Characterizing the Protein Isoforms of foraging (for), the PKGI Ortholog in Drosophila melanogaster.

The foraging (for) gene of Drosophila melanogaster encodes a cGMP-dependent protein kinase (PKG), which is a major effector of the cGMP signaling pathway involved in the regulation of behaviour and metabolic traits. Despite being well studied at the transcript level, little is known about the for gene at the protein level. Here, we provide a detailed characterization of the for gene protein (FOR) products and present new tools for their study, including five isoform-specific antibodies and a transgenic strain that carries an HA-labelled for allele (forBAC::HA). Our results showed that multiple FOR isoforms were expressed in the larval and adult stages of D. melanogaster and that the majority of whole-body FOR expression arises from three (P1, P1α, and P3) of eight putative protein isoforms. We found that FOR expression differed between the larval and adult stages and between the dissected larval organs we analyzed, which included the central nervous system (CNS), fat body, carcass, and intestine. Moreover, we showed that the FOR expression differed between two allelic variants of the for gene, namely, fors (sitter) and forR (rover), that are known to differ in many food-related traits. Together, our in vivo identification of FOR isoforms and the existence of temporal, spatial, and genetic differences in their expression lay the groundwork for determining their functional significance.

Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster.

BACKGROUND: The social life of animals depends on communication between individuals. Recent studies in Drosophila melanogaster demonstrate that various behaviors are influenced by social interactions. For example, courtship is a social interaction mediated by pheromonal signaling that occurs more frequently during certain times of the day than others. In adult flies, sex pheromones are synthesized in cells called oenocytes and displayed on the surface of the cuticle. Although the role of Drosophila pheromones in sexual behavior is well established, little is known about the timing of these signals or how their regulation is influenced by the presence of other flies. RESULTS: We report that oenocytes contain functional circadian clocks that appear to regulate the synthesis of pheromones by controlling the transcription of desaturase1 (desat1), a gene required for production of male cuticular sex pheromones. Moreover, levels of these pheromones vary throughout the day in a pattern that depends on the clock genes and most likely also depends on the circadian control of desat1 in the oenocytes. To assess group dynamics, we manipulated the genotypic composition of social groups (single versus mixed genotypes). This manipulation significantly affects clock gene transcription both in the head and oenocytes, and it also affects the pattern of pheromonal accumulation on the cuticle. Remarkably, we found that flies in mixed social groups mate more frequently than do their counterparts in uniform groups. CONCLUSIONS: These results demonstrate that social context exerts a regulatory influence on the expression of chemical signals, while modulating sexual behavior in the fruit fly.

Secretome-based identification and characterization of potential biomarkers in thyroid cancer.

In search of thyroid cancer biomarkers, proteins secreted by thyroid cancer cell lines, papillary-derived TPC-1 and anaplastic-derived CAL62, were analyzed using liquid chromatography-tandem mass spectrometry. Of 46 high-confidence identifications, 6 proteins were considered for verification in thyroid cancer patients' tissue and blood. The localization of two proteins, nucleolin and prothymosin-α (PTMA), was confirmed in TPC-1 and CAL62 cells by confocal microscopy and immunohistochemically in xenografts of TPC-1 cells in NOD/SCID/γ mice and human thyroid cancers (48 tissues). Increased nuclear and cytoplasmic expression of PTMA was observed in anaplastic compared to papillary and poorly differentiated carcinomas. Nuclear expression of nucleolin was observed in all subtypes of thyroid carcinomas, along with faint cytoplasmic expression in anaplastic cancers. Importantly, PTMA, nucleolin, clusterin, cysteine-rich angiogenic inducer 61, enolase 1, and biotinidase were detected in thyroid cancer patients' sera, warranting future analysis to confirm their potential as blood-based thyroid cancer markers. In conclusion, we demonstrated the potential of secretome analysis of thyroid cancer cell lines to identify novel proteins that can be independently verified in cell lines, xenografts, tumor tissues, and blood samples of thyroid cancer patients. These observations support their potential utility as minimally invasive biomarkers for thyroid carcinomas and their application in management of these diseases upon future validation.

A multiprotein bicarbonate dehydration complex essential to carboxysome function in cyanobacteria.

Carboxysomes are proteinaceous biochemical compartments that constitute the enzymatic "back end" of the cyanobacterial CO2-concentrating mechanism. These protein-bound organelles catalyze HCO3- dehydration and photosynthetic CO2 fixation. In Synechocystis sp. strain PCC6803 these reactions involve the beta-class carbonic anhydrase (CA), CcaA, and Form 1B ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The surrounding shell is thought to be composed of proteins encoded by the ccmKLMN operon, although little is known about how structural and catalytic proteins integrate to form a functional carboxysome. Using biochemical activity assays and molecular approaches we have identified a catalytic, multiprotein HCO3- dehydration complex (BDC) associated with the protein shell of Synechocystis carboxysomes. The complex was minimally composed of a CcmM73 trimer, CcaA dimer, and CcmN. Larger native complexes also contained RbcL, RbcS, and two or three immunologically identified smaller forms of CcmM (62, 52, and 36 kDa). Yeast two-hybrid analyses indicated that the BDC was associated with the carboxysome shell through CcmM73-specific protein interactions with CcmK and CcmL. Protein interactions between CcmM73 and CcaA, CcmM73 and CcmN, or CcmM73 and itself required the N-terminal gamma-CA-like domain of CcmM73. The specificity of the CcmM73-CcaA interaction provided both a mechanism to integrate CcaA into the fabric of the carboxysome shell and a means to recruit this enzyme to the BDC during carboxysome biogenesis. Functionally, CcaA was the catalytic core of the BDC. CcmM73 bound H14CO3- but was unable to catalyze HCO3- dehydration, suggesting that it may potentially regulate BDC activity.

Biotinidase is a novel marker for papillary thyroid cancer aggressiveness.

Biotinidase was identified in secretome analysis of thyroid cancer cell lines using proteomics. The goal of the current study was to analyze the expression of biotinidase in thyroid cancer tissues and fine needle aspiration (FNA) samples to evaluate its diagnostic and prognostic potential in thyroid cancer. Immunohistochemical analysis of biotinidase was carried out in 129 papillary thyroid cancer (PTC, 34 benign thyroid tissues and 43 FNA samples and correlated with patients' prognosis. Overall biotinidase expression was decreased in PTC compared to benign nodules (p = 0.001). Comparison of aggressive and non-aggressive PTC showed decrease in overall biotinidase expression in the former (p = 0.001). Loss of overall biotinidase expression was associated with poor disease free survival (p = 0.019, Hazards ratio (HR) = 3.1). We examined the effect of subcellular compartmentalization of nuclear and cytoplasmic biotinidase on patient survival. Decreased nuclear expression of biotinidase was observed in PTC as compared to benign tissues (p<0.001). Upon stratification within PTC, nuclear expression was reduced in aggressive as compared to non-aggressive tumors (p<0.001). Kaplan-Meier survival analysis showed significant association of loss of nuclear biotinidase expression with reduced disease free survival (p = 0.014, HR = 5.4). Cytoplasmic biotinidase expression was reduced in aggressive thyroid cancers in comparison with non-aggressive tumors (p = 0.002, Odds ratio (OR) = 0.29) which was evident by its significant association with advanced T stage (p = 0.003, OR = 0.28), nodal metastasis (p<0.001, OR = 0.16), advanced TNM stage (p<0.001, OR = 0.21) and extrathyroidal extension (p = 0.001, OR = 0.23). However, in multivariate analysis extrathyroidal extension emerged as the most significant prognostic marker for aggressive thyroid carcinomas (p = 0.015, HR = 12.8). In conclusion, loss of overall biotinidase expression is a novel marker for thyroid cancer aggressiveness.

An Ep-ICD based index is a marker of aggressiveness and poor prognosis in thyroid carcinoma.

BACKGROUND: Nuclear accumulation of the intracellular domain of epithelial cell adhesion molecule (Ep-ICD) in tumor cells was demonstrated to predict poor prognosis in thyroid carcinoma patients in our earlier study. Here, we investigated the clinical significance of Ep-ICD subcellular localization index (ESLI) in distinguishing aggressive papillary thyroid carcinoma (PTC) from non-aggressive cases. METHODS: Using domain specific antibodies against the intracellular (Ep-ICD) and extracellular (EpEx) domains of epithelial cell adhesion molecule, 200 archived tissues from a new cohort of patients with benign thyroid disease as well as malignant aggressive and non aggressive PTC were analyzed by immunohistochemistry (IHC). ESLI was defined as sum of the IHC scores for accumulation of nuclear and cytoplasmic Ep-ICD and loss of membranous EpEx; ESLI = [Ep-ICD(nuc) + Ep-ICD(cyt) + loss of membranous EpEx]. RESULTS: For the benign thyroid tissues, non-aggressive PTC and aggressive PTC, the mean ESLI scores were 4.5, 6.7 and 11 respectively. Immunofluorescence double staining confirmed increased nuclear Ep-ICD accumulation and decreased membrane EpEx expression in aggressive PTC. Receiver-operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.841, 70.2% sensitivity and 83.9% specificity for nuclear Ep-ICD for differentiating aggressive PTC from non-aggressive PTC. ESLI distinguished aggressive PTC from non-aggressive cases with improved AUC of 0.924, 88.4% sensitivity and 85.5% specificity. Our study confirms nuclear accumulation of Ep-ICD and loss of membranous EpEx occurs in aggressive PTC underscoring the potential of Ep-ICD and ESLI to serve as diagnostic markers for aggressive PTC. Kaplan Meier survival analysis revealed significantly reduced disease free survival (DFS) for ESLI positive (cutoff >10) PTC (p<0.05), mean DFS=133 months as compared to 210 months for patients who did not show positive ESLI. CONCLUSION: ESLI scoring improves the identification of aggressive PTC and thereby may serve as a useful index for defining aggressiveness and poor prognosis among PTC patients.

Nuclear and cytoplasmic accumulation of Ep-ICD is frequently detected in human epithelial cancers.

BACKGROUND: We previously demonstrated that nuclear and cytoplasmic accumulation of the intracellular domain (Ep-ICD) of epithelial cell adhesion molecule (EpCAM) accompanied by a reciprocal reduction of its extracellular domain (EpEx), occurs in aggressive thyroid cancers. This study was designed to determine whether similar accumulation of Ep-ICD is a common event in other epithelial cancers. METHODOLOGY AND RESULTS: Ten epithelial cancers were immunohistochemically analyzed using Ep-ICD and EpEx domain-specific antibodies. The subcellular localization of EpEx and Ep-ICD in the human colon adenocarcinoma cell line CX-1 was observed using immunofluorescence. Nuclear and cytoplasmic Ep-ICD expression was increased in cancers of the breast (31 of 38 tissues, 82%), prostate (40 of 49 tissues, 82%), head and neck (37 of 57 tissues, 65%) and esophagus (17 of 46 tissues, 37%) compared to their corresponding normal tissues that showed membrane localization of the protein. Importantly, Ep-ICD was not detected in the nuclei of epithelial cells in most normal tissues. High nuclear and cytoplasmic Ep-ICD accumulation also occurred in the other six epithelial cancer types analyzed - lung, colon, liver, bladder, pancreatic, and ovarian. A concomitant reduction in membrane EpEx expression was observed in a subset of all cancer types. Receiver operating characteristic curve analysis revealed nuclear Ep-ICD distinguished breast cancers with 82% sensitivity and 100% specificity and prostate cancers with 82% sensitivity and 78% specificity. Similar findings were observed for cytoplasmic accumulation of Ep-ICD in these cancers. We provide clinical evidence of increased nuclear and cytoplasmic Ep-ICD accumulation and a reduction in membranous EpEx in these cancers. CONCLUSIONS: Increased nuclear and cytoplasmic Ep-ICD was observed in all epithelial cancers analyzed and distinguished them from normal tissues with high-sensitivity, specificity, and AUC. Development of a robust high throughput assay for Ep-ICD will facilitate the determination of its diagnostic, prognostic and therapeutic relevance in epithelial cancers.

Involvement of the cynABDS operon and the CO2-concentrating mechanism in the light-dependent transport and metabolism of cyanate by cyanobacteria.

The cyanobacteria Synechococcus elongatus strain PCC7942 and Synechococcus sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO-) to CO2 and NH3 through the action of a cytosolic cyanase which required HCO3- as a second substrate. The ability to metabolize NCO- relied on three essential elements: proteins encoded by the cynABDS operon, the biophysical activity of the CO2-concentrating mechanism (CCM), and light. Inactivation of cynS, encoding cyanase, and cynA yielded mutants unable to decompose cyanate. Furthermore, loss of CynA, the periplasmic binding protein of a multicomponent ABC-type transporter, resulted in loss of active cyanate transport. Competition experiments revealed that native transport systems for CO2, HCO3-, NO3-, NO2-, Cl-, PO4(2-), and SO4(2-) did not contribute to the cellular flux of NCO- and that CynABD did not contribute to the flux of these nutrients, implicating CynABD as a novel primary active NCO- transporter. In the S. elongatus strain PCC7942 DeltachpX DeltachpY mutant that is defective in the full expression of the CCM, mass spectrometry revealed that the cellular rate of cyanate decomposition depended upon the size of the internal inorganic carbon (Ci) (HCO3- + CO2) pool. Unlike wild-type cells, the rate of NCO- decomposition by the DeltachpX DeltachpY mutant was severely depressed at low external Ci concentrations, indicating that the CCM was essential in providing HCO3- for cyanase under typical growth conditions. Light was required to activate and/or energize the active transport of both NCO- and Ci. Putative cynABDS operons were identified in the genomes of diverse Proteobacteria, suggesting that CynABDS-mediated cyanate metabolism is not restricted to cyanobacteria.

Natural polymorphism affecting learning and memory in Drosophila.

Knowing which genes contribute to natural variation in learning and memory would help us understand how differences in these cognitive traits evolve among populations and species. We show that a natural polymorphism at the foraging (for) locus, which encodes a cGMP-dependent protein kinase (PKG), affects associative olfactory learning in Drosophila melanogaster. In an assay that tests the ability to associate an odor with mechanical shock, flies homozygous for one natural allelic variant of this gene (forR) showed better short-term but poorer long-term memory than flies homozygous for another natural allele (fors). The fors allele is characterized by reduced PKG activity. We showed that forR-like levels of both short-term learning and long-term memory can be induced in fors flies by selectively increasing the level of PKG in the mushroom bodies, which are centers of olfactory learning in the fly brain. Thus, the natural polymorphism at for may mediate an evolutionary tradeoff between short- and long-term memory. The respective strengths of learning performance of the two genotypes seem coadapted with their effects on foraging behavior: forR flies move more between food patches and so could particularly benefit from fast learning, whereas fors flies are more sedentary, which should favor good long-term memory.

Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase.

This study links natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses.

Construction of a cDNA-based microarray for Drosophila melanogaster: a comparison of gene transcription profiles from SL2 and Kc167 cells.

We have constructed a DNA microarray that represents approximately 6900 of the estimated 13,598 genes in the Drosophila melanogaster genome. The microarray contains 5756 target cDNAs from the Berkeley Drosophila Genome Project, 1078 cDNAs from the National Institutes of Health Drosophila testis cDNA library, and 546 gene fragments that were amplified from genomic DNA. The methods for DNA amplification and microarray manufacture are presented. Academic researchers can obtain the microarray from the Canadian Drosophila Microarray Centre. To evaluate the utility of these arrays, we compared the gene transcription profiles of two commonly used Drosophila cell lines. Analysis revealed that 5412 spot pairs gave signals consistently above the average background in Kc167 cells, whereas 5636 spot pairs met this criterion in SL2 cells. When the expression profiles of the cell lines were compared, 1437 genes displayed at least a 1.5-fold difference, and 170 genes had a threefold or greater difference between the two cell lines. In each case, with respect to Kc167 when compared with SL2 cells, the number of genes that were upregulated was nearly equal to the number of downregulated genes. This result demonstrates that despite the similar embryonic derivation of both cell lines, their transcriptional profiles are very different.

Characterization of the C-terminal extension of carboxysomal carbonic anhydrase from Synechocystis sp. PCC6803.

Sequence analysis of the carboxysomal carbonic anhydrase (CcaA) from Synechocystis PCC6803, Synechococcus PCC7942 and Nostoc ATCC29133, indicated high sequence identity to the ß class of plant and bacterial carbonic anhydrases (CA), and conservation of the active site region. However, the cyanobacterial enzyme has a C-terminal extension of about 75 amino acids (aa) not found in the plant enzymes, and largely absent from other bacterial enzymes. Using recombinant DNA technology, genes encoding C-terminal truncation products of up to 127 aa were overexpressed in E. coli, and partially purified lysates were analysed for CA-mediated exchange of 18O between 13C18O2and H216O. Recombinant CcaA proteins with up to 60 aa removed (CcaAΔ60) were catalytically competent, but beyond this there was an abrupt loss of activity. CcaAΔ0, along with CcaAΔ40 and CcaAΔ60, also catalysed the hydrolysis of carbon oxysulfide (COS; an isoelectronic structural analogue of CO2), but CcaAΔ63 and CcaAΔ127 did not, indicating that truncations greater than 62 aa resulted in a general loss of catalytic competency. Analysis of protein-protein interaction using the yeast two-hybrid system revealed that CcaA did not interact with the large or small Rubisco subunits (RbcL and RbcS, respectively) of Synechocystis, but there was strong CcaA-CcaA interaction. This protein interaction also ceased with C-terminal truncations in CcaA greater than 60 aa. The correlation between loss of CcaA-CcaA interaction and CcaA catalytic activity suggests that the proximal portion of the C-terminal extension is required for oligomerization, and that this oligomerization is essential for catalysis by the cyanobacterial enzyme. Thus, the C-terminal extension may play an important role in the function of CA within cyanobacterial carboxysomes, which is not required by the higher plant enzymes.

Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803.

A fully-segregated mutant (ccaA::kanR) defective in the ccaA gene, encoding a carboxysome-associated beta-carbonic anhydrase (CA), was generated in the cyanobacterium Synechocystis sp. PCC6803 by insertional mutagenesis. Immunoblot analysis indicated that the CcaA polypeptide was absent from the carboxysome-enriched fraction obtained from ccaA::kanR, but was present in wild-type (WT) cells. The carboxysome-enriched fraction isolated from WT cells catalyzed 18O exchange between 13C18O2 and H2O, indicative of CA activity, while ccaA::kanR carboxysomes did not. Transmission and immunogold electron microscopy revealed that carboxysomes of WT and ccaA::kanR were of similar size, shape and cellular distribution, and contained most of the cellular complement of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ccaA::kanR cells were substantially smaller than WT and were unable to grow autotrophically at air levels of CO2. However, cell division occurred at near-WT rates when ccaA::kanR was supplied with 5% CO2 (v/v) in air. The apparent photosynthetic affinity of the mutant for inorganic carbon (Ci) was 500-fold lower than that of WT cells although intracellular Ci accumulation was comparable to WT measurements. Mass spectrometric analysis revealed that the CA-like activity associated with the active CO2 transport system was retained by ccaA::kanR cells and was inhibited by H2S, indicating that CO2 transport was distinct from the CcaA-mediated dehydration of intracellular HCO3-. The data suggest that the ccaA mutant was unable to efficiently utilize the internal Ci pool for carbon fixation and that the high-CO2-requiring phenotype of ccaA::kanR was due primarily to an inability to generate enough CO2 in the carboxysomes to sustain normal rates of photosynthesis.

Effects of carbon nutrition on the physiological expression of HCO3- transport and the CO2-concentrating mechanism in the Cyanobacterium chlorogloeopsis sp. ATCC 27193.

We have examined the effect of inorganic and organic carbon nutrition on the physiological expression of HCO3- transport and the CO2-concentrating mechanism (CCM) in the nutritionally versatile cyanobacterium Chlorogloeopsis sp. ATCC 27193. Cells grown under photoautotrophic conditions in the presence of limiting or replete levels of inorganic carbon (Ci), or grown under mixotrophic (light) or chemoheterotrophic (dark) conditions in the presence of sucrose retained both active CO2 and Na(+)-independent HCO3- transport activity. However, two distinct effects on the kinetic properties of HCO3- transport were observed, which segregated on the basis of phototrophic and chemoheterotrophic growth in the dark. In the former, the apparent substrate affinity of the HCO3- transport system (K0.5) varied (12-fold) in response to the growth Ci or mixotrophy while the maximum rate of HCO3- transport was approximately constant. In the latter case, the K0.5 value was unchanged from the starting value (35 microM) of Ci-limited photoautotrophic cells used to initiate the dark-grown cultures, but transport capacity declined 3-fold. Modulation of the K0.5 (HCO3- transport) value required light. Cellular carboxysome content was unaffected by growth under any of the regimes employed and these structures were the predominant location of ribulose-1,5-bisphosphate carboxylase/oxygenase, as indicated by immunogold electron microscopy. Mixotrophic and chemoheterotrophic growth resulted in a diminished ability to concentrate Ci internally and a reduction in Ci accumulation ratios at low external Ci concentrations. The relationship between photosynthetic carbon fixation and the internal Ci pool varied by 2-fold, with high-Ci-grown cells being the most efficient and mixotrophically grown cells the least, indicating that there was limited capacity to modulate this relationship in response to changes in carbon nutrition. Within broad limits this relationship appeared to be a fixed trait of the strain and an important factor in determining growth rate.

A novel evolutionary lineage of carbonic anhydrase (epsilon class) is a component of the carboxysome shell.

A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.