, we investigate structure–activity relationships in the bacterial membrane disruptor βpep-25, a peptide 33-mer. For insight into which amino acid residues are functionally important, we synthesized alanine-scanning variants of βpep-25 and assessed their ability to kill bacteria (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and to neutralize LPS (lipopolysaccharide). Activity profiles were found to vary with the bacterial strain examined. Specific cationic and smaller hydrophobic alkyl residues were crucial to optimal bactericidal activity against the Gram-negative bacteria, whereas larger hydrophobic and cationic residues mediated optimal activity against Gram-positive Staph. aureus. Lysine-substituted norleucine (n-butyl group) variants demonstrated that both charge and alkyl chain length mediate optimal activity. In terms of LPS neutralization, activity profiles were essentially the same against four species of LPS (E. coli 055 and 0111, Salmonella enterica serotype Typhimurium and Klebsiella pneumoniae), and different for two others (Ps. aeruginosa and Serratia marcescens), with specific hydrophobic, cationic and, surprisingly, anionic residues being functionally important. Furthermore, disulfide-bridged analogues demonstrated that an anti parallel β-sheet structure is the bioactive conformation of βpep-25 in terms of its bactericidal, but not LPS endotoxin neutralizing, activity. Moreover, βpep-25 variants, like the parent peptide, do not lyse eukaryotic cells. This research contributes to the development and design of novel antibiotics.
Assembly of a membrane receptor complex: roles of the uroplakin II prosequence in regulating uroplakin bacterial receptor oligomerization
Authors: Hu Chih-Chi Andrew , Bachmann Thomas , Zhou Ge , Liang Feng-Xia , Ghiso Jorge , Kreibich Gert , Sun Tung-Tien
Published: June 2008

The apical surface of the mammalian urothelium is almost completely covered by two-dimensional protein crystals (known as urothelial plaques) of hexagonally packed 16 nm particles consisting of two UP (uroplakin) heterodimers, i.e. UPs Ia/II and Ib/III pairs. UPs are functionally important as they contribute to the urothelial permeability barrier function, and UPIa may serve as the receptor for the uropathogenic Escherichia coli that causes over 90% of urinary tract infections. We study here how the UP proteins are assembled and targeted to the urothelial apical surface, paying special attention to the roles of the prosequence of UPII in UP oligomerization. We show that (i) the formation of the UPIa/UPII heterodimer, necessary for ER (endoplasmic reticulum) exit, requires disulfide formation in the prosequence domain of proUPII (the immature form of UPII still containing its prosequence); (ii) differentiation-dependent N-glycosylation of the prosequence leads to UP stabilization; (iii) a failure to form tetramers in cultured urothelial cells, in part due to altered glycosylation of the prosequence, may block two-dimensional crystal formation; and (iv) the prosequence of UPII remains attached to the mature protein complex on the urothelial apical surface even after it has been cleaved by the trans-Golgi-network-associated furin. Our results indicate that proper secondary modifications of the prosequence of UPII play important roles in regulating the oligomerization and function of the UP protein complex.
A chemical screen in diverse breast cancer cell lines reveals genetic enhancers and suppressors of sensitivity to PI3K isoform-selective inhibition
Authors: Torbett Neil E. , Luna-Moran Antonio , Knight Zachary A. , Houk Andrew , Moasser Mark , Weiss William , Shokat Kevan M. , Stokoe David
Published: June 2008

The PI3K (phosphoinositide 3-kinase) pathway regulates cell proliferation, survival and migration and is consequently of great interest for targeted cancer therapy. Using a panel of small-molecule PI3K isoform-selective inhibitors in a diverse set of breast cancer cell lines, we have demonstrated that the biochemical and biological responses were highly variable and dependent on the genetic alterations present. p110α inhibitors were generally effective in inhibiting the phosphorylation of PKB (protein kinase B)/Akt and S6, two downstream components of PI3K signalling, in most cell lines examined. In contrast, p110β-selective inhibitors only reduced PKB/Akt phosphorylation in PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutant cell lines, and was associated with a lesser decrease in S6 phosphorylation. PI3K inhibitors reduced cell viability by causing cell-cycle arrest in the G1 phase, with multi-targeted inhibitors causing the most potent effects. Cells expressing mutant Ras were resistant to the cell-cycle effects of PI3K inhibition, which could be reversed using inhibitors of Ras signalling pathways. Taken together, our data indicate that these compounds, alone or in suitable combinations, may be useful as breast cancer therapeutics, when used in appropriate genetic contexts.
Rab geranylgeranylation occurs preferentially via the pre-formed REP–RGGT complex and is regulated by geranylgeranyl pyrophosphate
Authors: Baron Rudi A. , Seabra Miguel C.
Published: June 2008

Prenylation (or geranylgeranylation) of Rab GTPases is catalysed by RGGT (Rab geranylgeranyl transferase) and requires REP (Rab escort protein). In the classical pathway, REP associates first with unprenylated Rab, which is then prenylated by RGGT. In the alternative pathway, REP associates first with RGGT; this complex then binds and prenylates Rab proteins. In the present paper we show that REP mutants defective in RGGT binding (REP1 F282L and REP1 F282L/V290F) are unable to compete with wild-type REP in the prenylation reaction in vitro. When over-expressed in cells, REP wild-type and mutants are unable to form stable cytosolic complexes with endogenous unprenylated Rabs. These results suggest that the alternative pathway may predominate in vivo. We also extend previous suggestions that GGPP (geranylgeranyl pyrophosphate) acts as an allosteric regulator of the prenylation reaction. We observed that REP–RGGT complexes are formed in vivo and are unstable in the absence of intracellular GGPP. RGGT increases the ability of REP to extract endogenous prenylated Rabs from membranes in vitro by stabilizing a soluble REP–RGGT–Rab-GG (geranylgeranylated Rab) complex. This effect is regulated by GGPP, which promotes the dissociation of RGGT and REP–Rab-GG to allow delivery of prenylated Rabs to membranes.
Transaldolase deficiency influences the pentose phosphate pathway, mitochondrial homoeostasis and apoptosis signal processing
Authors: Qian Yueming , Banerjee Sanjay , Grossman Craig E. , Amidon Wendy , Nagy Gyorgy , Barcza Maureen , Niland Brian , Karp David R. , Middleton Frank A. , Banki Katalin , Perl Andras
Published: June 2008

TAL (transaldolase) was originally described in the yeast as an enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall reason for its existence is unclear. Recently, deletion of Ser171 (TALΔS171) was found in five patients causing inactivation, proteasome-mediated degradation and complete deficiency of TAL. In the present study, microarray and follow-up Western-blot, enzyme-activity and metabolic studies of TALΔS171 TD (TAL-deficient) lymphoblasts revealed co-ordinated changes in the expression of genes involved in the PPP, mitochondrial biogenesis, oxidative stress, and Ca2+ fluxing. Sedoheptulose 7-phosphate was accumulated, whereas G6P (glucose 6-phosphate) was depleted, indicating a failure to recycle G6P for the oxidative branch of the PPP. Nucleotide analysis showed depletion of NADPH and NAD+ and accumulation of ADP-ribose. TD cells have diminished Δψm (mitochondrial transmembrane potential) and increased mitochondrial mass associated with increased production of nitric oxide and ATP. TAL deficiency resulted in enhanced spontaneous and H2O2-induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses NAD+ into ADP-ribose, a trigger of Ca2+ release from the endoplasmic reticulum that, in turn, facilitated CD20-induced apoptosis. By contrast, TD cells were resistant to CD95/Fas-induced apoptosis, owing to a dependence of caspase activity on redox-sensitive cysteine residues. Normalization of TAL activity by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca2+ levels, suppressed H2O2- and CD20-induced apoptosis and enhanced Fas-induced cell death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca2+ fluxing and apoptosis.
Structural and mechanistic insights into type II trypanosomatid tryparedoxin-dependent peroxidases
Authors: Alphey Magnus S. , König Janine , Fairlamb Alan H.
Published: June 2008

TbTDPX (Trypanosoma brucei tryparedoxin-dependent peroxidase) is a genetically validated drug target in the fight against African sleeping sickness. Despite its similarity to members of the GPX (glutathione peroxidase) family, TbTDPX2 is functional as a monomer, lacks a selenocysteine residue and relies instead on peroxidatic and resolving cysteine residues for catalysis and uses tryparedoxin rather than glutathione as electron donor. Kinetic studies indicate a saturable Ping Pong mechanism, unlike selenium-dependent GPXs, which display infinite Km and Vmax values. The structure of the reduced enzyme at 2.1 Å (0.21 nm) resolution reveals that the catalytic thiol groups are widely separated [19 Å (0.19 nm)] and thus unable to form a disulphide bond without a large conformational change in the secondary-structure architecture, as reported for certain plant GPXs. A model of the oxidized enzyme structure is presented and the implications for small-molecule inhibition are discussed.
Characterization of the topology and functional domains of RKTG
Authors: Luo Xiaolin , Feng Lin , Jiang Xiaomeng , Xiao Fei , Wang Zhenzhen , Feng Gen-Sheng , Chen Yan
Published: June 2008

RKTG (Raf kinase trapping to Golgi) is exclusively localized at the Golgi apparatus and functions as a spatial regulator of Raf-1 kinase by sequestrating Raf-1 to the Golgi. Based on the structural similarity with adiponectin receptors, RKTG was predicted to be a seven-transmembrane protein with a cytosolic N-terminus, distinct from classical GPCRs (G-protein-coupled receptors). We analysed in detail the topology and functional domains of RKTG in this study. We determined that the N-terminus of RKTG is localized on the cytosolic side. Two short stretches of amino acid sequences at the membrane proximal to the N- and C-termini (amino acids 61–71 and 299–303 respectively) were indispensable for Golgi localization of RKTG, but were not required for the interaction with Raf-1. The three loops facing the cytosol between the transmembrane domains had different roles in Golgi localization and Raf-1 interaction. While the first cytosolic loop was only important for Golgi localization, the third cytosolic loop was necessary for both Golgi localization and Raf-1 sequestration. Taken together, these findings suggest that RKTG is a type III membrane protein with its N-terminus facing the cytosol and multiple sequences are responsible for its localization at the Golgi apparatus and Raf-1 interaction. As RKTG is the first discovered Golgi protein with seven transmembrane domains, the knowledge derived from this study would not only provide structural information about the protein, but also pave the way for future characterization of the unique functions of RKTG in the regulation of cell signalling.
The pancreatic β-cell: birth, life and death
Authors: Rutter Guy A. , Wong F. Susan
Published: June 2008

Defective insulin secretion is a hallmark of all forms of diabetes. Whereas Type 1 diabetes has long been known to result from the immune-mediated destruction of β-cells, Type 2 diabetes appears to involve both loss of β-cell mass and glucose sensitivity in the face of extrapancreatic insulin resistance. We summarize here the proceedings of a Biochemical Society Focused Meeting, held at the St Thomas campus of King's College London in December 2007, which highlighted recent research advances targeting the β-cell.
EGF receptor in pancreatic β-cell mass regulation
Authors: Miettinen Päivi , Ormio Päivi , Hakonen Elina , Banerjee Meenal , Otonkoski Timo
Published: June 2008

Pancreatic islet development is impaired in mice lacking EGFRs (epidermal growth factor receptors). Even partial tissue-specific attenuation of EGFR signalling in the islets leads to markedly reduced β-cell proliferation and development of diabetes during the first weeks after birth. Out of the many EGFR ligands, betacellulin has been specifically associated with positive effects on β-cell growth, through both increased proliferation and neogenesis. EGFR action is also necessary for the β-cell mitogenic activity of the gut hormone GLP-1 (glucagon-like peptide 1). Finally, in vitro models demonstrate a central role for EGFR in transdifferentiation of pancreatic acinar and ductal cells into endocrine islet cells. EGFR thus plays an essential role in β-cell mass regulation, but its mechanisms of action remain poorly understood.
Experimental models of β-cell regeneration
Authors: Bonal Claire , Avril Isabelle , Herrera Pedro L.
Published: June 2008

The control of glucose metabolism by pancreatic endocrine cells throughout life relies on a tight regulation of the mass of insulin-producing β-cells. How this homoeostasis is achieved is not well understood. Over the last few years, experimental rodent models with altered β-cell mass, and, more recently, new transgenic approaches designed to tackle this problem, have provided abundant information. Processes such as β-cell proliferation and apoptosis, or even β-cell differentiation from poorly characterized progenitor cells, whether immature or differentiated, appear to be implicated. A complex picture is thus emerging in which the nature of the pancreatic lesion appears to determine the kind of regenerative response. The environment formed by acinar and ductal cells, and also by vascular and neuronal structures, which surround islets and penetrate into their β-cell core, might play crucial roles so far unsuspected, which should be explored in the near future.
Glucokinase (GCK) and other susceptibility genes for β-cell dysfunction: the candidate approach
Authors: Gloyn Anna L. , Tribble Nicholas D. , Bunt Martijn van de , Barrett Amy , Johnson Paul R.V.
Published: June 2008

There are well-documented examples in the literature of where determining the genetic aetiology of a disorder has provided insights into important regulatory pathways and protein interactions, and, more recently, has led to improved treatment options for patients. The studies of monogenic forms of β-cell dysfunction are no exception. Naturally occurring mutations in the gene for the β-cell enzyme glucokinase (GCK) result in both hyper- and hypo-glycaemia. Over 200 mutations have been described, and careful study of the mutational mechanisms for a number of these has provided important insights into glucokinase regulation. Increased understanding of post-translational regulatory mechanisms holds the promise of novel pharmacotherapeutic options for the treatment of T2DM (Type 2 diabetes mellitus). It is well established that common genetic variation in genes involved in monogenic forms of β-cell dysfunction contributes to susceptibility to T2DM. Recent genome-wide scans for association have identified a number of novel T2DM susceptibility genes which probably influence β-cell mass and/or function. Their identification allows the investigation of the role of rare mutations in monogenic β-cell dysfunction. Current results indicate the importance of these genes in pancreatic development and suggest that mutations which result in a severe functional defect could be lethal.
Commonality in the genetic control of Type 1 diabetes in humans and NOD mice: variants of genes in the IL-2 pathway are associated with autoimmune diabetes in both species
Authors: Rainbow Dan B. , Esposito Laura , Howlett Sarah K. , Hunter Kara M. , Todd John A. , Peterson Laurence B. , Wicker Linda S.
Published: June 2008

Variants within the IL-2 (interleukin 2) and CD25 genes are associated with T1DM (Type 1 diabetes mellitus) in mice and humans respectively. Both gene products are essential for optimal immune tolerance and a partial failure to tolerize is linked to the autoimmune responses to insulin and other β-cell proteins that precede T1DM onset. Gene variants that contribute to common disease susceptibility often alter gene expression only modestly. Small expression changes can be technically challenging to measure robustly, especially since biological variation usually contributes negatively to this goal. The present review focuses on allele-specific expression assays that can be used to quantify genotype-determined expression differences such as those observed for IL-2, where the susceptibility allele is transcribed 2-fold less than the resistance allele.
Use of a systems biology approach to understand pancreatic β-cell death in Type 1 diabetes
Authors: Eizirik Decio L. , Moore Fabrice , Flamez Daisy , Ortis Fernanda
Published: June 2008

Accumulating evidence indicates that β-cells die by apoptosis in T1DM (Type 1 diabetes mellitus). Apoptosis is an active gene-directed process, and recent observations suggest that β-cell apoptosis depends on the parallel and/or sequential up- and down-regulation of hundreds of genes controlled by key transcription factors such as NF-κB (nuclear factor κB) and STAT-1 (signal transducer and activator of transcription 1). Understanding the regulation of these gene networks, and how they modulate β-cell death and the ‘dialogue’ between β-cells and the immune system, will require a systems biology approach to the problem. This will hopefully allow the search for a cure for T1DM to move from a ‘trial-and-error’ approach to one that is really mechanistically driven.
Cytokine signalling in the β-cell: a dual role for IFNγ
Authors: Gysemans Conny , Callewaert Hanne , Overbergh Lutgart , Mathieu Chantal
Published: June 2008

IFNγ (interferon γ), a cytokine typically secreted by infiltrating immune cells in insulitis in Type 1 diabetes, is by itself not detrimental to β-cells, but, together with other cytokines, such as IL-1β (interleukin 1β) and TNFα (tumour necrosis factor α), or dsRNA (double-stranded RNA), it induces β-cell apoptosis. The complex gene and protein networks that are altered by the combination of cytokines clearly point towards synergisms between these agents. IFNγ acts mostly via JAK (Janus kinase) activation, with the transcription factors STAT-1 (signal transducer and activator of transcription-1) and IRF-1 (IFNγ regulatory factor-1) playing a central role in the downstream pathway. The study of mice with a disruption of these transcription factors has revealed a possible dual role for IFNγ in β-cell destruction by cytokines or dsRNA. We demonstrated that the absence of STAT-1 from β-cells completely protects against IFNγ+IL-1β- and IFNγ+dsRNA-mediated β-cell death in vitro, whereas absence of IRF-1 does not prevent cytokine-induced β-cell apoptosis. In vivo, a lack of the IRF-1 gene in pancreatic islets even promotes low-dose streptozotocin-induced diabetes, whereas lack of STAT-1 confers resistance against β-cell death following low-dose streptozotocin-induced diabetes. Additionally, IRF-1−/− islets are more sensitive to PNF (primary islet non-function) after transplantation in spontaneously diabetic NOD (non-obese diabetic) mice, whereas STAT-1−/− islets are fully protected. Moreover, proteomic analysis of β-cells exposed to IFNγ or IFNγ+IL-1β confirms that very different pathways are activated by IFNγ alone compared with the combination. We conclude that IFNγ may play a dual role in immune-induced β-cell destruction. Transcription factors drive this dual role, with STAT-1 driving β-cell destruction and IRF-1 possibly playing a role in up-regulation of protective pathways induced by IFNγ.
Role of NF-κB in β-cell death
Authors: Melloul Danielle
Published: June 2008

Apoptotic β-cell death appears to be central to the pathogenesis of Type 1 diabetes mellitus and in islet graft rejection. The β-cell destruction is partially mediated by cytokines, such as IL-1β (interleukin 1β), TNFα (tumour necrosis factor α) and IFN-γ (interferon γ). IL-1β and TNFα mediate activation of the transcription factor NF-κB (nuclear factor κB) pathway. Use of a degradation-resistant NF-κB protein inhibitor (ΔNIκBα), specifically expressed in β-cells, significantly reduced IL-1β+IFN-γ-induced apoptosis. Moreover, in vivo, it protected against multiple low-dose streptozocin-induced diabetes, with reduced intra-islet lymphocytic infiltration. Thus β-cell-specific activation of NF-κB is a key event in the progressive loss of β-cells in diabetes. Inhibition of this process could be a potential effective strategy for β-cell protection.
Oxidative stress: the vulnerable β-cell
Authors: Lenzen Sigurd
Published: June 2008

Antioxidative defence mechanisms of pancreatic β-cells are particularly weak and can be overwhelmed by redox imbalance arising from overproduction of reactive oxygen and reactive nitrogen species. The consequences of this redox imbalance are lipid peroxidation, oxidation of proteins, DNA damage and interference of reactive species with signal transduction pathways, which contribute significantly to β-cell dysfunction and death in Type 1 and Type 2 diabetes mellitus. Reactive oxygen species, superoxide radicals (O2•−), hydrogen peroxide (H2O2) and, in a final iron-catalysed reaction step, the most reactive and toxic hydroxyl radicals (OH•) are produced during both pro-inflammatory cytokine-mediated β-cell attack in Type 1 diabetes and glucolipotoxicity-mediated β-cell dysfunction in Type 2 diabetes. In combination with NO•, which is toxic in itself, as well as through its reaction with the O2•− and subsequent formation of peroxynitrite, reactive species play a central role in β-cell death during the deterioration of glucose tolerance in the development of diabetes.