Lupeol and its ester ameliorate the cyclophosphamide provoked cardiac lysosomal damage studied in rat

Cyclophosphamide (CP), an alkylating agent widely used in cancer chemotherapy causes fatal cardiotoxicity. Lupeol, a pentacyclic triterpene, isolated from Crataeva nurvala stem bark and its ester, lupeol linoleate possess a wide range of medicinal properties. The effect of lupeol and its ester was evaluated in CP-induced myocardial toxicity in rats. Male albino rats of Wistar strain were categorized into six groups. Group I served as control. Rats in groups II, V and VI animals were injected intraperitoneally with a single dose of CP (200 mg/kg body weight) dissolved in saline. CP-treated groups V and VI received lupeol and lupeol linoleate (50 mg/kg body weight), respectively, dissolved in olive oil for 10 days by oral gavage. CP-administered rats showed a significant increase (p < 0.001) in the activities of lysosomal hydrolases in serum and heart, a decrease (p < 0.001) in the levels of cellular thiols and myofibres were swollen with loss of myofilaments in electron microscopical analysis in heart. Lupeol and its ester showed reversal of the above alterations induced by CP. These findings demonstrate that the supplementation with lupeol and its ester could preserve lysosomal integrity, improve thiol levels, highlighting their protective effect against CP-induced cardiotoxicity.     

Molecular modeling of cornulin (CRNN) for docking with phytocompounds from Justicia adhatoda L.

Cornulin (CRNN) is linked with tumour progression. Therefore, it is of interest to document data on the molecular modeling of cornulin (CRNN) for docking with phytocompounds (Pyrazinamide, Anisotine, Vasicinone, Vasicoline) from Justicia adhatoda L. Thus, we document the optimal binding features of these compounds with the cornulin model for further consideration.     

Regulation of the Phosphoinositide Cascade by Polyamines in Brain

Abstract: The endogenous polyamines spermidine and spermine enhanced guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S)-stimulated phosphoinositide turnover with EC₅₀ values of 100 ± 30 and 50 ± 15 µM, respectively, whereas the synthetic polyamines N,N′-bis(3-aminopropyl)-1,3-propanediamine and -ethylenediamine inhibited GTP-γ-S-stimulated phosphoinositide turnover, with maximal inhibition at 1 mM. Kinetic analysis of GTP-γ-S-stimulated phosphoinositide turnover in the absence and presence of spermidine showed that the K_m for GTP-γ-S was not changed (1,303 ± 270 and 1,069 ± 214 nM, respectively), whereas the V_max was increased by 206% (1,566 ± 141 and 4,792 ± 84 cpm, respectively), indicating that spermidine and GTP-γ-S acted at different sites. Spermidine also enhanced Ca²⁺-stimulated phosphoinositide turnover in the absence of GTP-γ-S by decreasing the Ca²⁺ requirement of the phosphoinositide-specific phospholipase C. Arcaine and agmatine, polyamine antagonists at the NMDA receptor complex, did not block the effects of spermidine on GTP-γ-S- and Ca²⁺-induced phosphoinositide turnover, suggesting that the spermidine effects are not mediated through these specific polyamine sites. Furthermore, spermidine increased the level of [³H]phosphatidylinositol 4-phosphate (EC₅₀ = 120 ± 10 µM), without affecting significantly the levels of [³H]phosphatidylinositol and [³H]phosphatidylinositol 4,5-bisphosphate. Collectively these data indicate that the enhanced phosphoinositide turnover induced by spermidine in the presence of GTP-γ-S or Ca²⁺ is mediated through multiple levels of the phosphoinositide turnover cascade.     

Evidence of physical interactions of puroindoline proteins using the yeast two-hybrid system

The puroindoline proteins PINA and PINB play key roles in determining wheat grain texture and also have potential antimicrobial roles. Many recent studies show that their roles in grain texture involve some interaction or interdependence, and their antimicrobial activity may also involve formation of protein complexes. The issue of whether any homo- and/or heteromeric associations occur amongst the PIN proteins is thus critical for understanding their biological functions and exploiting them for grain texture modifications or antimicrobial applications, but is as yet unresolved. This work has utilised the well-established yeast two-hybrid system to directly address this issue. The results confirm occurrence of in vivo interactions between the two PIN proteins for the first time, and show that PINB interacts with itself and also interacts, although somewhat weakly, with PINA, while PINA is a weaker interactor. The results explain the many reported observations suggesting a co-operative interaction between the two proteins and provide a rapid and efficient tool for testing the effects of various alleles/mutations on the interactions and lipid binding properties of these proteins, which are of functional significance to grain texture and antimicrobial defence functions.     

Ouabain decreases sarco(endo)plasmic reticulum calcium ATPase activity in rat hearts by a process involving protein oxidation

The effect of cardiac glycosides to increase cardiac inotropy by altering Ca(2+) cycling is well known but still poorly understood. The studies described in this report focus on defining the effects of ouabain signaling on sarcoplasmic reticulum Ca(2+)-ATPase function. Rat cardiac myocytes treated with 50 microM ouabain demonstrated substantial increases in systolic and diastolic Ca(2+) concentrations. The recovery time constant for the Ca(2+) transient, tau(Ca(2+)), was significantly prolonged by ouabain. Exposure to 10 microM H(2)O(2), which causes an increase in intracellular reactive oxygen species similar to that of 50 microM ouabain, caused a similar increase in tau(Ca(2+)). Concurrent exposure to 10 mM N-acetylcysteine or an aqueous extract from green tea (50 mg/ml) both prevented the increases in tau(Ca(2+)) as well as the changes in systolic or diastolic Ca(2+) concentrations. We also observed that 50 microM ouabain induced increases in developed pressure in addition to diastolic dysfunction in the isolated perfused rat heart. Coadministration of ouabain with N-acetylcysteine prevented these increases. Analysis of sarcoplasmic reticulum Ca(2+)-ATPase protein revealed increases in both the oxidation and nitrotyrosine content in the ouabain-treated hearts. Liquid chromatography-mass spectrometric analysis confirmed that the sarcoplasmic reticulum Ca(2+)-ATPase protein from ouabain-treated hearts had modifications consistent with oxidative and nitrosative stress. These data suggest that ouabain induces oxidative changes of the sarcoplasmic reticulum Ca(2+)-ATPase structure and function that may, in turn, produce some of the associated changes in Ca(2+) cycling and physiological function.     

Heat and chemical shock potentiation of glucocorticoid receptor transactivation requires heat shock factor (HSF) activity. Modulation of HSF by vanadate and wortmannin

Heat shock and other forms of stress increase glucocorticoid receptor (GR) activity in cells, suggesting cross-talk between the heat shock and GR signal pathways. An unresolved question concerning this cross-talk is whether heat shock factor (HSF1) activity is required for this response. We addressed this issue by modulating HSF1 activity with compounds acting by distinct mechanisms: sodium vanadate (SV), an inhibitor of protein phosphatases; and wortmannin, an inhibitor of DNA-dependent protein kinase. Using HSF1- and GR-responsive CAT reporters, we demonstrate that SV inhibits both HSF1 activity and the stress potentiation of GR, while having no effect on the hormone-free GR or HSF1. Paradoxically, SV increased hormone-induced GR activity in the absence of stress. In contrast, wortmannin increased HSF1 activity in stressed cells and had no effect on HSF1 in the absence of stress. Using the pMMTV-CAT reporter containing the negative regulatory element 1 site for DNA-dependent protein kinase, wortmannin was found to increase the GR response. However, in cells expressing a minimal promoter lacking negative regulatory element 1 sites, wortmannin had no effect on the GR in the absence of stress but increased the stress potentiation of GR. Our results show that the mechanism by which GR activity is increased in stressed cells requires intrinsic HSF1 activity.     

Non‐invasive sentinel lymph node mapping and needle guidance using clinical handheld photoacoustic imaging system in small animal

Translating photoacoustic imaging (PAI) into clinical setup is a challenge. Handheld clinical real‐time PAI systems are not common. In this work, we report an integrated photoacoustic (PA) and clinical ultrasound imaging system by combining light delivery with the ultrasound probe for sentinel lymph node imaging and needle guidance in small animal. The open access clinical ultrasound platform allows seamless integration of PAI resulting in the development of handheld real‐time PAI probe. Both methylene blue and indocyanine green were used for mapping the sentinel lymph node using 675 and 690 nm wavelength illuminations, respectively. Additionally, needle guidance with combined ultrasound and PAI was demonstrated using this imaging system. Up to 1.5 cm imaging depth was observed with a 10 Hz laser at an imaging frame rate of 5 frames per second, which is sufficient for future translation into human sentinel lymph node imaging and needle guidance for fine needle aspiration biopsy.      

Hyperinsulinemia-induced vascular smooth muscle cell (VSMC) migration and proliferation is mediated by converging mechanisms of mitochondrial dysfunction and oxidative stress

Atherosclerosis is one of the major complications of diabetes and involves endothelial dysfunction, matrix alteration, and most importantly migration and proliferation of vascular smooth muscle cells (VSMCs). Although hyperglycemia and hyperinsulinemia are known to contribute to atherosclerosis, little is known about the specific cellular signaling pathways that mediate the detrimental hyperinsulinemic effects in VSMCs. Therefore, we investigated the cellular mechanisms of hyperinsulinemia-induced migration and proliferation of VSMCs. VSMCs were treated with insulin (100 nM) for 6 days and subjected to various physiological and molecular investigations. VSMCs subjected to hyperinsulinemia exhibited increased migration and proliferation, and this is paralleled by oxidative stress [increased NADPH oxidase activity, NADPH oxidase 1 mRNA expression, and reactive oxygen species (ROS) generation], alterations in mitochondrial physiology (membrane depolarization, decreased mitochondrial mass, and increased mitochondrial ROS), changes in mitochondrial biogenesis-related genes (mitofusin 1, mitofusin 2, dynamin-related protein 1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, peroxisome proliferator-activated receptor gamma coactivator 1-beta, nuclear respiratory factor 1, and uncoupling protein 2), and increased Akt phosphorylation. Diphenyleneiodonium, a known NADPH oxidase inhibitor significantly inhibited migration and proliferation of VSMCs and normalized all the above physiological and molecular perturbations. This study suggests a plausible crosstalk between mitochondrial dysfunction and oxidative stress under hyperinsulinemia and emphasizes counteracting mitochondrial dysfunction and oxidative stress as a novel therapeutic strategy for atherosclerosis.     

A cytosolic glutathione s-transferase,GST-theta from freshwater prawn Macrobrachium rosenbergii: molecular and biochemical properties

Glutathione S-transferases play an important role in cellular detoxification and may have evolved to protect cells against reactive oxygen metabolites. In this study, we report the molecular characterization of glutathione s-transferase-theta (GST-θ) from freshwater prawn Macrobrachium rosenbergii. A full length cDNA of GSTT (1417 base pairs) was isolated and characterized bioinformatically. Exposure to virus (white spot syndrome baculovirus or M. rosenbergii nodovirus), bacteria (Aeromonas hydrophila or Vibrio harveyi) or heavy metals (cadmium or lead) significantly increased the expression of GSTT (P < 0.05) in hepatopancreas. Recombinant GST-θ with monochlorobimane substrate had an optimum activity at pH 7.5 and 35 °C. Furthermore recombinant GST-θ activity was abolished by the denaturants triton X-100, Gua-HCl, Gua-thiocyanate, SDS and urea in a dose-dependent manner. Overall, the results suggest a potential role for M. rosenbergii GST-θ in detoxification and possibly conferring immune protection.     

Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (-/-) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice.