Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.

Proteomic analysis of banana fruit reveals proteins that are differentially accumulated during ripening

Bananas (Musa spp.) are highly perishable fruit of notable economic and nutritional relevance. Because the identification of proteins involved in metabolic pathways could help to extend green-life and improve the quality of the fruit, this study aimed to compare the proteins of banana pulp at the pre-climacteric and climacteric stages. The use of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) revealed 50 differentially expressed proteins, and comparing those proteins to the Mass Spectrometry Protein Sequence Database (MSDB) identified 26 known proteins. Chitinases were the most abundant types of proteins in unripe bananas, and two isoforms in the ripe fruit have been implicated in the stress/defense response. In this regard, three heat shock proteins and isoflavone reductase were also abundant at the climacteric stage. Concerning fruit quality, pectate lyase, malate dehydrogenase, and starch phosphorylase accumulated during ripening. In addition to the ethylene formation enzyme amino cyclo carboxylic acid oxidase, the accumulation of S-adenosyl-L-homocysteine hydrolase was needed because of the increased ethylene synthesis and DNA methylation that occurred in ripening bananas. Differential analysis provided information on the ripening-associated changes that occurred in proteins involved in banana flavor, texture, defense, synthesis of ethylene, regulation of expression, and protein folding, and this analysis validated previous data on the transcripts during ripening. In this regard, the differential proteomics of fruit pulp enlarged our understanding of the process of banana ripening. (C) 2012 Elsevier B.V. All rights reserved.

Differential Proteomic Analysis of Noncardia Gastric Cancer from Individuals of Northern Brazil

Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets.

Psychophysical analysis of contrast processing segregated into magnocellular and parvocellular systems in asymptomatic carriers of 11778 Leber`s hereditary optic neuropathy

We examined achromatic contrast discrimination in asymptomatic carriers of 11778 Leber`s hereditary optic neuropathy (LHON 18 controls) and 18 age-match were also tested. To evaluate magnocellular (MC) and Parvocellular (PC) contrast discrimination, we used a version of Pokorny and Smith`s (1997) Pulsed/steady-pedestal paradigms (PPP/SPP) thought to be detected via PC and MC pathways, respectively. A luminance pedestal (four 1 degrees x 1 degrees squares) was presented on a 12 cd/m(2) surround. The luminance of one of the squares (trial square, TS) was randomly incremented for either 17 or 133 ms. Observers had to detect the TS, in a forced-choice task, at each duration, for three pedestal levels: 7, 12, 19 cd/m(2). In the SPP, the pedestal was fixed, and the TS was modulated. For the PPP, all four pedestal squares pulsed for 17 or 133 ms, and the TS was simultaneously incremented or decremented. We found that contrast discrimination thresholds of LHON carriers were significantly higher than controls` in the condition with the highest luminance of both paradigms, implying impaired contrast processing with no evidence of differential sensitivity losses between the two systems. Carriers` thresholds manifested significantly longer temporal integration than controls in the SPP, consistent with slowed MC responses. The SPP and PPP paradigms can identify contrast and temporal processing deficits in asymptomatic LHON carriers, and thus provide an additional tool for early detection and characterization of the disease.

Determination of Tetradifon in Brazilian Green Bell Pepper by Differential Pulse Voltammetry

Tetradifon, a potentially carcinogenic and mutagenic pesticide, can contribute to environmental and human contamination when applied to green bell pepper crops. In this context, in this work, a reliable and sensitive method for determination of tetradifon in Brazilian green bell pepper samples involving a differential pulse voltammetry (DPV) technique on a glassy carbon electrode is proposed. The electrochemical behavior of tetradifon as followed by cyclic voltammetry (CV) suggests that its reduction occurs via an irreversible five-electron transfer vs. Ag vertical bar AgCl, KCl 3 M reference electrode. Very well-resolved diffusion controlled voltammetric peaks have been obtained in a supporting electrolyte solution composed of a mixture of 40% dimethylformamide (DMF), 30% methanol, and 30% NaOH 0.3 mol L-1 at -1.43, -1.57, -1.73, -1.88, and -2.05 V. The proposed DPV method has a good linear response in the 3.00 - 10.0 mu mol L-1 range, with a limit of detection (L.O.D) of 0.756 mu mol L-1 and 0.831 mu mol L-1 in the absence and in the presence of the matrix, respectively. Moreover, improved L.O.D results (0.607 mu mol L-1) have been achieved in the absence of DMF from the supporting electrolyte solution. Recovery has been evaluated in five commercial green bell pepper samples, and recovery percentages ranging from 91.0 to 109 have been obtained for tetradifon determinations. The proposed voltammetric method has also been tested for reproducibility, repeatability, and potential interferents, and the results obtained for these three analytical parameters are satisfactory for electroanalytical purposes. (C) 2012 The Electrochemical Society. [DOI: 10.1149/2.024207jes] All rights reserved.

Gene copy number and differential gene expression in haploid and diploid males of the stingless bee, Melipona quadrifasciata

Complementary sex determination in Hymenoptera implies that heterozygosity at the sex locus leads to the development of diploid females, whereas hemizygosity results in haploid males. Diploid males can arise through inbreeding. In social species, these pose a double burden on colony fitness, from significant reduction in its worker force and through being less viable and fertile than haploid males. Apart from being "misfits", diploid males are of interest to assess molecular correlates for possibly ploidy-related bionomic differences. Herein, we generated suppression subtractive cDNA libraries from newly emerged haploid and diploid males of the stingless bee Melipona quadrifasciata to enrich for differentially expressed genes. Gene Ontology classification revealed that in haploid males more DEGs were related to stress responsiveness, biosynthetic processes, reproductive processes and spermatogenesis, whereas in diploid ones differentially expressed genes were associated with cellular organization, nervous system development and amino acid transport were prevalent. Furthermore, both libraries contained over 40 % ESTs representing possibly novel transcripts. Quantitative RT-PCR analyses confirmed the differential expression of a representative DEG set in newly emerged males. Several muscle formation and energy metabolism-related genes were under-expressed in diploid males. On including 5-day-old males in the analysis, changes in transcript abundance during sexual maturation were revealed.

Differential expression of toll-like receptor mRNAs in recurrent aphthous ulceration

BACKGROUND: Toll-like receptors (TLR) are membrane proteins that recognize conserved molecules derived from bacterial, viral, fungal or host tissues. They are responsible for promoting the production of cytokines and chemokines, increasing the expression of costimulatory molecules and influencing the T Helper response (Th) toward either a Th1 or Th2 profile, thereby modulating the regulatory T cell response and controlling the integrity of the epithelial barrier. The key factors responsible for increased susceptibility to recurrent aphthous ulceration (RAU) are unclear, and because TLRs are involved in both immune regulation and control of the epithelial barrier, a deficiency in TLR activity is likely to cause increased susceptibility. METHODS: We investigated the gene expression of TLRs one through 10 in tissue samples and peripheral blood mononuclear cells (PBMC) of RAU patients in comparison to healthy controls using real-time quantitative reverse transcription PCR. RESULTS: The analysis of mRNA expression levels in oral lesion showed significant (P < 0.01) overexpression of the TLR2(similar to 6-fold) gene and decreased expression of the TLR3 (similar to 5-fold) and TLR5 (similar to 6-fold) genes in comparison with healthy oral mucosa. The analysis of mRNA expression in PBMC indicated a down-regulation of TLR5 gene expression in the cells from RAU patients (P < 0.05; similar to 2-fold). CONCLUSION: Our results support the hypothesis that a subset of RAU patients has fewer TLR expression that have been tentatively implicated in antiinflammatory effects. This derangement of TLR gene expression may cause an overlay exuberant inflammation reaction in situations where normal individuals are resistant. J Oral Pathol Med (2012) 41: 8085

Transcriptome Analysis of Renal Ischemia/Reperfusion Injury and Its Modulation by Ischemic Pre-Conditioning or Hemin Treatment

Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of the molecular mechanisms involved in renal IRI and counter protection promoted by ischemic pre-conditioning (IPC) or Hemin treatment is an important milestone that needs to be accomplished in this research area. We examined, through an oligonucleotide microarray protocol, the renal differential transcriptome profiles of mice submitted to IRI, IPC and Hemin treatment. After identifying the profiles of differentially expressed genes observed for each comparison, we carried out functional enrichment analysis to reveal transcripts putatively involved in potential relevant biological processes and signaling pathways. The most relevant processes found in these comparisons were stress, apoptosis, cell differentiation, angiogenesis, focal adhesion, ECM-receptor interaction, ion transport, angiogenesis, mitosis and cell cycle, inflammatory response, olfactory transduction and regulation of actin cytoskeleton. In addition, the most important overrepresented pathways were MAPK, ErbB, JAK/STAT, Toll and Nod like receptors, Angiotensin II, Arachidonic acid metabolism, Wnt and coagulation cascade. Also, new insights were gained about the underlying protection mechanisms against renal IRI promoted by IPC and Hemin treatment. Venn diagram analysis allowed us to uncover common and exclusively differentially expressed genes between these two protective maneuvers, underscoring potential common and exclusive biological functions regulated in each case. In summary, IPC exclusively regulated the expression of genes belonging to stress, protein modification and apoptosis, highlighting the role of IPC in controlling exacerbated stress response. Treatment with the Hmox1 inducer Hemin, in turn, exclusively regulated the expression of genes associated with cell differentiation, metabolic pathways, cell cycle, mitosis, development, regulation of actin cytoskeleton and arachidonic acid metabolism, suggesting a pleiotropic effect for Hemin. These findings improve the biological understanding of how the kidney behaves after IRI. They also illustrate some possible underlying molecular mechanisms involved in kidney protection observed with IPC or Hemin treatment maneuvers.

Periodic solutions of abstract neutral functional differential equations

We characterize the existence of periodic solutions of some abstract neutral functional differential equations with finite and infinite delay when the underlying space is a UMD space. (C) 2011 Elsevier Inc. All rights reserved.

Differential MiRNA expression in childhood acute lymphoblastic leukemia and association with clinical and biological features

The present study aimed to analyze the expression profile of the microRNAs previously described as associated with childhood ALL, miR-92a, miR-100, miR-125a-5p, miR-128a, miR-181b, miR-196b and let-7e, and their association with biological/prognostic features in 128 consecutive samples of childhood acute lymphoblastic leukemia (ALL) by quantitative real-time PCR. A significant association was observed between higher expression levels of miR-196b and T-ALL, miR-100 and patients with low white blood cell count at diagnosis and t(12;21) positive ALL. These findings suggest a potential activity of these microRNAs in pediatric ALL biology. (C) 2011 Elsevier Ltd. All rights reserved.

Proteomic analysis of papaya fruit ripening using 2DE-DIGE

Papayas have a very short green life as a result of their rapid pulp softening as well as their susceptibility to physical injury and mold growth. The ripening-related changes take place very quickly, and there is a continued interest in the reduction of postharvest losses. Proteins have a central role in biological processes, and differential proteomics enables the discrimination of proteins affected during papaya ripening. A comparative analysis of the proteomes of climacteric and pre-climacteric papayas was performed using 2DE-DIGE. Third seven proteins corresponding to spots with significant differences in abundance during ripening were submitted to MS analysis, and 27 proteins were identified and classified into six main categories related to the metabolic changes occurring during ripening. Proteins from the cell wall (alpha-galactosidase and invertase), ethylene biosynthesis (methionine synthase), climacteric respiratory burst, stress response, synthesis of carotenoid precursors (hydroxymethylbutenyl 4-diphosphate synthase, GcpE), and chromoplast differentiation (fibrillin) were identified. There was some correspondence between the identified proteins and the data from previous transcript profiling of papaya fruit, but new, accumulated proteins were identified, which reinforces the importance of differential proteomics as a tool to investigate ripening and provides potentially useful information for maintaining fruit quality and minimizing postharvest losses. (C) 2011 Elsevier B.V. All rights reserved.

Thermal Analysis and Raman Spectra of Different Phases of the Ionic Liquid Butyltrimethylammonium Bis(trifluoromethylsulfonyl)imide

The ionic liquid butyltrimethylammonium bis(trifluoromethylsulfonyl)imide, [C4C1C1C1N][Tf2N], is a glass-forming liquid that exhibits partial crystallization depending on the cooling rate. Differential scanning calorimetry (DSC) indicates crystallization at T-c = 227 K, melting at T-m = 258 K, glass transition at T-g similar to 191 K, and also cold crystallization at T-cc similar to 219 K. Raman spectroscopy shows that the crystalline structure obtained by slow cooling is formed with [Tf2N](-) in cisoid conformation, whereas [Tf2N](-) in transoid conformation results from fast cooling. No preferred conformation of the butyl chain of the [C4C1C1C1N](+) cation is favored by slow or fast cooling of [C4C1C1C1N][Tf2N]. Low-frequency Raman spectroscopy shows that crystalline domains developing in the supercooled liquid result in a glacial state made of a mixture of crystallites and amorphous phase. However, these crystalline structures obtained by slow cooling or cold crystallization are not the same because anion-cation interactions promote local structures with distinct conformations of the [Tf2N](-) anion.

Analysis of phase transition and dehydration processes of nevirapine

Solid-state characterization of crystalline drugs is an important pre-formulation step for the development and design of solid dosage forms, such as pellets and tablets. In this study, phase transition and dehydration processes of nevirapine have been studied by differential scanning calorimetry and thermogravimetry differential thermal analysis to overcome the problems of drug formulation, namely poor solubility and poor content uniformity. Phase solubility studies elucidated the mechanism of enhanced nevirapine solubility.

Simultaneous optical signal-to-noise ratio and differential group delay monitoring based on degree of polarization measurements in optical communications systems

We present a simultaneous optical signal-to-noise ratio (OSNR) and differential group delay (DGD) monitoring method based on degree of polarization (DOP) measurements in optical communications systems. For the first time in the literature (to our best knowledge), the proposed scheme is demonstrated to be able to independently and simultaneously extract OSNR and DGD values from the DOP measurements. This is possible because the OSNR is related to maximum DOP, while DGD is related to the ratio between the maximum and minimum values of DOP. We experimentally measured OSNR and DGD in the ranges from 10 to 30 dB and 0 to 90 ps for a 10 Gb/s non-return-to-zero signal. A theoretical analysis of DOP accuracy needed to measure low values of DGD and high OSNRs is carried out, showing that current polarimeter technology is capable of yielding an OSNR measurement within 1 dB accuracy, for OSNR values up to 34 dB, while DGD error is limited to 1.5% for DGD values above 10 ps. For the first time to our knowledge, the technique was demonstrated to accurately measure first-order polarization mode dispersion (PMD) in the presence of a high value of second-order PMD (as high as 2071 ps(2)). (C) 2012 Optical Society of America

SAGE analysis highlights the putative role of underexpression of ribosomal proteins in GH-secreting pituitary adenomas

Background: Although the molecular pathogenesis of pituitary adenomas has been assessed by several different techniques, it still remains partially unclear. Ribosomal proteins (RPs) have been recently related to human tumorigenesis, but they have not yet been evaluated in pituitary tumorigenesis. Objective: The aim of this study was to introduce serial analysis of gene expression (SAGE), a high-throughput method, in pituitary research in order to compare differential gene expression. Methods: Two SAGE cDNA libraries were constructed, one using a pool of mRNA obtained from five GH-secreting pituitary tumors and another from three normal pituitaries. Genes differentially expressed between the libraries were further validated by real-time PCR in 22 GH-secreting pituitary tumors and in 15 normal pituitaries. Results: Computer-generated genomic analysis tools identified 13 722 and 14 993 exclusive genes in normal and adenoma libraries respectively. Both shared 6497 genes, 2188 were underexpressed and 4309 overexpressed in tumoral library. In adenoma library, 33 genes encoding RPs were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by real-time PCR. Conclusion: We report the first SAGE library from normal pituitary tissue and GH-secreting pituitary tumor, which provide quantitative assessment of cellular transcriptome. We also validated some downregulated genes encoding RPs. Altogether, the present data suggest that the underexpression of the studied RP genes possibly collaborates directly or indirectly with other genes to modify cell cycle arrest, DNA repair, and apoptosis, leading to an environment that might have a putative role in the tumorigenesis, introducing new perspectives for further studies on molecular genesis of somatotrophinomas.