Vacuolar ATP Synthase Subunit Research Articles

Giardia intestinalis coiled-coil cytolinker protein 259 interacts with actin and tubulin.

Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.

In silico analyses of molecular interactions between groundnut bud necrosis virus and its vector, Thrips palmi.

Groundnut bud necrosis virus (GBNV) is an economically important tospovirus transmitted by Thrips palmi (Thysanoptera: Thripidae). The current understanding of thrips-tospovirus interactions is largely based on the tomato spotted wilt virus-Frankliniella occidentalis relationship. Only limited information is available for the GBNV-T. palmi system. In the present study, available genome data of T. palmi and GBNV were used to predict the protein partners that may play a crucial role in the internalization of GBNV virions into thrips cells. Computational analyses showed that the GBNV precursor glycoprotein bears a signal peptide of 24 amino acids and a secondary cleavage site at position 434-435 separates the amino-terminal mature glycoprotein (GN) from the carboxyl-terminal glycoprotein (GC). Potential interactions of GBNV glycoproteins were predicted with T. palmi enolase, cathepsin, C-type lectin, clathrin and vacuolar ATP synthase subunit E. The in silico analyses suggested that C-type lectin is the primary cellular receptor to interact with GBNV-GN. After receptor binding, virus particles probably enter vector cells by clathrin-mediated endocytosis. This is the first in silico evidence of GBNV-T. palmi protein interaction.

An EPIC journey to locate single‐copy nuclear markers in sea anemones

AbstractSea anemones (Order Actiniaria) are among the most diverse members of the subclass Hexacorallia and are an emerging model system. Unfortunately, defining species and creating robust phylogenies remain a serious challenge due to simple body plans that reduce the number of available morphological characters, slow mitochondrial sequence evolution and a complete absence of nuclear markers in the actiniarian molecular toolkit other than the ribosomal cistron. Defining species boundaries and phylogenetic relationships is imperative for advancing our understanding of sea anemone taxonomy and systematics. Herein, we utilized EPIC (exon‐priming intron‐crossing) primers, in addition to non‐standard PCR profiling conditions, to obtain sequence data for seven nuclear introns located within Calmodulin, Calpain, Nck Associated Protein 1 Homolog, Pescadillo Homolog, Signal Recognition Particle 54‐kDa Subunit, Transferase and Vacuolar ATP Synthase Subunit B. These new markers were employed to evaluate whether morphological variability in marginal tentacle protuberances is indicative of intraspecific variation or cryptic species in the shallow‐water anemone Phymanthus crucifer. Variability within these nuclear introns was compared to mitochondrial 12S, 16S and cox3, and nuclear 18S and 28S. PCR products of all nuclear introns were cloned to determine copy number and the resulting variability among clones was consistent with single‐copy markers. We also amplified and sequenced six of the seven introns in a sister species, Phymanthus loligo, to determine the degree of interspecific variation. In addition, we evaluate the applicability of a subset of these markers within Actinostola and conclude with a review of putative single‐copy markers used in octocoral and scleractinian phylogenetics.

Transcriptomic alterations in Sitophilus zeamais in response to allyl isothiocyanate fumigation

To study the fumigation mechanisms of Allyl isothiocyanate (AITC) a promising biorational alternative to present fumigants (phosphine and methyl bromide), and provide theoretical basis for its further development in the control of stored grain pests, this research presents a transcriptome analysis of Sitophilus zeamais fumigated with AITC at the concentration of LC50 (5.69μg/mL) and control over 8h. 21,869,022 and 23,873,110 clean reads in insects fumigated with AITC and control were gained, respectively. The results of RNA-seq were confirmed by qRT-PCR determination of the expression levels of NADH dehydrogenase subunit 6 and Vacuolar ATP synthase subunit B in the insects fumigated with AITC at different concentrations. After enrichment analysis of differentially expressed genes, 117 over-expressed and 271 down-regulated transcripts were gained. Following GO enrichment, these transcripts were classified into 38 GO subgroups (at level 2), and the majority enriched GO terms were “Binding” “Cell process” and “metabolic”. KEGG enrichment analysis showed that the majority enriched pathway were “Folding, sorting and degradation”, “Transport and catabolism”, “Energy metabolism”, and “Carbohydrate metabolism”. Connected with previous researches on mechanisms of isothiocyanates, cytoskeleton collapse and mitochondria dysfunction are proposed to be significant lethal mechanisms of AITC.

Quantitative proteomic analysis of the rice (Oryza sativa L.) salt response.

Salt stress is one of most serious limiting factors for crop growth and production. An isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approach was used to analyze proteomic changes in rice shoots under salt stress in this study. A total of 56 proteins were significantly altered and 16 of them were enriched in the pathways of photosynthesis, antioxidant and oxidative phosphorylation. Among these 16 proteins, peroxiredoxin Q and photosystem I subunit D were up-regulated, while thioredoxin M-like, thioredoxin x, thioredoxin peroxidase, glutathione S-transferase F3, PSI subunit H, light-harvesting antenna complex I subunits, chloroplast chaperonin, vacuolar ATP synthase subunit H, and ATP synthase delta chain were down-regulated. Moreover, physiological data including total antioxidant capacity, peroxiredoxin activity, chlorophyll a/b content, glutathione S-transferase activity, reduced glutathione content and ATPase activity were consistent with changes in the levels of these proteins. The levels of the mRNAs encoding these proteins were also analyzed by real-time quantitative reverse transcription PCR, and approximately 86% of the results were consistent with the iTRAQ data. Importantly, our data suggest the important role of PSI in balancing energy supply and ROS generation under salt stress. This study provides information for an improved understanding of the function of photosynthesis and PSI in the salt-stress response of rice.

RNA interference tools for the western flower thrips, Frankliniella occidentalis

The insect order Thysanoptera is exclusively comprised of small insects commonly known as thrips. The western flower thrips, Frankliniella occidentalis, is an economically important pest amongst thysanopterans due to extensive feeding damage and tospovirus transmission to hundreds of plant species worldwide. Geographically-distinct populations of F. occidentalis have developed resistance against many types of traditional chemical insecticides, and as such, management of thrips and tospoviruses are a persistent challenge in agriculture. Molecular methods for defining the role(s) of specific genes in thrips–tospovirus interactions and for assessing their potential as gene targets in thrips management strategies is currently lacking. The goal of this work was to develop an RNA interference (RNAi) tool that enables functional genomic assays and to evaluate RNAi for its potential as a biologically-based approach for controlling F. occidentalis. Using a microinjection system, we delivered double-stranded RNA (dsRNA) directly to the hemocoel of female thrips to target the vacuolar ATP synthase subunit B (V-ATPase-B) gene of F. occidentalis. Gene expression analysis using real-time quantitative reverse transcriptase-PCR (qRT-PCR) revealed significant reductions of V-ATPase-B transcripts at 2 and 3days post-injection (dpi) with dsRNA of V-ATPase-B compared to injection with dsRNA of GFP. Furthermore, the effect of knockdown of the V-ATPase-B gene in females at these two time points was mirrored by the decreased abundance of V-ATPase-B protein as determined by quantitative analysis of Western blots. Reduction in V-ATPase-B expression in thrips resulted in increased female mortality and reduced fertility, i.e., number of viable offspring produced. Survivorship decreased significantly by six dpi compared to the dsRNA-GFP control group, which continued decreasing significantly until the end of the bioassay. Surviving female thrips injected with dsRNA-V-ATPase-B produced significantly fewer offspring compared to those in the dsRNA-GFP control group. Our findings indicate that an RNAi-based strategy to study gene function in thrips is feasible, can result in quantifiable phenotypes, and provides a much-needed tool for investigating the molecular mechanisms of thrips–tospovirus interactions. To our knowledge, this represents the first report of RNAi for any member of the insect order Thysanoptera and demonstrates the potential for translational research in the area of thrips pest control.

Evaluation of anti-coccidial effects of 1-[4-(4-nitrophenoxy)phenyl]propane-1-one and identification of its potential target proteins in Toxoplasma gondii.

Coccidiosis affects many vertebrates worldwide, but treatment with known anti-coccidial drugs causes several adverse side effects. There is a critical need for the development and evaluation of new drugs. The anti-coccidial effect of 1-[4-(4-nitrophenoxy)phenyl]propane-1-one (NPPP), a synthetic compound, was studied in vitro and in vivo. Treatment with NPPP showed anti-Toxoplasma activity in vitro with a lower EC50 value than pyrimethamine. In ICR mice infected with Toxoplasma gondii, oral administration of NPPP for 4days showed statistically significant anti-Toxoplasma activity with lower numbers of tachyzoite than those of the negative control (p<0.01). NPPP also exhibited strong anti-Eimeria activity in Eimeria tenella-infected chickens when treated for 4days with orally administered NPPP at a dose of 100mg/kg. Potential target proteins of NPPP were analyzed by proteomic profiles of T. gondii tachyzoites. Two hypothetical proteins were identified as possible targets of NPPP, a putative ortholog of vacuolar ATP synthase subunit C and a class I S-adenosylmethionine-dependent methyltransferase. Our data show that the NPPP might be an anti-coccidial drug candidate for clinical application against coccidial infections. Future investigations will focus on identifying the function of proteins regulated by NPPP.

Proteomic Analysis of Differentially Expressed Proteins in Fenneropenaeus chinensis Hemocytes upon White Spot Syndrome Virus Infection

To elucidate molecular responses of shrimp hemocytes to white spot syndrome virus (WSSV) infection, two-dimensional gel electrophoresis was applied to investigate differentially expressed proteins in hemocytes of Chinese shrimp (Fenneropenaeus chinensis) at 24 h post infection (hpi). Approximately 580 protein spots were detected in hemocytes of healthy and WSSV-infected shrimps. Quantitative intensity analysis revealed 26 protein spots were significantly up-regulated, and 19 spots were significantly down-regulated. By mass spectrometry, small ubiquitin-like modifier (SUMO) 1, cytosolic MnSOD, triosephosphate isomerase, tubulin alpha-1 chain, microtubule-actin cross-linking factor 1, nuclear receptor E75 protein, vacuolar ATP synthase subunit B L form, inositol 1,4,5-trisphosphate receptor, arginine kinase, etc., amounting to 33 differentially modulated proteins were identified successfully. According to Gene Ontology annotation, the identified proteins were classified into nine categories, consisting of immune related proteins, stimulus response proteins, proteins involved in glucose metabolic process, cytoskeleton proteins, DNA or protein binding proteins, proteins involved in steroid hormone mediated signal pathway, ATP synthases, proteins involved in transmembrane transport and ungrouped proteins. Meanwhile, the expression profiles of three up-regulated proteins (SUMO, heat shock protein 70, and arginine kinase) and one down-regulated protein (prophenoloxidase) were further analyzed by real-time RT-PCR at the transcription level after WSSV infection. The results showed that SUMO and heat shock protein 70 were significantly up-regulated at each sampling time point, while arginine kinase was significantly up-regulated at 12 and 24 hpi. In contrast, prophenoloxidase was significantly down-regulated at each sampling time point. The results of this work provided preliminary data on proteins in shrimp hemocytes involved in WSSV infection.

Molecular characterization and serological reactivity of a vacuolar ATP synthase subunit ε-like protein from Clonorchis sinensis

The vacuolar ATPase enzyme complex (V-ATPase) pumps protons across membranes, energized by hydrolysis of ATP. Extensive investigations on structural and biochemical features of these molecules have implied their importance in the physiological process. In this study, a full-length sequence encoding a vacuolar ATP synthase subunit ε-like protein of Clonorchis sinensis (CsATP-ε) was isolated from our cDNA library. The hypothetical 226 amino acid sequence shared 76% identity with ATP-ε proteins of Schistosoma japonicum and above 55% identity with ATP-ε proteins from human and other eukaryotes. Characteristic Asp₁₄₀ amino acid residues and seven B-cell epitopes were predicted in this sequence. The complete coding sequence of the gene was expressed in Escherichia coli. Recombinant CsATP-ε (rCsATP-ε) protein could be probed by anti-rCsATP-ε rat serum and C.sinensis-infected human serum in Western blotting experiment, indicating that it is an antigen of strong antigenicity. The high level of antibody titers (1:204,800) showed that CsATP-ε has a powerful immunogenicity. Both the increased level and the change trend of IgG1/IgG2a subtypes in serum showed that the rCsATP-ε can induce strong combined Th1/Th2 immune responses in rats and stimulate the immune response changes to the dominant Th2 from Th1 along with long time infection. The results of immunoblot and immunolocalization demonstrated that CsATP-ε was consecutively expressed at various developmental stages of the parasite, which was supported by real-time PCR analysis. In immunohistochemistry, CsATP-ε was localized on the intestine, vitellarium, and testicle of an adult worm and excretory bladder of metacercaria, implying that CsATP-ε may relate to energy intake and metabolism. This fundamental study would contribute to further researches that are related to growth and development and immunomodulation of C. sinensis.

Co‐immunoprecipitation‐based identification of putative BAX INHIBITOR‐1‐interacting proteins involved in cell death regulation and plant–powdery mildew interactions

The endoplasmic reticulum (ER)-resident BAX INHIBITOR-1 (BI-1) protein is one of a few cell death suppressors known to be conserved in animals and plants. The function of BI-1 proteins in response to various biotic and abiotic stress factors is well established. However, little is known about the underlying mechanisms. We conducted co-immunoprecipitation (co-IP) experiments to identify Arabidopsis thaliana BI-1-interacting proteins to obtain a potentially better understanding of how BI-1 functions during plant-pathogen interactions and as a suppressor of cell death. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 95 proteins co-immunoprecipitated with green fluorescing protein (GFP)-tagged BI-1. Five selected candidate proteins, a RIBOPHORIN II (RPN2) family protein, VACUOLAR ATP SYNTHASE SUBUNIT A (VHA-A), cytochrome P450 83A1 (CYP83A1), H(+) -ATPASE 1 (AHA1) and PROHIBITIN 2 (PHB2), were further investigated with regard to their role in BI-1-associated processes. To this end, we analysed a set of Arabidopsis mutants in the interaction with the adapted powdery mildew fungus Erysiphe cruciferarum and on cell death-inducing treatments. Two independent rpn2 knock-down mutants tended to better support powdery mildew, and a phb2 mutant showed altered responses to cell death-inducing Alternaria alternata f.sp. lycopersici (AAL) toxin treatment. Two independent cyp83a1 mutants showed a strong powdery mildew resistance phenotype and enhanced sensitivity to AAL toxin. Moreover, co-localization studies and fluorescence resonance energy transfer (FRET) experiments suggested a direct interaction of BI-1 with CYP83A1 at the ER.

Comparison of proteomes between wheat-rye translocations and their recurrent parents

2BS.2RL wheat-rye translocations have presented phenotypes of improved agronomic performance, such as Hessian fly resistance. The main objective of this work was to use two-dimensional electrophoresis to identify the proteomic differences between 2BS.2RL wheat-rye translocations and their recurrent parents. The investigation of seeds revealed line-specific protein spots, such as α-amylase inhibitor 0.19. More diverse expression patterns were observed in leaves than in seeds. Protein spots found specifically in 2BS.2RL, but not in non-2RL translocations were identified as β-glucosidase and vacuolar ATP synthase subunit B2, demonstrating the effects of translocated rye chromatin 2RL on common wheat genetic background. When the leaf protein spots were compared in the control and Hessian fly-infested near-isogenic line (NIL) (2BS.2RL), many down-regulated proteins and specific proteins, such as β-glucosidase, were detected in the latter.

RNA interference in Nilaparvata lugens (Homoptera: Delphacidae) based on dsRNA ingestion

An efficient and convenient RNA interference (RNAi) technique involving double-stranded RNA (dsRNA) ingestion is useful for gene function studies of non-model insects. Three dsRNAs targeting different sites within a gene encoding vacuolar ATP synthase subunit E (V-ATPase-E, 21E01) were synthesised for RNAi in Nilaparvata lugens. dsRNA was found to be stable in 0.1 g mL(-1) sucrose solution, but unstable in artificial fodder. Therefore, dsRNAs were orally delivered into N. lugens in 0.1 g mL(-1) sucrose solution. RNAi was induced by all three of the dsRNAs at 0.05 µg µL(-1) in N. lugens. Time dynamics analysis of gene silencing indicated that significant suppression of the target gene began as early as 2 days after ingestion of ds2-21E01 and ds3-21E01. However, significant repressive effects were recorded up to 10 days after exposure to ds1-21E01. The maximum reduction in target gene mRNA was observed after 10 days of treatment, with suppression ratios induced by ds1-21E01, ds2-21E01 and ds3-21E01 of 41, 55 and 48% respectively. An efficient and convenient RNAi technique involving dsRNA ingestion has been successfully developed for N. lugens. This will be a useful tool for further functional genomic investigation in this organism.

Comparative Proteome Analysis of Silkworm in Its Susceptibility and Resistance Responses to Bombyx mori Densonucleosis Virus

Bombyx mori densonucleosis virus (BmDNV) is one of the most disastrous viruses in cocoon production. Silkworm resistance to BmDNV has been examined previously using a number of traditional biochemical and molecular techniques. In this study, a near isogenic line, BC₆, was constructed to eliminate the difference in inherited background, which has 99.9% identity with the susceptible strain but carries a resistant gene. We utilized a proteomic approach involving two-dimensional differential gel electrophoresis and mass spectrometry to examine changes in the midgut proteins from the susceptible and resistant silkworm larvae infected with BmDNV. The protein profiles were compared and 9 differentially expressed proteins were identified by mass spectrometry. In the resistant strains, the heat-shock 70-kDa protein cognate, cytochrome P450, vacuolar ATP synthase subunit B, arginine kinase, vacuolar ATP synthase subunit D and glutathione S-transferase sigma were strongly upregulated and α-tubulin was downregulated. Our results imply that these upregulated genes and the downregulated genes might be involved in B. mori immune responses against BmDNV-Z infection.

Proteomic analysis of differentially expressed proteins in lymphoid organ of Fenneropenaeus chinensis response to Vibrio anguillarum stimulation

To gain an insight into the function of shrimp lymphoid organ at protein level, we analyzed the proteome of lymphoid organ in healthy Chinese shrimp Fenneropenaeus chinensis ( F. chinensis) through two-dimensional gel electrophoresis (2-DE) based proteomic approach. A total of 95 spots representing 75 protein entries were identified by liquid chromatography tandem mass spectrometry (LC–MS/MS) with both online and in-house database. According to Gene Ontology (GO) annotation of biological process, the identified proteins were classified into 13 categories. Among them, approximately 36% of proteins related to cytoskeleton are noticeable. Then, a comparative proteomic approach was employed to investigate the differentially expressed proteins in lymphoid organ of Vibrio anguillarum-challenged F. chinensis. At 24 h post-injection (hpi), 17 differentially expressed protein spots were successfully identified, including 4 up-regulated protein spots (represent 4 proteins: cathepsin L, protein similar to squid CG16901-PC, protein kinase C and protein similar to T-complex Chaperonin 5 CG8439-PA), and 13 down-regulated protein spots (represent 9 proteins: actin, beta-actin, cytoplasmic actin CyII, alpha tubulin, beta tubulin, protein similar to proteasome delta, vacuolar ATP synthase subunit B, elongation factor 2, carboxypeptidase B). These data may help us to understand the function of lymphoid organ and the molecular immune mechanism of shrimp responsive to pathogen infection.

Proteomic analysis of novel Cry1Ac binding proteins in Helicoverpa armigera (Hübner)

Aminopeptidase N (APN) and cadherin-like proteins have been previously identified as Cry1Ac-binding proteins in Helicoverpa armigera (Hübner). In this study, a proteomic approach was used to identify novel Cry1Ac-binding proteins in H. armigera. Brush border membrane vesicles (BBMV) of H. armigera were extracted and separated by two-dimensional gel electrophoresis (2-DE). Cry1Ac-binding proteins were detected using antisera against Cry1Ac. Peptide mass fingerprinting (PMF) was used to identify Cry1Ac-binding proteins. In total, four proteins were identified as candidate Cry1Ac-binding proteins in H. armigera: vacuolar ATP synthase (V-ATPase) subunit B, actin, heat shock cognate protein (HSCP), and a novel protein.

Biomarker discovery: A proteomic approach for brain cancer profiling

Gliomas in the form of astrocytomas, anaplastic astrocytomas and glioblastomas are the most common brain tumors in humans. Early detection of these cancers is crucial for successful treatment. Proteomics promises the discovery of biomarkers and tumor markers for early detection and diagnosis. In the current study, a differential gel electrophoresis technology coupled with matrix-assisted laser desorption/ionization-time of flight and liquid chromatography-tandem mass spectroscopy was used to investigate tumor-specific changes in the proteome of human brain cancer. Fifty human brain tissues comprising varying diagnostic groups (non-tumor, grade I, grade II, grade III and grade IV) were run in duplicate together with an internal pool sample on each gel. The proteins of interest were automatically picked, in-gel digested and mass spectrometry fingerprinted. Two hundred and eleven protein spots were identified successfully and were collapsed into 91 unique proteins. Approximately 20 of those 91 unique proteins had, to our knowledge, not been reported previously as differentially expressed in human brain cancer. Alb protein, peroxiredoxin 4 and SH3 domain-binding glutamic acid-rich-like protein 3 were upregulated in glioblastoma multiform versus non-tumor tissues. However, aldolase C fructose-biphosphate, creatine kinase, B chain dihydrolipoyl dehydrogenase, enolase 2, fumarate hydratase, HSP60, lactoylglutathione lyase, lucine aminopeptidase, Mu-crystallin homolog, NADH-UO 24, neurofilament triplet L protein, septin 2, stathmin and vacuolar ATP synthase subunit E were downregulated in glioblastoma multiform compared with non-tumor tissues. These differentially expressed proteins provided novel information on the differences existing between normal brain and gliomas, and thus might prove to be useful molecular indicators of diagnostic or prognostic value.

Hippocampus protein profiling reveals aberration of malate dehydrogenase in chlorpromazine/clozapine treated rats

Chlorpromazine and clozapine are antipsychotic agents used to relieve symptoms of early-diagnosed schizophrenia. In this study, we used proteomics technology to screen the protein aberration in Sprague–Dawley rats treated with these two antipsychotic agents. Our goal was to identify the effects of antipsychotics on hippocampal proteins in rats. Protein expression profiles were compared in each experimental group using two-dimensional gel electrophoresis and identified using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Malate dehydrogenase, peroxiredoxin 3, vacuolar ATP synthase subunit β and mitogen-activated protein kinase kinase 1 were found to have altered expression levels in the groups treated with antipsychotics compared with the matched controls. These findings should contribute to identifying new targets for disease preventing pharmacological agents and be beneficial for the development of more effective agents.

Clustering the annotation space of proteins

BackgroundCurrent protein clustering methods rely on either sequence or functional similarities between proteins, thereby limiting inferences to one of these areas.ResultsHere we report a new approach, named CLAN, which clusters proteins according to both annotation and sequence similarity. This approach is extremely fast, clustering the complete SwissProt database within minutes. It is also accurate, recovering consistent protein families agreeing on average in more than 97% with sequence-based protein families from Pfam. Discrepancies between sequence- and annotation-based clusters were scrutinized and the reasons reported. We demonstrate examples for each of these cases, and thoroughly discuss an example of a propagated error in SwissProt: a vacuolar ATPase subunit M9.2 erroneously annotated as vacuolar ATP synthase subunit H. CLAN algorithm is available from the authors and the CLAN database is accessible at ConclusionsCLAN creates refined function-and-sequence specific protein families that can be used for identification and annotation of unknown family members. It also allows easy identification of erroneous annotations by spotting inconsistencies between similarities on annotation and sequence levels.

Up-regulated expression of the MAT-8 gene in prostate cancer and its siRNA-mediated inhibition of expression induces a decrease in proliferation of human prostate carcinoma cells

In order to analyze differential gene expression of putative prostate tumor markers we compared the expression levels of >400 cancer-related genes using the cDNA array technique in a set of prostate tumors and matched normal prostate tissues. Up-regulated expression of mammary tumor 8 kDa protein (MAT-8), complement component C1S (C1S), ferritin heavy chain (FTH1), peptidyl-prolyl cis-trans isomerase A (PPIA), RNA-binding protein regulatory subunit DJ-1 protein (DJ-1) and vacuolar ATP synthase subunit F (ATP6V1F) was determined in prostate carcinoma and confirmed by using quantitative real-time RT-PCR analyses. Furthermore, quantitative real time RT-PCR on intact RNAs from 11 paired laser microdissected epithelial tissue samples confirmed up-regulated MAT-8 expression in 6 out of 11 prostate tumors. To determine the function of MAT-8 in vitro, human PC-3 and LNCaP prostate carcinoma cells were transfected with small interfering double-stranded RNA (siRNA) oligonucleotides against the MAT-8 gene leading to a specific down-regulation of MAT-8 expression. In addition, suppression of MAT-8 expression caused a significant decrease in cellular proliferation of both prostate cancer cell lines, whereas invasive capacity and cellular apoptosis remained unaffected. Taken together, our results indicate that the human MAT-8 gene contains the potential to serve as a prostate cancer expression marker and that MAT-8 plays an important role in cellular growth of prostate carcinomas.

Identification and quantitative expression analysis of genes that are differentially expressed during conidial germination in Pyrenophora teres.

Net blotch, caused by Pyrenophora teres, is a common disease of barley ( Hordeum vulgareL.). Two PCR-based differential screening techniques, cDNA-amplified fragment length polymorphism (cDNA-AFLP) and suppression subtractive hybridisation (SSH), were employed to clone cDNA copies of transcripts that are up-regulated during conidial germination. The nucleotide sequences of 35 transcripts were analysed, and the amino acid sequences of their predicted products were compared with entries in databases. Eleven of these clones showed homology to genes from other ascomycetes coding for a transcription factor, two regulatory proteins, a putative transposase, a protein required for the biogenesis of cytochrome C oxidase, a threonine synthase, a probable subunit of a phenylalanine-tRNA synthetase, a subunit of RNA polymerase I, a cation transport protein, a vacuolar ATP synthase subunit, and an RNA processing protein. One conserved hypothetical protein was found and 23 sequences could not be functionally classified. The relative expression of five transcripts at 0, 1, 2, 3, 6, 12 and 24 h after induction of germination was determined by real-time RT-PCR using 18S rRNA as the endogenous reference sequence. All transcripts showed a significant increase in expression during early stages of germination. The maximum change in expression relative to ungerminated conidia ranged between 2.6- and 6-fold. The characterisation of genes involved in biochemical processes during the germination of conidia could be useful for target-specific development of new antifungal agents.