Nitrate enhancement of CAM activity in two Kalanchoë species is associated with increased vacuolar proton transport capacity

Among species that perform CAM photosynthesis, members of the genus Kalanchoë have been studied frequently to investigate the effect of environmental factors on the magnitude of CAM activity. In particular, different nitrogen sources have been shown to influence the rate of nocturnal CO₂ fixation and organic‐acid accumulation in several species of Kalanchoë. However, there has been little investigation of the interrelationship between nitrogen source (nitrate versus ammonium), concentration and the activity of the vacuolar proton pumps responsible for driving nocturnal organic‐acid accumulation in these species. In the present study with Kalanchoë laxiflora and Kalanchoë delagoensis cultivated on different nitrogen sources, both species were found to show highest total nocturnal organic‐acid accumulation and highest rates of ATP‐ and PPi‐dependent vacuolar proton transport on 2.5 mM nitrate, whereas plants cultivated on 5.0 mM ammonium showed the lowest values. In both species malate was the principal organic‐acid accumulated during the night, but the second‐most accumulated organic‐acid was fumarate for K. laxiflora and citrate for K. delagoensis. Higher ATP‐ and PPi‐dependent vacuolar proton transport rates and greater nocturnal acid accumulation were observed in K. delagoensis compared with K. laxiflora. These results show that the effect of nitrogen source on CAM activity in Kalanchoë species is reflected in corresponding differences in activity of the tonoplast proton pumps responsible for driving sequestration of these acids in the vacuole of CAM‐performing cells.     

Antioxidant properties of mesenchymal stem cells against oxidative stress in a murine model of colitis

Objective

To investigate the effects of oxidative stress injury in dextran sulfate sodium (DSS)-induced colitis in mice treated with mesenchymal stem cells (MSC).

Results

Mice exposed to oral administration of 2% DSS over 7 days presented a high disease activity index and an intense colonic inflammation. Systemic infusion of MSC protected from severe colitis, reducing weight loss and diarrhea while lowering the infiltration of inflammatory cells. Moreover, toxic colitis injury increased oxidative stress. Administration of DSS decreased reduced glutathione (GSH) and superoxide dismutase (SOD) activity, and increased thiobarbituric acid-reactive substances levels in the colon. No alteration was found in catalase (CAT) and glutathione peroxidase (GPx) activity. Otherwise, MSC transplantation was able to prevent the decrease of GSH levels and SOD activity suggestive of an antioxidant property of MSC.

Conclusion

The oxidative stress is a pathomechanism underlying the pathophysiology of colitis and MSC play an important role in preventing the impairment of antioxidants defenses in inflamed colon.

     

Interactome disassembly during apoptosis occurs independent of caspase cleavage

Protein–protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas‐mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome‐wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases.     

A panel of the most suitable reference genes for RT-qPCR expression studies of coffee: screening their stability under different conditions

The reliability of analyses using real-time quantitative polymerase chain reaction (RT-qPCR) depends on the selection of appropriate reference genes to correct for sample-to-sample and run-to-run variations. The aim of the present study was to select the most suitable reference genes for gene expression analyses in tissue samples from coffee, Coffea arabica L. (Arabica) grown under well-watered (WW) and water-deficit (WD) conditions and C. canephora Pierre ex A. Froehner (Robusta) grown under WW conditions. Expression profiles and stabilities were evaluated for 12 reference genes in different tissues from C. arabica and for 8 genes in tissues from C. canephora. The web-based RefFinder tool, which combines the geNorm, NormFinder, Bestkeeper, and Delta-Ct algorithms, was employed to assess the stability of the tested genes. The most stable reference genes identified for all tissues grouped (WW/WD) of C. arabica were clathrin adaptor protein medium subunit (AP47), ubiquitin (UBQ), 60S ribosomal protein L39 (RPL39), and elongation factor 1α (EF1α), while class III alcohol dehydrogenase (ADH2), β-actin (ACT), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and ubiquitin (UBQ) genes were the most stable for all tissues grouped (WW) of C. canephora tissues. Validation by the expression level analysis of CaACO-like demonstrated that the use of the best and the worst set of reference genes produced different expression results. The results reinforce the general assumption that there is no universal reference gene and that it is essential to select the most appropriate gene for each individual experiment to apply adequate normalization procedures of RT-qPCR data.     

Wnt8 is required for growth-zone establishment and development of opisthosomal segments in a spider

The Wnt genes encode secreted glycoprotein ligands that regulate many developmental processes from axis formation to tissue regeneration [1]. In bilaterians, there are at least 12 subfamilies of Wnt genes [2]. Wnt3 and Wnt8 are required for somitogenesis in vertebrates [3-7] and are thought to be involved in posterior specification in deuterostomes in general [8]. Although TCF and beta-catenin have been implicated in the posterior patterning of some short-germ insects [9, 10], the specific Wnt ligands required for posterior specification in insects and other protostomes remained unknown. Here we investigated the function of Wnt8 in a chelicerate, the common house spider Achaearanea tepidariorum[11]. Knockdown of Wnt8 in Achaearanea via parental RNAi caused misregulation of Delta, hairy, twist, and caudal and resulted in failure to properly establish a posterior growth zone and truncation of the opisthosoma (abdomen). In embryos with the most severe phenotypes, the entire opisthosoma was missing. Our results suggest that in the spider, Wnt8 is required for posterior development through the specification and maintenance of growth-zone cells. Furthermore, we propose that Wnt8, caudal, and Delta/Notch may be parts of an ancient genetic regulatory network that could have been required for posterior specification in the last common ancestor of protostomes and deuterostomes.     

Quantitative Proteome-Property Relationships (QPPRs). Part 1: finding biomarkers of organic drugs with mean Markov connectivity indices of spiral networks of blood mass spectra

Numerical parameters of the molecular networks, also referred as Topological Indices or Connectivity Indices (CIs), have been used in Bioorganic and Medicinal Chemistry to find Quantitative Structure-Activity, Property or Toxicity Relationship (QSAR, QSPR and QSTR) models. QSPR models generally use CIs as inputs to predict the biological activity of compounds. However, the literature does not evidence a great effort to find QSAR-like models for other biologically and chemically relevant systems. For instance, blood proteome constitutes a protein-rich information reservoir, since the serum proteome Mass Spectra (MS) represents a potential information source for the early detection of Biomarkers for diseases and/or drug-induced toxicities. The concept of mass spectrum network (MS network) for a single protein is already well-known. However, there are no reported results on the use of CIs for a MS network of a whole proteome to explore MS patterns. In this work, we introduced for the first time a novel network representation and the CIs for the MS of blood proteome samples. The new network bases on Randic's Spiral network have been previously introduced for protein sequences. The new MS CIs, called here Spiral Markov Connectivity (SMC(k)) of the MS Spiral graph can be calculated with the software MARCH-INSIDE, combining network and Markov model theory. The SMC(k) values could be used to seek QSAR-like models, called in this work Quantitative Proteome-Property Relationships (QPPRs). We calculate the SMC(k) values for 62 blood samples and fit a QPPR model by discriminating proteome MS, typical of individuals susceptible to suffer drug-induced cardiotoxicity from control samples. The accuracy, sensitivity, and specificity values of the QPPR model were between 73.08% and 87.5% in training and validation series. This work points to QPPR models as a powerful tool for MS detection of biomarkers in proteomics.     

Systematic approaches to using the FOX hunting system to identify useful rice genes

Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23 000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis . This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.     

Identification of a New Functional Domain in Angiopoietin-like 3 (ANGPTL3) and Angiopoietin-like 4 (ANGPTL4) Involved in Binding and Inhibition of Lipoprotein Lipase (LPL)

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln²⁹–His⁵³, which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu³²–His⁵⁵. We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3^-/- mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.Lipoprotein lipase (LPL)⁵ plays a pivotal role in lipid metabolism by catalyzing the hydrolysis of plasma triglycerides (TGs). LPL is likely to be regulated by mechanisms that depend on nutritional status and on the tissue in which it is expressed (1–3). Two secreted proteins, angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), play important roles in the regulation of LPL activity (4, 5). ANGPTL3 and ANGPTL4 consist of a signal peptide, an N-terminal segment containing coiled-coil domains, and a C-terminal fibrinogen-like domain. The N-terminal segment as well as full-length ANGPTL3 and ANGPTL4 have been shown to inhibit LPL activity, and deletion of the N-terminal segment of ANGPTL3 and ANGPTL4 resulted in total loss of LPL-inhibiting activity (6, 7). These observations clearly indicate that the N-terminal region of ANGPTL4 contains the functional domain that inhibits LPL and affects plasma lipid levels. The coiled-coil domains have been proposed to be responsible for oligomerization (8); however, it is not known whether the coiled-coil domains directly mediate the inhibition of LPL activity.To define the physiological role of ANGPTL4 more clearly, we characterized the pharmacological consequences of ANGPTL4 inhibition in mice treated with the ANGPTL4-neutralizing monoclonal antibody (mAb) 14D12 (9). Injection of mAb 14D12 significantly lowered fasting TG levels in C57BL/6J mice relative to levels in C57BL/6J mice treated with an isotype-matched anti-KLH control (KLH) mAb (9). These reduced TG values were similar to decreases in fasting plasma TG levels measured in Angptl4 knock-out (-/-) mice. This study demonstrated that mAb 14D12 is a potent ANGPTL4-neutralizing antibody that is able to inhibit systemic ANGPTL4 activity and thereby recapitulate the reduced lipid phenotype found in Angptl4^-/- mice. The readily apparent pharmacological effect of mAb 14D12 prompted new questions about the epitope recognized by mAb 14D12 and how this antibody-antigen binding event affected ANGPTL4 function as an LPL inhibitor.Although ANGPTL4 is able to interact directly with LPL (10), it is not clear which amino acids within ANGPTL4 mediate this interaction. Here we show that amino acids Gln²⁹–His⁵³ of mANGPTL4 contain the epitope for mAb 14D12. This region, hereby designated specific epitope 1 (SE1), also defines a domain that mediates the interaction between ANGPTL4 and LPL and the subsequent inactivation of LPL. With this information we present evidence that ANGPTL3 also contains an SE1 region, and with antibodies specifically reactive with ANGPTL3 SE1 we examine whether the ANGPTL3 SE1 region is involved in LPL binding and inhibition. We also determined whether treatment of C57BL/6 mice with an anti-ANGPTL3 SE1 mAb can recapitulate the phenotype of lower serum TG and cholesterol levels found in Angptl3^-/- mice. Finally we tested the therapeutic potential of an anti-ANGPTL3 SE1 mAb for treatment of hyperlipidemia in apolipoprotein E^-/- (ApoE^-/-) or low density lipoprotein receptor^-/- (LDLr^-/-) mice.     

Differentiation of Gram-Negative Bacterial Aerosol Exposure Using Detected Markers in Bronchial-Alveolar Lavage Fluid

The identification of biosignatures of aerosol exposure to pathogens has the potential to provide useful diagnostic information. In particular, markers of exposure to different types of respiratory pathogens may yield diverse sets of markers that can be used to differentiate exposure. We examine a mouse model of aerosol exposure to known Gram negative bacterial pathogens, Francisella tularensis novicida and Pseudomonas aeruginosa. Mice were subjected to either a pathogen or control exposure and bronchial alveolar lavage fluid (BALF) was collected at four and twenty four hours post exposure. Small protein and peptide markers within the BALF were detected by matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and analyzed using both exploratory and predictive data analysis methods; principle component analysis and degree of association. The markers detected were successfully used to accurately identify the four hour exposed samples from the control samples. This report demonstrates the potential for small protein and peptide marker profiles to identify aerosol exposure in a short post-exposure time frame.