NMR study of in vivo RIF-1 tumors: Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the ³¹P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the ³¹P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The ¹H and ¹³C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vivo and in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.     

Localization and kinetics of protein kinase C-epsilon anchoring in cardiac myocytes

Protein kinase C-epsilon (PKC-epsilon) plays a central role in cardiac cell signaling, but mechanisms of translocation and anchoring upon activation are poorly understood. Conventional PKC isoforms rely on a rapid Ca2+-mediated recruitment to cell membranes, but this mechanism cannot be employed by PKC-epsilon or other PKC isoforms lacking a Ca2+-binding domain. In this study, we used recombinant green fluorescent protein (GFP) fusion constructs and confocal microscopy to examine the localization, kinetics, and reversibility of PKC-epsilon anchoring in permeabilized rat cardiac myocytes. PKC-epsilon-GFP bound with a striated pattern that co-localized with alpha-actinin, a marker of the Z-line of the sarcomere. Binding required activation of PKC and occurred slowly but reversibly with apparent rate constants of k(on) = 4.6 +/- 1.2 x 10(3) M(-1) x s(-1) and k(off) = 1.4 +/- 0.5 x 10(-3) s(-1) (t1/2 = 8 min) as determined by fluorescence recovery after photobleaching and by perfusion experiments. A truncated construct composed of the N-terminal 144-amino-acid variable region of PKC-epsilon (epsilonV1-GFP), but not an analogous N-terminal domain of PKC-delta, mimicked the Z-line decoration and slow binding rate of the full-length enzyme. These findings suggest that the epsilonV1 domain is important in determining PKC-epsilon localization and translocation kinetics in cardiac muscle. Moreover, PKC-epsilon translocation is not a diffusion-controlled binding process but instead may be limited by intramolecular conformational changes within the V1 domain. The k(off) for epsilonV1-GFP was two- to threefold faster than for full-length enzyme, indicating that other domains in PKC-epsilon contribute to anchoring by prolonging the bound state.     

The calmodulin binding region of the skeletal ryanodine receptor acts as a self-modulatory domain

A synthetic peptide (CaMBP) matching amino acids 3614-3643 of the skeletal ryanodine receptor (RyR1) binds to both Ca2+-free calmodulin (CaM) and Ca2+-bound CaM with nanomolar affinity [J. Biol. Chem. 276 (2001) 2069]. We report here that CaMBP increases [3H]ryanodine binding to RyR1 in a dose- and Ca2+-dependent manner; it also induces Ca2+ release from SR vesicles, and increases open probability (P(o)) of single RyR channels reconstituted in planar lipid bilayers. Further, CaMBP removes CaM associated with SR vesicles and increases [3H]ryanodine binding to purified RyR1, suggesting that its mechanism of action is two-fold: it removes endogenous inhibitors and also interacts directly with complementary regions in RyR1. Remarkably, the N-terminus of CaMBP activates RyRs while the C-terminus of CaMBP inhibits RyR activity, suggesting the presence of two discrete functional subdomains within this region. A ryr1 mutant lacking this region, RyR1-Delta3614-3643, was constructed and expressed in dyspedic myoblasts (RyR1-knockout). The depolarization-, caffeine- and 4-chloro-m-cresol (4-CmC)-induced Ca2+ transients in these cells were dramatically reduced compared with cells expressing wild type RyR1. Deletion of the 3614-3643 region also resulted in profound changes in unitary conductance and channel gating. We thus propose that the RyR1 3614-3643 region acts not only as the CaM binding site, but also as an important modulatory domain for RyR1 function.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Effects of the Dietary ω3:ω6 Fatty Acid Ratio on Body Fat and Inflammation in Zebrafish (Danio rerio)

The diets of populations in industrialized nations have shifted to dramatically increased consumption of ω6 polyunsaturated fatty acids (PUFA), with a corresponding decrease in the consumption of ω3 PUFA. This dietary shift may be related to observed increases in obesity, chronic inflammation, and comorbidities in the human population. We examined the effects of ω3:ω6 fatty acid ratios in the context of constant total dietary lipid on the growth, total body fat, and responses of key inflammatory markers in adult zebrafish (Danio rerio). Zebrafish were fed diets in which the ω3:ω6 PUFA ratios were representative of those in a purported ancestral diet (1:2) and more contemporary Western diets (1:5 and 1:8). After 5 mo, weight gain (fat free mass) of zebrafish was highest for those that received the 1:8 ratio treatment, but total body fat was lowest at this ratio. Measured by quantitative real-time RT–PCR, mRNA levels from liver samples of 3 chronic inflammatory response genes (C-reactive protein, serum amyloid A, and vitellogenin) were lowest at the 1:8 ratio. These data provide evidence of the ability to alter zebrafish growth and body composition through the quality of dietary lipid and support the application of this model to investigations of human health and disease related to fat metabolism.Abbreviations: LC-PUFA, long-chain PUFA; PUFA, polyunsaturated fatty acidsMost animals require specific (essential) dietary fatty acids, and deficiencies in these fatty acids typically exert a negative effect on their health at some level. The ω3 and ω6 families of fatty acids are essential polyunsaturated fatty acids (PUFA) or long-chain PUFA (LC-PUFA) for many animals, including humans; however, consensus regarding the recommended dietary levels of these PUFA has not been achieved for any species, including humans. Several studies have proposed that a disproportionately high intake of ω6 PUFA and LC-PUFA promotes inflammation, resulting in chronic inflammatory diseases associated with metabolic syndrome.^10,22 This ‘high’ intake is difficult to describe accurately because both individual as well as regional diversity in the dietary intake of ω3 and ω6 fatty acids exist globally. Over the last century, diets in the western hemisphere have shifted to a dramatically increased consumption of total lipids. This increase in total fat consumption is associated with increases in ω6 PUFA and ω6 LC-PUFA intakes and corresponding decreases in ω3 PUFA and ω3 LC-PUFA.¹⁶ The shift in the dietary ω3:ω6 ratio, toward ω6 and away from ω3 fatty acids, in industrialized societies has been proposed to be the major factor contributing to inflammatory diseases.²² This proinflammatory effect is often attributed to the production of arachidonic acid metabolites, which act as potent proinflammatory and plaque forming molecules, from ω6 fatty acids, like linoleic acid.⁷ However, many antiinflammatory mediators also are produced during the metabolism of ω6. Several studies support a possible association between a reduced risk of coronary heart disease and increased dietary ω6 PUFA.⁷ The American Heart Association Science Advisory Panel has stated, “At present, there is little direct evidence that supports a net proinflammatory, proatherogenic effect of linoleic acid (18:2 ω6) in humans.”¹¹ The authors of a recent review¹⁹ concluded that reducing the intake of dietary ω6 fatty acid did not change the levels of arachidonic acid in the plasma, serum, or erythrocytes of adults who consumed western-type, high-fat diets. Other scientists¹⁸ have suggested that specific proportional combinations of ω3 and ω6 fatty acids may actually decrease the concentrations of proinflammatory cytokines.Zebrafish continue to gain popularity as an animal model for cardiovascular disease.⁴ For example, blood vessel plaques formed in zebrafish that consumed a high-cholesterol (4%) diet, mimicking atherosclerosis in humans.²⁴ Recent advances in the area of zebrafish nutrition²⁵ allow the use of formulated diets, wherein the levels of specific nutrients, such as fatty acids, can be modified to evaluate response. The current study evaluated the effects of different dietary ω3:ω6 fatty acid ratios on weight gain, body composition, and inflammatory response proteins in the zebrafish.     

Th2 Cytokines Inhibit Lymphangiogenesis

Lymphangiogenesis is the process by which new lymphatic vessels grow in response to pathologic stimuli such as wound healing, inflammation, and tumor metastasis. It is well-recognized that growth factors and cytokines regulate lymphangiogenesis by promoting or inhibiting lymphatic endothelial cell (LEC) proliferation, migration and differentiation. Our group has shown that the expression of T-helper 2 (Th2) cytokines is markedly increased in lymphedema, and that these cytokines inhibit lymphatic function by increasing fibrosis and promoting changes in the extracellular matrix. However, while the evidence supporting a role for T cells and Th2 cytokines as negative regulators of lymphatic function is clear, the direct effects of Th2 cytokines on isolated LECs remains poorly understood. Using in vitro and in vivo studies, we show that physiologic doses of interleukin-4 (IL-4) and interleukin-13 (IL-13) have profound anti-lymphangiogenic effects and potently impair LEC survival, proliferation, migration, and tubule formation. Inhibition of these cytokines with targeted monoclonal antibodies in the cornea suture model specifically increases inflammatory lymphangiogenesis without concomitant changes in angiogenesis. These findings suggest that manipulation of anti-lymphangiogenic pathways may represent a novel and potent means of improving lymphangiogenesis.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.     

An efficient in vitro shoot regeneration from leaf petiolar explants and ex vitro rooting of Bixa orellana L.- A dye yielding plant

Physiology and Molecular Biology of Plants - Bixa orellana L. (Bixaceae) is a multipurpose tree grown for the production of commercially important dyes. In the present study, an efficient,...     

Glutathione signaling acts through NPR1-dependent SA-mediated pathway to mitigate biotic stress

Glutathione (GSH) has widely been known to be a multifunctional molecule especially as an antioxidant up until now, but has found a new role in plant defense signaling. Research from the past three decades indicate that GSH is a player in pathogen defense in plants, but the mechanism underlying this has not been elucidated fully. We have recently shown that GSH acts as a signaling molecule and mitigates biotic stress through non-expressor of PR genes 1 (NPR1)-dependent salicylic acid (SA)-mediated pathway. Transgenic tobacco with enhanced level of GSH (NtGB lines) was found to synthesize more SA, was capable of enhanced expression of genes belonging to NPR1-dependent SA-mediated pathway, were resistant to Pseudomonas syringae, the biotrophic pathogen and many SA-related proteins were upregulated. These results gathered experimental evidence on the mechanism through which GSH combats biotic stress. In continuation with our previous investigation we show here that the expression of glutathione S-transferase (GST), the NPR1-independent SA-mediated gene was unchanged in transgenic tobacco with enhanced level of GSH as compared to wild-type plants. Additionally, the transgenic plants were barely resistant to Botrytis cinerea, the necrotrophic pathogen. SA-treatment led to enhanced level of expression of pathogenesis-related protein gene (PR1) and PR4 as against short-chain dehydrogenase/reductase family protein (SDRLP) and allene oxide synthase (AOS). These data provided significant insight into the involvement of GSH in NPR1-dependent SA-mediated pathway in mitigating biotic stress.Key words: GSH, signaling molecule, biotrophic pathogen, NPR-1, PR-1, PR-4, transgenic tobaccoPlant responses to different environmental stresses are achieved through integrating shared signaling networks and mediated by the synergistic or antagonistic interactions with the phytohormones viz. SA, jasmonic acid (JA), ethylene (ET), abscisic acid (ABA) and reactive oxygen species (ROS).¹ Previous studies have shown that in response to pathogen attack, plants produce a highly specific blend of SA, JA and ET, resulting in the activation of distinct sets of defense-related genes.^2,3 Regulatory functions for ROS in defense, with a focus on the response to pathogen infection occur in conjunction with other plant signaling molecules, particularly with SA and nitric oxide (NO).^4–6 Till date, numerous physiological functions have been attributed to GSH in plants.^7–11 In addition to previous studies, recent study has also shown that GSH acts as a signaling molecule in combating biotic stress through NPR1-dependent SA-mediated pathway.^12,13Our recent investigation involved raising of transgenic tobacco overexpressing gamma-glutamylcysteine synthetase (γ-ECS), the rate-limiting enzyme of the GSH biosynthetic pathway.¹² The stable integration and enhanced expression of the transgene at the mRNA as well as protein level was confirmed by Southern blot, quantitative RT-PCR and western blot analysis respectively. The transgenic plants of the T₂ generation (Fig. 1), the phenotype of which was similar to that of wild-type plants were found to be capable of synthesizing enhanced amount of GSH as confirmed by HPLC analysis.Open in a separate windowFigure 1Transgenic tobacco of T₂ generation, (A) three-week-old plant, (B) mature plant.In the present study, the expression profile of GST was analyzed in NtGB lines by quantitative RT-PCR (qRT-PCR) and found that the expression level of this gene is unchanged in NtGB lines as compared to wild-type plants (Fig. 2). GST is known to be a NPR1-independent SA-related gene.¹⁴ This suggests that GSH does not follow the NPR1-independent SA-mediated pathway in defense signaling.Open in a separate windowFigure 2Expression pattern of GST in wild-type and NtGB lines.Disease test assay with NtGB lines and wild-type plants was performed using B. cinerea and the NtGB lines showed negligible rate of resistance to this necrotrophic pathogen (Fig. 3). SA signaling has been known to control defense against biotrophic pathogen in contrast, JA/ET signaling controls defense against necrotrophic pathogen.^1,15 Thus it has again been proved that GSH is not an active member in the crosstalk of JA-mediated pathway, rather it follows the SA-mediated pathway as has been evidenced earlier.¹²Open in a separate windowFigure 3Resistance pattern of wild-type and NtGB lines against Botrytis cinerea.Additionally, the leaves of wild-type and NtGB lines were treated with 1 mM SA and the expression of PR1, SDRLP, AOS and PR4 genes were analyzed and compared to untreated plants to simulate pathogen infection. The expression of PR1 increased after exogenous application of SA. In case of PR4, the ET marker, the expression level increased in NtGB lines. On the other hand, the level of SDRLP was nearly the same. However, the expression of AOS was absent in SA-treated leaves (Fig. 4). PR1 has been known to be induced by SA-treatment¹⁶ which can be corroborated with our results. In addition, ET is known to enhance SA/NPR1-dependent defense responses,¹⁷ which was reflected in our study as well. AOS, the biosynthetic pathway gene of JA, further known to be the antagonist of SA, was downregulated in SA-treated plants.Open in a separate windowFigure 4Gene expression pattern of PR1, SDRLP, PR4 and AOS in untreated and SA-treated wildtype and NtGB lines.Taken together, it can be summarized that this study provided new evidence on the involvement of GSH with SA in NPR1-dependent manner in combating biotic stress. Additionally, it can be claimed that GSH is a signaling molecule which takes an active part in the cross-communication with other established signaling molecules like SA, JA, ET in induced defense responses and has an immense standpoint in plant defense signaling.