bluuebunny/crossref_metadata_2025 huggingface dataset:
PhosphoPOINT: a comprehensive human kinase interactome and phospho-protein database
Authors: Yang Chia-Ying , Chang Chao-Hui , Yu Ya-Ling , Lin Tsu-Chun Emma , Lee Sheng-An , Yen Chueh-Chuan , Yang Jinn-Moon , Lai Jin-Mei , Hong Yi-Ren , Tseng Tzu-Ling , Chao Kun-Mao , Huang Chi-Ying F.
Published: August 2008

Motivation: To fully understand how a protein kinase regulates biological processes, it is imperative to first identify its substrate(s) and interacting protein(s). However, of the 518 known human serine/threonine/tyrosine kinases, 35% of these have known substrates, while 14% of the kinases have identified substrate recognition motifs. In contrast, 85% of the kinases have protein–protein interaction (PPI) datasets, raising the possibility that we might reveal potential kinase–substrate pairs from these PPIs.

Results: PhosphoPOINT, a comprehensive human kinase interactome and phospho-protein database, is a collection of 4195 phospho-proteins with a total of 15 738 phosphorylation sites. PhosphoPOINT annotates the interactions among kinases, with their down-stream substrates and with interacting (phospho)-proteins to modulate the kinase–substrate pairs. PhosphoPOINT implements various gene expression profiles and Gene Ontology cellular component information to evaluate each kinase and their interacting (phospho)-proteins/substrates. Integration of cSNPs that cause amino acids change with the proteins with the phosphoprotein dataset reveals that 64 phosphorylation sites result in a disease phenotypes when changed; the linked phenotypes include schizophrenia and hypertension. PhosphoPOINT also provides a search function for all phospho-peptides using about 300 known kinase/phosphatase substrate/binding motifs. Altogether, PhosphoPOINT provides robust annotation for kinases, their downstream substrates and their interaction (phospho)-proteins and this should accelerate the functional characterization of kinomemediated signaling.

Availability: PhosphoPOINT can be freely accessed in http://kinase.bioinformatics.tw/

Contact:  cyhuang5@ym.edu.tw; kmchao@csie.ntu.edu.tw

Supplementary information:  Supplementary data are available at Bioinformatics online.
HapCUT: an efficient and accurate algorithm for the haplotype assembly problem
Authors: Bansal Vikas , Bafna Vineet
Published: August 2008

Motivation: The goal of the haplotype assembly problem is to reconstruct the two haplotypes (chromosomes) for an individual using a mix of sequenced fragments from the two chromosomes. This problem has been shown to be computationally intractable for various optimization criteria. Polynomial time algorithms have been proposed for restricted versions of the problem. In this article, we consider the haplotype assembly problem in the most general setting, i.e. fragments of any length and with an arbitrary number of gaps.

Results: We describe a novel combinatorial approach for the haplotype assembly problem based on computing max-cuts in certain graphs derived from the sequenced fragments. Levy et al. have sequenced the complete genome of a human individual and used a greedy heuristic to assemble the haplotypes for this individual. We have applied our method HapCUTto infer haplotypes from this data and demonstrate that the haplotypes inferred using HapCUT are significantly more accurate (20–25% lower maximum error correction scores for all chromosomes) than the greedy heuristic and a previously published method, Fast Hare. We also describe a maximum likelihood based estimator of the absolute accuracy of the sequence-based haplotypes using population haplotypes from the International HapMap project.

Availability: A program implementing HapCUT is available on request.

Contact:  vibansal@cs.ucsd.edu
Inter-species normalization of gene mentions with GNAT
Authors: Hakenberg Jörg , Plake Conrad , Leaman Robert , Schroeder Michael , Gonzalez Graciela
Published: August 2008

Motivation: Text mining in the biomedical domain aims at helping researchers to access information contained in scientific publications in a faster, easier and more complete way. One step towards this aim is the recognition of named entities and their subsequent normalization to database identifiers. Normalization helps to link objects of potential interest, such as genes, to detailed information not contained in a publication; it is also key for integrating different knowledge sources. From an information retrieval perspective, normalization facilitates indexing and querying. Gene mention normalization (GN) is particularly challenging given the high ambiguity of gene names: they refer to orthologous or entirely different genes, are named after phenotypes and other biomedical terms, or they resemble common English words.

Results: We present the first publicly available system, GNAT, reported to handle inter-species GN. Our method uses extensive background knowledge on genes to resolve ambiguous names to EntrezGene identifiers. It performs comparably to single-species approaches proposed by us and others. On a benchmark set derived from BioCreative 1 and 2 data that contains genes from 13 species, GNAT achieves an F-measure of 81.4% (90.8% precision at 73.8% recall). For the single-species task, we report an F-measure of 85.4% on human genes.

Availability: A web-frontend is available at http://cbioc.eas.asu.edu/gnat/. GNAT will also be available within the BioCreative MetaService project, see http://bcms.bioinfo.cnio.es.

Contact:  joerg.hakenberg@asu.edu

Supplementary information: The test data set, lexica, and links to external data are available at http://cbioc.eas.asu.edu/gnat/
Optimal spliced alignments of short sequence reads
Authors: De Bona Fabio , Ossowski Stephan , Schneeberger Korbinian , Rätsch Gunnar
Published: August 2008

Motivation: Next generation sequencing technologies open exciting new possibilities for genome and transcriptome sequencing. While reads produced by these technologies are relatively short and error prone compared to the Sanger method their throughput is several magnitudes higher. To utilize such reads for transcriptome sequencing and gene structure identification, one needs to be able to accurately align the sequence reads over intron boundaries. This represents a significant challenge given their short length and inherent high error rate.

Results: We present a novel approach, called QPALMA, for computing accurate spliced alignments which takes advantage of the read's quality information as well as computational splice site predictions. Our method uses a training set of spliced reads with quality information and known alignments. It uses a large margin approach similar to support vector machines to estimate its parameters to maximize alignment accuracy. In computational experiments, we illustrate that the quality information as well as the splice site predictions help to improve the alignment quality. Finally, to facilitate mapping of massive amounts of sequencing data typically generated by the new technologies, we have combined our method with a fast mapping pipeline based on enhanced suffix arrays. Our algorithms were optimized and tested using reads produced with the Illumina Genome Analyzer for the model plant Arabidopsis thaliana.

Availability: Datasets for training and evaluation, additional results and a stand-alone alignment tool implemented in C++ and python are available at http://www.fml.mpg.de/raetsch/projects/qpalma.

Contact:  Gunnar.Raetsch@tuebingen.mpg.de
An environmental perspective on large-scale genome clustering based on metabolic capabilities
Authors: Kastenmüller Gabi , Gasteiger Johann , Mewes Hans-Werner
Published: August 2008

Motivation: In principle, an organism's ability to survive in a specific environment, is an observable result of the organism's regulatory and metabolic capabilities. Nonetheless, current knowledge about the global relation of the metabolisms and the niches of organisms is still limited.

Results: In order to further investigate this relation, we grouped species showing similar metabolic capabilities and systematically mapped their habitats onto these groups. For this purpose, we predicted the metabolic capabilities for 214 sequenced genomes. Based on these predictions, we grouped the genomes by hierarchical clustering. Finally, we mapped different environmental conditions and diseases related to the genomes onto the resulting clusters. This mapping uncovered several conditions and diseases that were unexpectedly enriched in clusters of metabolically similar species. As an example, Encephalitozoon cuniculi—a microsporidian causing a multisystemic disease accompanied by CNS problems in rabbits— occurred in the same metabolism-based cluster as bacteria causing similar symptoms in humans.

Supplementary information:  Supplementary data are available at Bioinformatics online.

Contact:  g.kastenmueller@helmholtz-muenchen.de
BioJava: an open-source framework for bioinformatics
Authors: Holland R. C. G. , Down T. A. , Pocock M. , Prlić A. , Huen D. , James K. , Foisy S. , Dräger A. , Yates A. , Heuer M. , Schreiber M. J.
Published: August 2008

Summary: BioJava is a mature open-source project that provides a framework for processing of biological data. BioJava contains powerful analysis and statistical routines, tools for parsing common file formats and packages for manipulating sequences and 3D structures. It enables rapid bioinformatics application development in the Java programming language.

Availability: BioJava is an open-source project distributed under the Lesser GPL (LGPL). BioJava can be downloaded from the BioJava website (http://www.biojava.org). BioJava requires Java 1.5 or higher.

Contact:  andreas.prlic@gmail.com. All queries should be directed to the BioJava mailing lists. Details are available at http://biojava.org/wiki/BioJava:MailingLists.
An Antioxidant Phytotherapy to Rescue Neuronal Oxidative Stress
Authors: Lin Zhihong , Zhu Danni , Yan Yongqing , Yu Boyang , Wang Qiujuan , Shen Pingniang , Ruan Kefeng
Published: August 2008

Oxidative stress is involved in the pathogenesis of ischemic neuronal injury. A Chinese herbal formula composed of Poria cocos (Chinese name: Fu Ling), Atractylodes macrocephala (Chinese name: Bai Zhu) and Angelica sinensis (Chinese names: Danggui, Dong quai, Donggui; Korean name: Danggwi) (FBD), has been proved to be beneficial in the treatment of cerebral ischemia/reperfusion (I/R).This study was carried out to evaluate the protective effect of FBD against neuronal oxidative stress in vivo and in vitro. Rat I/R were established by middle cerebral artery occlusion (MCAO) for 1 h, followed by 24 h reperfusion. MCAO led to significant depletion in superoxide dismutase and glutathione and rise in lipid peroxidation (LPO) and nitric oxide in brain. The neurological deficit and brain infarction were also significantly elevated by MCAO as compared with sham‐operated group. All the brain oxidative stress and damage were significantly attenuated by 7 days pretreatment with the aqueous extract of FBD (250 mg kg−1, p.o.). Moreover, cerebrospinal fluid sampled from FBD‐pretreated rats protected PC12 cells against oxidative insult induced by 0.2 mM hydrogen peroxide, in a concentration and time‐dependent manner (IC50 10.6%, ET50 1.2 h). However, aqueous extract of FBD just slightly scavenged superoxide anion radical generated in xanthine–xanthine oxidase system (IC50 2.4 mg ml−1) and hydroxyl radical generated in Fenton reaction system (IC50 3.6 mg ml−1). In conclusion, FBD was a distinct antioxidant phytotherapy to rescue neuronal oxidative stress, through blocking LPO, restoring endogenous antioxidant system, but not scavenging free radicals.
Anglerfish discard estimates and patterns in Spanish Northeast Atlantic trawl fisheries
Authors: Díaz Paz , Santos Juan , Velasco Francisco , Serrano Alberto , Pérez Nélida
Published: August 2008

Díaz, P., Santos, J., Velasco, F., Serrano, A., and Pérez, N. 2008. Anglerfish discard estimates and patterns in Spanish Northeast Atlantic trawl fisheries. – ICES Journal of Marine Science, 65: 1350–1361. Estimates of Spanish bottom trawl discards of anglerfish Lophius budegassa and Lophiuspiscatorius in the Northeast Atlantic Ocean are presented. Both species are commercially valuable in ICES Subareas VI and VII, and Divisions VIIIc and IXa. Information was obtained from observers on board during discard-monitoring programmes. Raising by effort, rather than by landings and target species, was used to determine the total annual discards by weight and number, the discard percentage, and the discard length distribution by stock throughout the dataseries. By weight and number, discards varied interannually, ranging from 4 to 600 t (∼28 000–7 000 000 fish) in northern stocks of anglerfish, and from 1 to 100 t (∼20 000–400 000 fish) in southern stocks. The discard percentage for both species and northern and southern stocks therefore ranged between 2 and 76% by number. Length distributions of the discarded anglerfish show that most were juveniles, although the pattern differed in years with high or with low rates of discarding. The number of juveniles caught apparently increased in most areas after 2000. Correlations between the quantities discarded and recruitment indices from French and Irish trawl surveys were found for northern stocks and with Spanish trawl survey indices for southern stocks.
Spawning groups of European anchovy: population structure and management implications
Authors: Sanz Núria , García-Marín José-Luis , Viñas Jordi , Roldán Marina , Pla Carles
Published: August 2008

Sanz, N., García-Marín, J-L., Viñas, J., Roldán, M., and Pla, C. 2008. Spawning groups of European anchovy: population structure and management implications. – ICES Journal of Marine Science, 65: 1635–1644. Samples of Engraulis encrasicolus from the western Mediterranean and the eastern Atlantic spawning groups were examined by protein electrophoresis to investigate their relevance in the genetic structure of European anchovy, and how they fit with the current definition of anchovy fisheries. Samples from an earlier study were also included in the analyses to assess the temporal genetic stability of spawning groups. Gene diversity analysis indicated greater differentiation between spawning groups within seas (GRS = 0.046) than between seas (GSEA = 0.022), and lesser differentiation between locations within spawning groups (GLR = 0.003), as well as genetic stability of spawning groups (GCL = 0.002). The six spawning groups apparently involved three genetic units according to Structure results, and the results of other analyses also suggested genetic differences between the two Atlantic spawning groups. The genetic substructure detected within the Atlantic agrees with the spawning groups, but does not fit easily with ICES Subdivisions. Based on these results, a revision of the current anchovy management in both Alboran Sea and Bay of Biscay areas is suggested.
Stringent promoter recognition and autoregulation by the group 3 σ-factor SigF in the cyanobacterium Synechocystis sp. strain PCC 6803
Authors: Asayama Munehiko , Imamura Sousuke
Published: August 2008

The cyanobacteirum Synechocystis sp. strain PCC 6803 possesses nine species of the sigma (σ)-factor gene for RNA polymerase (RNAP). Here, we identify and characterize the novel-type promoter recognized by a group 3 σ-factor, SigF. SigF autoregulates its own transcription and recognizes the promoter of pilA1 that acts in pilus formation and motility in PCC 6803. The pilA1 promoter (PpilA1-54) was recognized only by SigF and not by other σ-factors in PCC 6803. No PpilA1-54 activity was observed in Escherichia coli cells that possess RpoF (σ28) for fragellin and motility. Studies of in vitro transcription for PpilA1-54 identified the region from −39 to −7 including an AG-rich stretch and a core promoter with TAGGC (-32 region) and GGTAA (-12 region) as important for transcription. We also confirmed the unique PpilA1-54 architecture and further identified two novel promoters, recognized by SigF, for genes encoding periplasmic and phytochrome-like phototaxis proteins. These results and a phylogenetic analysis suggest that the PCC 6803 SigF is distinct from the E. coli RpoF or RpoD (σ70) type and constitutes a novel eubacterial group 3 σ- factor. We discuss a model case of stringent promoter recognition by SigF. Promoter types of PCC 6803 genes are also summarized.
Intragenic suppressors of temperature-sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli
Authors: Perwez Tariq , Hami Danyal , Maples Valerie F. , Min Zhao , Wang Bi-Cheng , Kushner Sidney R.
Published: August 2008

RNase E of Escherichia coli is an essential endoribonuclease that is involved in many aspects of RNA metabolism. Point mutations in the S1 RNA-binding domain of RNase E (rne-1 and rne-3071) lead to temperature-sensitive growth along with defects in 5S rRNA processing, mRNA decay and tRNA maturation. However, it is not clear whether RNase E acts similarly on all kinds of RNA substrates. Here we report the isolation and characterization of three independent intragenic second-site suppressors of the rne-1 and rne-3071 alleles that demonstrate for the first time the dissociation of the in vivo activity of RNase E on mRNA versus tRNA and rRNA substrates. Specifically, tRNA maturation and 9S rRNA processing were restored to wild-type levels in each of the three suppressor mutants (rne-1/172, rne-1/186 and rne-1/187), while mRNA decay and autoregulation of RNase E protein levels remained as defective as in the rne-1 single mutant. Each single amino acid substitution (Gly→ Ala at amino acid 172; Phe → Cys at amino acid 186 and Arg → Leu at amino acid 187) mapped within the 5′ sensor region of the RNase E protein. Molecular models of RNase E suggest how suppression may occur.
Interactions between branched DNAs and peptide inhibitors of DNA repair
Authors: Kepple Kevin V. , Patel Namita , Salamon Peter , Segall Anca M.
Published: August 2008

The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (Kd = 14 nM) and ~4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides’ mechanism of action will further increase their utility.
Innate Immunity Signaling: Cytosolic Ca2+ Elevation Is Linked to Downstream Nitric Oxide Generation through the Action of Calmodulin or a Calmodulin-Like Protein
Authors: Ma Wei , Smigel Andries , Tsai Yu-Chang , Braam Janet , Berkowitz Gerald A.
Published: August 2008

Ca2+ rise and nitric oxide (NO) generation are essential early steps in plant innate immunity and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this laboratory has demonstrated that a loss-of-function mutation of an Arabidopsis (Arabidopsis thaliana) plasma membrane Ca2+-permeable inwardly conducting ion channel impairs HR and that this phenotype could be rescued by the application of a NO donor. At present, the mechanism linking cytosolic Ca2+ rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca2+/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca2+ signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild-type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and the HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca2+ elevation, excluding the possibility of CaM acting upstream from Ca2+. The CaM antagonist and Ca2+ chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca2+/CaM dependent in vitro. The CaM-like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and [avirulent] pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogen-associated molecular pattern-mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca2+ influx into the cytosol activates CaM and/or a CML, which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade, leading to innate immune responses, including the HR.
LEAFY COTYLEDON1Is a Key Regulator of Fatty Acid Biosynthesis in Arabidopsis
Authors: Mu Jinye , Tan Helin , Zheng Qi , Fu Fuyou , Liang Yan , Zhang Jian , Yang Xiaohui , Wang Tai , Chong Kang , Wang Xiu-Jie , Zuo Jianru
Published: August 2008

In plants, fatty acids are de novo synthesized predominantly in plastids from acetyl-coenzyme A. Although fatty acid biosynthesis has been biochemically well studied, little is known about the regulatory mechanisms of the pathway. Here, we show that overexpression of the Arabidopsis (Arabidopsis thaliana) LEAFY COTYLEDON1 (LEC1) gene causes globally increased expression of fatty acid biosynthetic genes, which are involved in key reactions of condensation, chain elongation, and desaturation of fatty acid biosynthesis. In the plastidial fatty acid synthetic pathway, over 58% of known enzyme-coding genes are up-regulated in LEC1-overexpressing transgenic plants, including those encoding three subunits of acetyl-coenzyme A carboxylase, a key enzyme controlling the fatty acid biosynthesis flux. Moreover, genes involved in glycolysis and lipid accumulation are also up-regulated. Consistent with these results, levels of major fatty acid species and lipids were substantially increased in the transgenic plants. Genetic analysis indicates that the LEC1 function is partially dependent on ABSCISIC ACID INSENSITIVE3, FUSCA3, and WRINKLED1 in the regulation of fatty acid biosynthesis. Moreover, a similar phenotype was observed in transgenic Arabidopsis plants overexpressing two LEC1-like genes of Brassica napus. These results suggest that LEC1 and LEC1-like genes act as key regulators to coordinate the expression of fatty acid biosynthetic genes, thereby representing promising targets for genetic improvement of oil production plants.
Lipid Trafficking between the Endoplasmic Reticulum and the Plastid in Arabidopsis Requires the Extraplastidic TGD4 Protein
Authors: Xu Changcheng , Fan Jilian , Cornish Adam J. , Benning Christoph
Published: August 2008

The development of chloroplasts in Arabidopsis thaliana requires extensive lipid trafficking between the endoplasmic reticulum (ER) and the plastid. The biosynthetic enzymes for the final steps of chloroplast lipid assembly are associated with the plastid envelope membranes. For example, during biosynthesis of the galactoglycerolipids predominant in photosynthetic membranes, galactosyltransferases associated with these membranes transfer galactosyl residues from UDP-Gal to diacylglycerol. In Arabidopsis, diacylglycerol can be derived from the ER or the plastid. Here, we describe a mutant of Arabidopsis, trigalactosyldiacylglycerol4 (tgd4), in which ER-derived diacylglycerol is not available for galactoglycerolipid biosynthesis. This mutant accumulates diagnostic oligogalactoglycerolipids, hence its name, and triacylglycerol in its tissues. The TGD4 gene encodes a protein that appears to be associated with the ER membranes. Mutant ER microsomes show a decreased transfer of lipids to isolated plastids consistent with in vivo labeling data, indicating a disruption of ER-to-plastid lipid transfer. The complex lipid phenotype of the mutant is similar to that of the tgd1,2,3 mutants disrupted in components of a lipid transporter of the inner plastid envelope membrane. However, unlike the TGD1,2,3 complex, which is proposed to transfer phosphatidic acid through the inner envelope membrane, TGD4 appears to be part of the machinery mediating lipid transfer between the ER and the outer plastid envelope membrane. The extent of direct ER-to-plastid envelope contact sites is not altered in the tgd4 mutant. However, this does not preclude a possible function of TGD4 in those contact sites as a conduit for lipid transfer between the ER and the plastid.
Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction
Authors: Sun Kai‐Hui , De Pablo Yolanda , Vincent Fabien , Shah Kavita
Published: August 2008

Oxidative stress is one of the earliest events in Alzheimer’s disease (AD). A chemical genetic screen revealed that deregulated cyclin‐dependent kinase 5 (Cdk5) may cause oxidative stress by compromising the cellular anti‐oxidant defense system. Using novel Cdk5 modulators, we show the mechanism by which Cdk5 can induce oxidative stress in the disease’s early stage and cell death in the late stage. Cdk5 dysregulation upon neurotoxic insults results in reactive oxygen species (ROS) accumulation in neuronal cells because of the inactivation of peroxiredoxin I and II. Sole temporal activation of Cdk5 also increases ROS, suggesting its major role in this process. Cdk5 inhibition rescues mitochondrial damage upon neurotoxic insults, thereby revealing Cdk5 as an upstream regulator of mitochondrial dysfunction. As mitochondrial damage results in elevated ROS and Ca2+ levels, both of which activate Cdk5, we propose that a feedback loop occurs in late stage of AD and leads to cell death (active Cdk5 → ROS → excess ROS → mitochondrial damage → ROS → hyperactive Cdk5 → severe oxidative stress and cell injury → cell death). Cdk5 inhibition upon neurotoxic insult prevents cell death significantly, supporting this hypothesis. As oxidative stress and mitochondrial dysfunction play pivotal roles in promoting neurodegeneration, Cdk5 could be a viable therapeutic target for AD.
A coordinated approach to cutaneous wound healing: vibrational microscopy and molecular biology
Authors: Chan K. L. Andrew , Zhang Guojin , Tomic‐Canic Marjana , Stojadinovic Olivera , Lee Brian , Flach Carol R. , Mendelsohn Richard
Published: August 2008

The repair of cutaneous wounds in the adult body involves a complex series of spatially and temporally organized processes to prevent infection and restore homeostasis. Three characteristic phases of wound repair (inflammation, proliferation including re‐epithelialization and remodelling) overlap in time and space. We have utilized a human skin wound‐healing model to correlate changes in genotype and pheno‐type with infrared (IR) and confocal Raman spectroscopic images during the re‐epithelialization of excisional wounds. The experimental protocols validated as IR images clearly delineate the keratin‐rich migrating epithelial tongue from the collagen‐rich wound bed. Multivariate statistical analysis of IR datasets acquired 6 days post‐wounding reveal subtle spectral differences that map to distinct spatial distributions, which are correlated with immunofluorescent staining patterns of different keratin types. Images computed within collagen‐rich regions expose complementary spatial patterns and identify elastin in the wound bed. The temporal sequence of events is explored through a comparison of gene array analysis with confocal Raman microscopy. Our approach demonstrates the feasibility of acquiring detailed molecular structure information from the various proteins and their subclasses involved in the wound‐healing process.
Histochemical and ultrastructural characteristics of an interstitial cell type different from ICC and resident in the muscle coat of human gut
Authors: Pieri Laura , Vannucchi Maria Giuliana , Faussone‐Pellegrini Maria Simonetta
Published: August 2008

CD117 (or c‐kit) is expressed by the interstitial cells of Cajal (ICC), which are located within the gastrointestinal (GI) muscle coat and directly involved in its motility. CD34 is expressed by several cell types some of which have features and location resembling the ICC; however, a sure identification of these cells is still lacking. In order to establish whether the CD34‐positive cells of the human GI tract are to be considered as ICC subpopulation or a novel independent cell type, and to hypothesize their nature and role, we verified CD34 and CD117 receptor expression under light and fluorescence microscope and performed a routine and a CD34‐immuno‐electron microscopy. CD34‐positive cells were seen in the entire human GI tract. In the muscularis propria, shared morphologies similar to the c‐kit‐positive cells, in the submucosa, resembled fibroblasts. Their ultrastructure resembled that of the fibrocytes/fibroblasts and of the interstitial Cajal‐like cells (ICLC). Double labelling and immunoelectro‐microscopy demonstrated that they are unequivocally different to the ICC and, due to the similarities with the ICLC, we identified them as ICLC. The novelty of these results is that two types of interstitial cells are present in the GI muscle coat of humans: the ICC and the ICLC. We hypothesize a mechanical role for the septal ICLC, those at the myenteric plexus level and those bordering the muscle layers; a helping role in neurotransmission is proposed for the ICLC intercalated with the intramuscular ICC, possibly in spreading the slow waves generated by the ICC. Furthermore, the possibility that the ICLC represent the adult mesenchymal stromal cells able to guarantee the ICC renewal deserves to be considered.
One carbon metabolism disturbances and the C677T MTHFR gene polymorphism in children with autism spectrum disorders
Authors: Paşca Sergiu P. , Dronca Eleonora , Kaucsár Tamás , Crǎciun Elena C. , Endreffy Emõke , Ferencz Beatrix K. , Iftene Felicia , Benga Ileana , Cornean Rodica , Banerjee Ruma , Dronca Maria
Published: August 2008

Autism spectrum disorders (ASDs), which include the prototypic autistic disorder (AD), Asperger’s syndrome (AS) and pervasive developmental disorders not otherwise specified (PDD‐NOS), are complex neurodevelopmental conditions of unknown aetiology. The current study investigated the metabolites in the methionine cycle, the transsulphuration pathway, folate, vitamin B12 and the C677T polymorphism of the MTHFR gene in three groups of children diagnosed with AD (n= 15), AS (n= 5) and PDD‐NOS (n= 19) and their age‐ and sex‐matched controls (n= 25). No metabolic disturbances were seen in the AS patients, while in the AD and PDD‐NOS groups, lower plasma levels of methionine (P= 0.01 and P= 0.03, respectively) and α‐aminobutyrate were observed (P= 0.01 and P= 0.001, respectively). Only in the AD group, plasma cysteine (P= 0.02) and total blood glutathione (P= 0.02) were found to be reduced. Although there was a trend towards lower levels of serine, glycine, N, N‐dimethylglycine in AD patients, the plasma levels of these metabolites as well as the levels of homocysteine and cystathionine were not statistically different in any of the ASDs groups. The serum levels of vitamin B12 and folate were in the normal range. The results of the MTHFR gene analysis showed a normal distribution of the C677T polymorphism in children with ASDs, but the frequency of the 677T allele was slightly more prevalent in AD patients. Our study indicates a possible role for the alterations in one carbon metabolism in the pathophysiology of ASDs and provides, for the first time, preliminary evidence for metabolic and genetic differences between clinical subtypes of ASDs.
Proteomics analysis of protein expression and specific protein oxidation in human papillomavirus transformed keratinocytes upon UVB irradiation
Authors: Perluigi Marzia , Giorgi Alessandra , Blarzino Carla , De Marco Federico , Foppoli Cesira , Di Domenico Fabio , Butterfield D. Allan , Schininà M. Eugenia , Cini Chiara , Coccia Raffaella
Published: August 2008

Increasing evidence supports the role of oxidative stress in cancer development. Ultraviolet (UV) irradiation is one of the major sources of oxidative stress through the generation of reactive oxygen species (ROS). Besides the physiological function of ROS in cellular homeostasis, accumulating reports suggest that ROS are involved in all stages of multistep cancer development. In order to investigate the involvement of oxidative damage into the mechanisms of tumour progression, we used a parallel proteomic approach to analyse the protein expression profile and to identify oxidatively modified proteins in human papillomavirus (HPV)‐transformed keratinocytes (HK‐168 cells) upon ultraviolet B (UVB) exposure. The HK‐168 cells were obtained from normal human epidermal keratinocytes transfected with the whole genome of the high‐risk HPV type 16, unanimously recognized as an etiological agent of cervical carcinoma. Because of its year‐long latency, this tumour offers a convenient model to study the role of environmental concurring agents in the multistep malignant progression. By the protein expression profile, we identified 21 proteins that showed different expression levels in HK‐168 cells treated with UVB in comparison with untreated cells. Focusing on the oxidative modifications occurring at the protein level, we identified five proteins that showed elevated protein carbonyls levels: α‐enolase, heat shock protein 75, annexin 2, elongation factor Tu and elongation factor γ. Our results indicate that UVB‐induced oxidative stress perturbs the normal redox balance and shifts HPV‐transformed keratinocytes into a state in which the carbonylation of specific proteins is systematically induced. We suggest that UVB‐induced modulation of protein expression combined with oxidative modification lead to protein dysfunction that might contribute to the malignant progression of transformed cells.
PREB regulates transcription of pancreatic glucokinase in response to glucose and cAMP
Authors: Muraoka Tomie , Murao Koji , Imachi Hitomi , Yu Xiao , Li Junhua , Wong Norman CW , Ishida Toshihiko
Published: August 2008

Prolactin regulatory element binding (PREB) is a transcription factor that regulates prolactin promoter activity in rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the pancreas. We have recently reported that in pancreatic β‐cells, PREB regulates the transcription of the insulin gene in response to glucose stimulation. In the current study, we have examined the role of PREB in regulating glucokinase (GK) in pancreatic β‐cells. To analyse the effects of PREB on GK gene transcription, we employed a reporter gene assay. In the cells expressing or with knocked down PREB, GK expression was determined. GK expression was regulated by glucose and cAMP, and both glucose and cAMP stimulated the expression of PREB in a dose‐dependent manner. Conversely, overexpression of PREB using a PREB‐expressing adenovirus increased the expression of the GK protein. GK enzymatic activity was also significantly increased in the cells that stably expressed PREB. In addition, PREB induced GK promoter activity. Chromatin immunoprecipitation (ChIP) analyses showed that PREB mediated its transcriptional effect by binding to the PREB‐responsive cis‐element of the GK promoter. Finally, we used siRNA to inhibit PREB expression in cells and demonstrated that the knockdown of PREB attenuated the effects of glucose and cAMP on GK expression. Our data show that in pancreatic β‐cells, PREB regulates the transcription of the GK gene in response to glucose and cAMP.
Acute Perturbations in Golgi Organization Impact De Novo Sphingomyelin Synthesis
Authors: Chandran Suchismita , Machamer Carolyn E.
Published: August 2008

The mammalian Golgi apparatus is composed of multiple stacks of cisternal membranes organized laterally into a ribbon‐like structure, with close apposition of trans Golgi regions with specialized endoplasmic reticulum (ER) membranes. These contacts may be the site of ceramide transfer from its site of synthesis (ER) to sphingomyelin (SM) synthase through ceramide transfer protein (CERT). CERT extracts ceramide from the ER and transfers it to Golgi membranes but the role of overall Golgi structure in this process is unknown. We show here that localization of CERT in puncta around the Golgi complex requires both ER‐ and Golgi‐binding domains of CERT. To examine how Golgi structure contributes to SM synthesis, we treated cells with Golgi‐perturbing drugs and measured newly synthesized SM. Interestingly, disruption of Golgi morphology with nocodazole, but not ilimaquinone inhibited SM synthesis. Decreased localization of CERT with a Golgi marker correlated with decreased SM synthesis. We propose that some Golgi structural perturbations interfere with efficient ceramide trafficking through CERT, and thus SM synthesis. The organization of the mammalian Golgi ribbon together with CERT may promote specific ER–Golgi interactions for efficient delivery of ceramide for SM synthesis.
The Peroxisome RING‐Finger Complex is Required for Meiocyte Formation in the Fungus Podospora anserina
Authors: Peraza‐Reyes Leonardo , Zickler Denise , Berteaux‐Lecellier Véronique
Published: August 2008

Peroxisomes are involved in a variety of metabolic pathways and developmental processes. In the filamentous fungus Podospora anserina, absence of different peroxins implicated in peroxisome matrix protein import leads to different developmental defects. Lack of the RING‐finger complex peroxin PEX2 blocks sexual development at the dikaryotic stage, while in absence of both receptors, PEX5 and PEX7, karyogamy and meiosis can proceed and sexual spores are formed. This suggests a complex role for PEX2 that prompted us to study the developmental involvement of the RING‐finger complex. We show that, like PEX2, the two other proteins of the complex, PEX10 and PEX12, are equally implicated in peroxisome biogenesis and that absence of each or all these proteins lead to the same developmental defect. Moreover, we demonstrate that peroxisome localization of PEX2 is not drastically affected in the absence of PEX10 and PEX12 and that the upregulation of these latter RING‐finger peroxins does not compensate for the lack of a second one, suggesting that the three proteins work together in development but independent of their function in peroxisome biogenesis.
Rapid Recycling of β2‐Adrenergic Receptors is Dependent on the Actin Cytoskeleton and Myosin Vb
Authors: Millman Ellen E. , Zhang Haibin , Zhang Haixia , Godines Veronica , Bean Andrew J. , Knoll Brian J. , Moore Robert H.
Published: August 2008

For the β2‐adrenergic receptor (β2AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of β2AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, β2AR internalization into transferrin‐positive vesicles was not altered but both agents significantly decreased the rate at which β2ARs recycled to the cell surface. In LB‐treated cells, nonrecycled β2ARs were localized to early embryonic antigen 1‐positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP‐positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of β2ARs to the RE, while the expression of various CART fragments or the depletion of actinin‐4 had no detectable effect on β2AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of β2ARs, a process that likely is dependent on myosin Vb.
Palmitoylation Controls Recycling in Lysosomal Sorting and Trafficking
Authors: McCormick Peter J. , Dumaresq‐Doiron Karine , Pluviose Anne‐Sophie , Pichette Vincent , Tosato Giovanna , Lefrancois Stephane
Published: August 2008

For the efficient trafficking of lysosomal proteins, the cationic‐dependent and ‐independent mannose 6‐phosphate receptors and sortilin must bind cargo in the Golgi apparatus, be packaged into clathrin‐coated trafficking vesicles and traffic to the endosomes. Once in the endosomes, the receptors release their cargo into the endosomal lumen and recycle back to the Golgi for another round of trafficking, a process that requires retromer. In this study, we demonstrate that palmitoylation is required for the efficient retrograde trafficking of sortilin, and the cationic‐independent mannose 6‐phosphate as palmitoylation‐deficient receptors remain trapped in the endosomes. Importantly, we also show that palmitoylation is required for receptor interaction with retromer as nonpalmitoylated receptor did not interact with retromer. In addition, we have identified DHHC‐15 as the palmitoyltransferase responsible for this modification. In summary, we have shown the functional significance of palmitoylation in lysosomal receptor sorting and trafficking.
High Data Output and Automated 3D Correlative Light–Electron Microscopy Method
Authors: Vicidomini Giuseppe , Gagliani Maria C. , Canfora Michela , Cortese Katia , Frosi Fabio , Santangelo Clara , Di Fiore Pier Paolo , Boccacci Patrizia , Diaspro Alberto , Tacchetti Carlo
Published: August 2008

Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections. We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of our method are (i) the possibility to correlate several hundreds of events at the same time, (ii) the possibility to perform three‐dimensional (3D) correlation, (iii) the possibility to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the possibility to combine the high data analysis capability of FLM with the high precision–accuracy of transmission electron microscopy in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach.
Relative torque contribution of vastus medialis muscle at different knee angles
Authors: De Ruiter C. J. , Hoddenbach J. G. , Huurnink A. , De Haan A.
Published: August 2008

Aim:  We investigated the relative contribution of the vastus medialis (VM) muscle to total isometric knee extension torque at 10°, 30°, 60° and 90° knee flexion. In the past a more prominent role of the VM muscle at more extended knee angles has been put forward. However, different components of the quadriceps muscle converge via a common distal tendon. We therefore hypothesized that the relative contribution of the VM to total knee extension torque would be similar across angles.

Methods:  At each knee angle the EMG isometric torque relations [20%, 25%, 30%, 35% maximal voluntary contraction (MVC)] of the rectus femoris (RF), vastus lateralis (VL) and VM muscle were established in 10 healthy male subjects; rectified surface EMG was normalized to M‐wave area. Subsequently, the VM was functionally eliminated by selective electrical surface stimulation with occluded blood flow.

Results:  There was no evidence for preferential activation of VM at any of the knee angles. Following VM elimination, total knee extension torque during maximal femoral nerve stimulation (three pulses at 300Hz) at 10°, 30°, 60° and 90°, respectively, decreased (P < 0.05) to (mean ± SD): 75.7 ± 12.2, 75.1 ± 9.3, 78.2 ± 7.2 and 76.0 ± 5.8% (P > 0.05 among knee angles). In addition, during voluntary contractions at 20% MVC the increases in torque output of RF and VL compensating for the loss of VM function were calculated from the increases in EMG and found to be similar (P > 0.05) at 10°, 30°, 60° and 90° values (%MVC), respectively, were: 9.1 ± 6.8, 7.5 ± 2.9, 5.9 ± 3.7 and 6.9 ± 3.4.

Conclusion:  The present findings support our hypothesis that the VM contributes similarly to total knee extension torque at different knee angles.
“
Candidatus
Midichloria” Endosymbionts Bloom after the Blood Meal of the Host, the Hard Tick
Ixodes ricinus
Authors: Sassera Davide , Lo Nathan , Bouman Edwin A. P. , Epis Sara , Mortarino Michele , Bandi Claudio
Published: August 2008

“
Candidatus
Midichloria mitochondrii,” an intracellular symbiont of the tick
Ixodes ricinus
, is the only described organism able to invade the mitochondria of any multicellular organism. We used quantitative PCR to examine cycles of bacterial growth and death throughout the host's development and found that they correspond with the phases of engorgement and molt, respectively.
Detection of
Bifidobacterium animalis
subsp.
lactis
(Bb12) in the Intestine after Feeding of Sows and Their Piglets
Authors: Solano-Aguilar Gloria , Dawson Harry , Restrepo Marta , Andrews Kate , Vinyard Bryan , Urban Joseph F.
Published: August 2008

A real-time PCR method has been developed to distinguish
Bifidobacterium animalis
subspecies in the gastrointestinal tracts of pigs. Identification of a highly conserved single-copy
tuf
gene encoding the elongation factor Tu involved in bacterial protein biosynthesis was used as a marker to differentiate homologous
Bifidobacterium animalis
subsp.
lactis
(strain Bb12) from
Bifidobacterium animalis
subsp.
animalis
, as well as
Bifidobacterium suis
,
Bifidobacterium breve
,
Bifidobacterium longum
, several species of
Lactobacillus
, and
Enterococcus faecium
. Real-time PCR detection of serially diluted DNA extracted from a pure culture of Bb12 was linear for bacterial numbers ranging from 10 to 10,000
tuf
gene copies per PCR (
r
2
= 0.99). Relative differences in Bb12 bacterial numbers in pigs fed daily with Bb12 were determined after detection of Bb12
tuf
gene copies in DNA extracted from the intestinal contents. Piglets treated with Bb12 immediately after birth maintained a high level of Bb12 in their large intestines with continuous daily administration of Bb12. Piglets born to Bb12-treated sows during the last third of their gestation and also treated with Bb12 at birth (T/T group) had a higher number of Bb12 organisms per gram of intestinal contents compared to placebo-treated piglets born to placebo-treated sows (C/C group), Bb12-treated sows (T/C group), or piglets born to placebo sows but treated with Bb12 immediately after birth (C/T group). In addition, there was a significant increase in gene expression for Toll-like receptor 9 (TLR9) in piglets from the T/T group, with no change in TLR2 and TLR4. These findings suggest that the
tuf
gene represents a specific and functional marker for detecting
Bifidobacterium animalis
subsp.
lactis
strain Bb12 within the microbiota of the intestine.