Isolation and characterization of a 29-kDa glycoprotein with antifungal activity from bulbs of Urginea indica

In this study an antifungal protein from Urginea indica bulbs was purified to homogeneity by acid precipitation, Diol 300 Gel-filtration, and C 18 reverse phase HPLC. Its molecular mass was estimated to be 29kDa and periodic acid-Schiff (PAS) staining showed that identified antifungal molecule is a glycoprotein. The neutralization of antifungal activity after periodate oxidation of 29kDa glycoprotein suggests that the glycan part of the molecule appears to be involved in antifungal activity. N-terminal amino acid sequence of the purified protein was determined as SQLKAXIXDF. This sequence had no sequence similarity with any antifungal proteins. A polyclonal antiserum was raised against purified protein and used in immunolocalization analysis. Results suggest that it is localized to the cell wall of the bulb. Antifungal tests have demonstrated that U. indica protein exerts a fungistatic effect. It completely inhibits the germination of spores and hyphal growth of Fusarium oxysporum. © 2003 Elsevier Inc. All rights reserved

Inhibition of calmodulin-dependent enzymes in rat-brain by hexachlorocyclohexane

Ca2+ homeostasis is one of the major regulatory mechanisms operating in the nervous system, with calmodulin translating the Ca2+ message into cellular response. To check if hexachlorocyclohexane (HCH) acts as a calmodulin antagonist in the nervous system of rats, the in‐vitro effect of HCH on calmodulin‐dependent Ca2+‐ATPase and cAMP‐phosphodiesterase (PDE) in rat brain has been studied. In the membrane fraction from rat brain, a basal activity of Ca2+‐ATPase was obtained in the absence of Ca2+. Inclusion of Ca2+ (1 mM) increased the enzyme activity by 70%. Further, addition of fluphenazine, a potent calmodulin antagonist, inhibited the Ca2+‐dependent enzyme activity (IC50 = 85 μM), demonstrating the calmodulin dependence of the enzyme activity. The Ca2+‐ and calmodulin‐dependent Ca2+‐ATPase was inhibited by HCH in a dose‐dependent manner (IC50 = 80–90 μM). Ca2+‐ and calmodulin‐dependent cAMP‐PDE from the cytosolic fraction of rat brain was inhibited by HCH (340 μM) by 79%. Addition of excess calmodulin reversed the inhibitory effects of HCH or fluphenazine on Ca2+‐ATPase and cAMP‐PDE, suggesting their direct interaction with calmodulin. By fluorescence interaction studies it has been shown that HCH interacts directly with calmodulin. These studies show that HCH may modulate the intracellular concentration of Ca2+ and cAMP, by decreasing the effectiveness of calmodulin towards its effector enzymes, resulting in an altered signal transduction in the nervous syste

Mechanism of inhibition by cyclic AMP of protein kinase C-triggered respiratory burst in Ehrlich ascites tumour cells

The superoxide anion generation in Ehrlich ascites tumour (EAT) cells increased more than two-fold in the presence of the tumour promoter, tetradecanoyl phorbol myristate acetate (TPA). Epinephrine and dibutryl cAMP (Bt2 cAMP) inhibited in a dose-dependent manner, both basal and TPA-triggered superoxide generation in EAT cells. The kinetics of inhibition of superoxide generation showed a maximum inhibition between 30 and 40 min of preincubation with epinephrine or Bt2 cAMP of EAT cells and coincided with an increase in activity of a phosphoprotein phosphatase. In TPA-treated EAT cells, epinephrine or Bt2 cAMP increased the phosphatase activity in a dose-dependent manner. In vitro EGTA, EDTA and sodium fluoride inhibited phosphatase activity. Superoxide generation in response to TPA in Triton-permeabilized EAT cells was inhibited by inclusion of the phosphatase in the assay. Taken together, these results clearly suggest that the phosphatase activity in EAT cells develops as a result of protein kinase A (PKA) and protein kinase C (PKC)-mediated phosphorylation of the phosphatase which then mediates dephosphorylation of the PKC-triggered phosphorylation of proteins to inhibit respiratory burst. A cross-talk between PKA and PKC pathways negatively modulates superoxide generation in EAT cells

Cross-talk between protein kinase C and protein kinase a down-regulates the respiratory burst in polymorphonuclear leukocytes

In this paper it has been shown that increase in intracellular cAMP by epinephrine or its analogue dibutyryl cAMP (Bt2cAMP) abolishes in a dose-dependent manner the protein kinase C (PKC)-mediated respiratory burst in polymorphonuclear leukocytes. The mechanism of inhibition has been shown to involve induction of cytosolic phosphoprotein phosphatase activity specific to cells receiving dual signals (PKC, PKA), as minimum respiratory burst was associated with cells with maximum phosphatase activity. Inclusion of specific PKA inhibitor completely restricted the development of dual signal-induced phosphatase activity in vitro. demonstrating the requirement of multisite phosphorylation of the phosphatase for the development of its activity. Purified phosphatase had a molecular weight of 78,000 and could exert its inhibitory effect on PKC-triggered respiratory burst in permeabilized cells, clearly showing that down-regulation of oxidase activity involved dephosphorylation by the phosphatase. Interaction of the purified phosphatase with eight-fold purified NADPH oxidase as revealed by fluorescence studies further confirmed the role of the phosphatase in the respiratory burst event. Taken together, we have been able to establish that cross-talk between protein kinase C and protein kinase A is essential to `turn off' generation of reactive oxygen species

Effect of capsaicin on phospholipase A2 activity and superoxide generation in macrophages

The mechanism of inhibition of Ca2+ - triggered phospholipase A(2) (PLA(2)) activity and respiratory burst in macrophages by shown that capsaicin inhibits calcium-ionophore stimulated pro-inflammatory responses in macrophages such as generation of superoxide anion, PLA(2) activity (IC50 = 20 uM) and membrane liquid peroxidation (IC50 = 10 uM). Both capsaicin and PLA(2) and dose dependent manner. Arachidonic acid, linoleic acid and SDS restored capsaicin inhibited respiratory burst. Capsaicin and known PLA(2) inhibitors, dexamethasone and indomethacin, inhibited Ca2+-dependent PLA(2) activity in vitro from macrophages. Inhibition of PLA(2) activity by capsaicin is independent of Ca2+ and substrate concentration. Fluorescence studies studies suggest that capsaicin interacts directly with partially purified macrophage PLA(2). Finally, the antioxidant property of capsaicin was comparable to that of butylated hydroxy toludine (BHT). Taken together these results show that capsaicin an antiinflammatory agent with potential clinical application

Targeted Brain Tumor Treatment-Current Perspectives

Brain tumor is associated with poor prognosis. The treatment option is severely limited for a patient with brain tumor, despite great advances in understanding the etiology and molecular biology of brain tumors that have lead to breakthroughs in developing pharmaceutical strategies, and ongoing NCI/Pharma-sponsored clinical trials. We reviewed the literature on molecular targeted agents in preclinical and clinical studies in brain tumor for the past decade, and observed that the molecular targeting in brain tumors is complex. This is because no single gene or protein can be affected by single molecular agent, requiring the use of combination molecular therapy with cytotoxic agents. In this review, we briefly discuss the potential molecular targets, and the challenges of targeted brain tumor treatment. For example, glial tumors are associated with over-expression of calcium-dependent potassium (K Ca ) channels, and high grade glioma express specific K Ca channel gene (gBK) splice variants, and mutant epidermal growth factor receptors (EGFRvIII). These specific genes are promising targets for molecular targeted treatment in brain tumors. In addition, drugs like Avastin and Gleevec target the molecular targets such as vascular endothelial cell growth factor receptor, platelet-derived growth factor receptors, and BRC-ABL/Akt. Recent discovery of non-coding RNA, specifically microRNAs could be used as potential targeted drugs. Finally, we discuss the role of anti-cancer drug delivery to brain tumors by breaching the blood-brain tumor barrier. This non-invasive strategy is particularly useful as novel molecules and humanized monoclonal antibodies that target receptor tyrosine kinase receptors are rapidly being developed

Targeted Brain Tumor Treatment: Current Perspectives

Brain tumor is associated with poor prognosis. The treatment option is severely limited for a patient with brain tumor, despite great advances in understanding the etiology and molecular biology of brain tumors that have lead to breakthroughs in developing pharmaceutical strategies, and ongoing NCI/Pharma-sponsored clinical trials. We reviewed the literature on molecular targeted agents in preclinical and clinical studies in brain tumor for the past decade, and observed that the molecular targeting in brain tumors is complex. This is because no single gene or protein can be affected by single molecular agent, requiring the use of combination molecular therapy with cytotoxic agents. In this review, we briefly discuss the potential molecular targets, and the challenges of targeted brain tumor treatment. For example, glial tumors are associated with over-expression of calcium-dependent potassium (KCa) channels, and high grade glioma express specific KCa channel gene (gBK) splice variants, and mutant epidermal growth factor receptors (EGFRvIII). These specific genes are promising targets for molecular targeted treatment in brain tumors. In addition, drugs like Avastin and Gleevec target the molecular targets such as vascular endothelial cell growth factor receptor, platelet-derived growth factor receptors, and BRC-ABL/Akt. Recent discovery of non-coding RNA, specifically microRNAs could be used as potential targeted drugs. Finally, we discuss the role of anti-cancer drug delivery to brain tumors by breaching the blood-brain tumor barrier. This non-invasive strategy is particularly useful as novel molecules and humanized monoclonal antibodies that target receptor tyrosine kinase receptors are rapidly being developed