Dermal excisional wound healing in pigs following treatment with topically applied pure oxygen

Hypoxia, caused by disrupted vasculature and peripheral vasculopathies, is a key factor that limits dermal wound healing. Factors that can increase oxygen delivery to the regional tissue, such as supplemental oxygen, warmth, and sympathetic blockade, can accelerate healing. Clinical experience with adjunctive hyperbaric oxygen therapy (HBOT) in the treatment of chronic wounds have shown that wound hyperoxia may increase granulation tissue formation and accelerate wound contraction and secondary closure. However, HBOT is not applicable to all wound patients and may pose the risk of oxygen toxicity. Thus, the efficacy of topical oxygen treatment in an experimental setting using the pre-clinical model involving excisional dermal wound in pigs was assessed. Exposure of open dermal wounds to topical oxygen treatment increased tissue pO₂ of superficial wound tissue. Repeated treatment accelerated wound closure. Histological studies revealed that the wounds benefited from the treatment. The oxygen treated wounds showed signs of improved angiogenesis and tissue oxygenation. Topically applied pure oxygen has the potential of benefiting some wound types. Further studies testing the potential of topical oxygen in pre-clinical and clinical settings are warranted.     

Dual effects of copper-zinc superoxide dismutase

Copper-zinc superoxide dismutase (CuZnSOD) specifically catalyzes the removal of superoxide radicals to protect cellular function against the generation of superoxide-dependent hydroxyl radicals ((.)OH). However, an unexpected observation reveals that denatured CuZnSOD (dCuZnSOD) itself induces (.)OH formation. This dCuZnSOD-dependent (.)OH generation was not inhibited by active CuZnSOD, suggesting that it is a superoxide-independent process. Sodium cyanide, histidine, and N,N'-diethyldithiocarbamate abolished (.)OH generation, implying that Cu may be responsible for dCuZnSOD-induced (.)OH formation. Catalase eliminated ()OH generation, suggesting that hydrogen peroxide may be involved in the mechanism of dCuZnSOD-mediated (.)OH production. Furthermore, nitric oxide ((.)NO) completely inhibited dCuZnSOD-induced (.)OH radical generation, indicating that (.)NO is an important (.)OH radical scavenger. Our results shed new light on the effect of dysfunctional CuZnSOD and suggest that structural disorder of the enzyme may be one of the endogenous pathways of toxic (.)OH formation in biological systems.     

Kinetic analysis-based quantitation of free radical generation in EPR spin trapping

Because short-lived reactive oxygen radicals such as superoxide have been implicated in a variety of disease processes, methods to measure their production quantitatively in biological systems are critical for understanding disease pathophysiology. Electron paramagnetic resonance (EPR) spin trapping is a direct and sensitive technique that has been used to study radical formation in biological systems. Short-lived oxygen free radicals react with the spin trap and produce paramagnetic adducts with much higher stability than that of the free radicals. In many cases, the quantity of the measured adduct is considered to be an adequate measure of the amount of the free radical generated. Although the intensity of the EPR signal reflects the magnitude of free radical generation, the actual quantity of radicals produced may be different due to modulation of the spin adduct kinetics caused by a variety of factors. Because the kinetics of spin trapping in biochemical and cellular systems is a complex process that is altered by the biochemical and cellular environment, it is not always possible to define all of the reactions that occur and the related kinetic parameters of the spin-trapping process. We present a method based on a combination of measured kinetic data for the formation and decay of the spin adduct alone with the parameters that control the kinetics of spin trapping and radical generation. The method is applied to quantitate superoxide trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). In principle, this method is broadly applicable to enable spin trapping-based quantitative determination of free radical generation in complex biological systems.     

Heat shock protects cardiac cells from doxorubicin-induced toxicity by activating p38 MAPK and phosphorylation of small heat shock protein 27

Doxorubicin (DOX) and its derivatives are used as chemotherapeutic drugs to treat cancer patients. However, production of DOX-mediated reactive oxygen species (ROS) by prolonged use of these drugs has been found to cause dilative cardiomyopathy and congestive heart failure. Thus various preventive modalities have been developed to avoid this side effect. We have found that the DOX-mediated oxidant-induced toxicity in cardiac cells could be minimized by hyperthermia-induced small heat shock protein 27 (HSP27); that is, this protein acts as an endogenous antioxidant against DOX-derived oxidants such as H(2)O(2). Heat shock-induced HSP27 was found to act as an antiapoptotic protein (reducing ROS and Bax-to-Bcl2 ratio) against DOX, and its phosphorylated isoforms stabilized F-actin remodeling in DOX-treated cardiac cells and, hence, attenuated the toxicity. Protein kinase assays and proteomic analyses suggested that higher expression of HSP27 and its phosphorylation are responsible for the protection in heat-shocked cells. Two-dimensional gel electrophoresis showed six isoforms (nonphosphorylated and phosphorylated) of HSP27. Matrix-assisted laser desorption/ionization time of flight analyses showed alpha- and beta-isoforms of HSP27, which are phosphorylated by various protein kinases. Ser(15) and Ser(85) phosphorylation of HSP27 by MAPK-assisted protein kinase 2 was found to be the key mechanism in reduction of apoptosis and facilitation of F-actin remodeling. The present study illustrates that hyperthermia protects cells from DOX-induced death through induction and phosphorylation of HSP27 and its antiapoptotic and actin-remodeling activities.     

Novel levamisole derivative induces extrinsic pathway of apoptosis in cancer cells and inhibits tumor progression in mice

Background

Levamisole, an imidazo(2,1-b)thiazole derivative, has been reported to be a potential antitumor agent. In the present study, we have investigated the mechanism of action of one of the recently identified analogues, 4a (2-benzyl-6-(4′-fluorophenyl)-5-thiocyanato-imidazo[2,1-b][1], [3], [4]thiadiazole).

Materials and Methods

ROS production and expression of various apoptotic proteins were measured following 4a treatment in leukemia cell lines. Tumor animal models were used to evaluate the effect of 4a in comparison with Levamisole on progression of breast adenocarcinoma and survival. Immunohistochemistry and western blotting studies were performed to understand the mechanism of 4a action both ex vivo and in vivo.

Results

We have determined the IC₅₀ value of 4a in many leukemic and breast cancer cell lines and found CEM cells most sensitive (IC₅₀ 5 µM). Results showed that 4a treatment leads to the accumulation of ROS. Western blot analysis showed upregulation of pro-apoptotic proteins t-BID and BAX, upon treatment with 4a. Besides, dose-dependent activation of p53 along with FAS, FAS-L, and cleavage of CASPASE-8 suggest that it induces death receptor mediated apoptotic pathway in CEM cells. More importantly, we observed a reduction in tumor growth and significant increase in survival upon oral administration of 4a (20 mg/kg, six doses) in mice. In comparison, 4a was found to be more potent than its parental analogue Levamisole based on both ex vivo and in vivo studies. Further, immunohistochemistry and western blotting studies indicate that 4a treatment led to abrogation of tumor cell proliferation and activation of apoptosis by the extrinsic pathway even in animal models.

Conclusion

Thus, our results suggest that 4a could be used as a potent chemotherapeutic agent.     

Oviposition deterrent, ovicidal and gravid mortality effects of ethanolic extract of Andrographis paniculata Nees against the malarial vector Anopheles stephensi Liston (Diptera: Culicidae)

Oviposition is an important phenomenon of mosquitoes and has recently become the focus in the concept of integrated vector control management. In the present study, we evaluated oviposition deterrent, ovicidal and mortality effects of ethanolic extract of Andrographis paniculata Nees against gravid and oviposited females of Anopheles stephensi Liston. Water treated with the ethanolic extract had a high deterrent activity in ovipositing females: oviposition activity index values for the test species were –0.28, –0.45, –0.49 and –0.59 for extract concentrations of 29, 35, 41 and 46 p.p.m., respectively. High degrees of mortality were observed with various concentrations of extract: 1.12 (control) to 11.70 for gravid females, and 0.65 (control) to 10.25 for oviposited females. The highest mortality in both gravid and oviposited females was observed soon after they came in contact with oviposition medium treated with the extract, and this was found to be significant at doses higher than 35 p.p.m., suggesting possible contact toxicity of the extract. The extract caused moderate ovicidal activity against various age groups of A. stephensi, but it inflicted delayed effects such as high larval, pupal and adult mortality. The age of the eggs and the duration of the extract treatment influenced the ovicidal activity observed. It is clear that ethanolic extract of A. paniculata Nees can affect the oviposition cycle of A. stephensi Liston, thereby suppressing the vector population and adversely influencing transmission of the disease pathogen.     

Redox-active antioxidant modulation of lipid signaling in vascular endothelial cells: vitamin C induces activation of phospholipase D through phospholipase A2, lipoxygenase, and cyclooxygenase

We have earlier reported that the redox-active antioxidant, vitamin C (ascorbic acid), activates the lipid signaling enzyme, phospholipase D (PLD), at pharmacological doses (mM) in the bovine lung microvascular endothelial cells (BLMVECs). However, the activation of phospholipase A(2) (PLA(2)), another signaling phospholipase, and the modulation of PLD activation by PLA(2) in the ECs treated with vitamin C at pharmacological doses have not been reported to date. Therefore, this study aimed at the regulation of PLD activation by PLA(2) in the cultured BLMVECs exposed to vitamin C at pharmacological concentrations. The results revealed that vitamin C (3-10 mM) significantly activated PLA(2) starting at 30 min; however, the activation of PLD resulted only at 120 min of treatment of cells under identical conditions. Further studies were conducted utilizing specific pharmacological agents to understand the mechanism(s) of activation of PLA(2) and PLD in BLMVECs treated with vitamin C (5 mM) for 120 min. Antioxidants, calcium chelators, iron chelators, and PLA(2) inhibitors offered attenuation of the vitamin C-induced activation of both PLA(2) and PLD in the cells. Vitamin C was also observed to significantly induce the formation and release of the cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites and to activate the AA LOX in BLMVECs. The inhibitors of PLA(2), COX, and LOX were observed to effectively and significantly attenuate the vitamin C-induced PLD activation in BLMVECs. For the first time, the results of the present study revealed that the vitamin C-induced activation of PLD in vascular ECs was regulated by the upstream activation of PLA(2), COX, and LOX through the formation of AA metabolites involving oxidative stress, calcium, and iron.     

Reduction of cyclic AMP phosphodiesterase activity of several subcellular fractions of rat brain after pretreatment with phospholipase C

Cyclic AMP phosphodiesterase (PDE) activity was assayed in the plasma membrane, mitochondrial and microsomal fractions of rat brain. The specific activity of the enzyme was highest in the plasma membrane fraction followed by mitochondrial and then the microsomal fraction. Phosphodiesterase activity of all three fractions was reduced after pretreatment with lecithinase C (PCase) from Clostridium perfringens but less markedly affected by the pretreatment with sphingomyelinase (SMase) from human placenta. The PDE activity of the plasma membrane fraction was more sensitive to PCase treatment compared with the other two particulate fractions, which showed only a slight loss of activity. Temperature seemed to affect PDE activity of the plasma membrane. The enzyme was quite stable at 30 degrees C but its activity dropped by approximately 46% at 37 degrees C after 90 min of incubation. Pretreatment of the plasma membrane at 30 degrees C with PCase at a concentration of more than 5 U caused a marked loss of PDE activity and the decrease in activity reached a plateau at concentrations above 10 U.     

Macrobrachium rosenbergii cathepsin L: Molecular characterization and gene expression in response to viral and bacterial infections

Cathepsin L (MrCathL) was identified from a constructed cDNA library of freshwater prawn Macrobrachium rosenbergii. MrCathL full-length cDNA is 1161 base pairs (bp) with an ORF of 1026 bp which encodes a polypeptide of 342 amino acid (aa) long. The eukaryotic cysteine proteases, histidine and asparagine active site residues were identified in the aa sequence of MrCathL at 143–154, 286–296 and 304–323, respectively. The pair wise clustalW analysis of MrCathL showed the highest similarity (97%) with the homologous cathepsin L from Macrobrachium nipponense and the lowest similarity (70%) from human. Phylogenetic analysis revealed two distinct clusters of the invertebrates and vertebrates cathepsin L in the phylogenetic tree. MrCathL and cathepsin L from M. nipponense were clustered together, formed a sister group to cathepsin L of Penaeus monodon, and finally clustered to Lepeophtheirus salmonis. High level of (P < 0.05) MrCathL gene expression was noticed in haemocyte and lowest in eyestalk. Furthermore, the MrCathL gene expression in M. rosenbergii was up-regulated in haemocyte by virus [M. rosenbergii nodovirus (MrNV) and white spot syndrome baculovirus (WSBV)] and bacteria (Vibrio harveyi and Aeromonas hydrophila). The recombinant MrCathL exhibited a wide range of activity in various pH between 3 and 10 and highest at pH 7.5. Cysteine proteinase (stefin A, stefin B and antipain) showed significant influence (100%) on recombinant MrCathL enzyme activity. The relative activity and residual activity of recombinant MrCathL against various metal ions or salts and detergent tested at different concentrations. These results indicated that the metal ions, salts and detergent had an influence on the proteinase activity of recombinant MrCathL. Conclusively, the results of this study imply that MrCathL has high pH stability and is fascinating object for further research on the function of cathepsin L in prawn innate immune system.