The combination of RAD001 and NVP-BEZ235 exerts synergistic anticancer activity against non-small cell lung cancer in vitro and in vivo

The phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit PI3K, mTOR or both are currently under development. The mTOR allosteric inhibitor, RAD001, and the PI3K/mTOR dual kinase inhibitor, BEZ235, are examples of these agents. We were interested in developing strategies to enhance mTOR-targeted caner therapy. In this study, we found that BEZ235 alone effectively inhibited the growth of rapamycin-resistant cancer cells. Interestingly, the combination of sub-optimal concentrations of RAD001 and BEZ235 exerted synergistic inhibition of the growth of human lung cancer cells along with induction of apoptosis and G1 arrest. Furthermore, the combination was also more effective than either agent alone in inhibiting the growth of lung cancer xenografts in mice. The combination showed enhanced effects on inhibiting mTOR signaling and reducing the expression of c-Myc and cyclin D1. Taken together, our results suggest that the combination of RAD001 and BEZ235 is a novel strategy for cancer therapy.     

Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica

Collagenase (matrix metalloproteinase-1, EC:3.4.24.7) was isolated from the hepatopancreas of Achatina fulica and characterized for its enzymatic activity and immunological properties. Procollagenase was isolated using ammonium sulphate precipitation and gel filtration, followed by purification by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid and by dialysis in neutral buffer. In the presence of SDS and beta-mercaptoethanol, the procollagenase resolved into two subunits with molecular masses of 63 and 28 kDa, respectively. The 63 kDa fragment retained its ability to bind and degrade gelatin, but the 28 kDa was inactive. Analysis by 2D gel electrophoresis revealed that the 63 kDa fragment was basic (pIs 7.6, 7.8 and 8.15), while the 28 kDa fragment was acidic (pI 4.7 and 5.1). Western blot analysis confirmed the identity of collagenase, as only matrix metalloproteinase-1 rabbit antibodies against human matrix metalloproteinase-1 (N-terminal region) recognized both the isolated procollagenase and the 63 kDa fragment.     

Alpha-1 Adrenergic Receptors in the Medial Preoptic Area are Involved in the Induction of Sleep

This paper reviews the recent studies that led to the conclusion that the noradrenergic neurons projecting to the medial preoptic area (mPOA) are hypnogenic and that they mediate this action through α₁ adrenergic receptors. Microinjection of noradrenaline (NA) into the mPOA induced arousal. Studies using α₂ adrenergic drugs showed that the arousal induced by intrapreoptic injection of NA was due to its action on presynaptic α₂ adrenergic receptors. A combination of lesion and chemical stimulation techniques demonstrated that when NA acted on the postsynaptic α₁ receptors in the mPOA, it induced sleep. Intrapreoptic injection of α₁ agonist, methoxamine could induce sleep, when the hypothermia, which was simultaneously produced, was behaviorally compensated for by the animal. Increased arousal produced by the destruction of noradrenergic fibers in the mPOA further confirmed the hypnogenic role of these fibers.     

Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target IRAK1 and MYD88, Two Components of the Toll-Like Receptor/Interleukin-1R Signaling Cascade,To Reduce Inflammatory-Cytokine Expression

Kaposi''s sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, an important AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs). We identified interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1) as a potential target of miR-K12-9 (miR-K9) in an array data set examining changes in cellular gene expression levels in the presence of KSHV miRNAs. Using 3′-untranslated region (3′UTR) luciferase reporter assays, we confirmed that miR-K9 and other miRNAs inhibit IRAK1 expression. In addition, IRAK1 expression is downregulated in cells transfected with miR-K9 and during de novo KSHV infection. IRAK1 is an important component of the Toll-like receptor (TLR)/IL-1R signaling cascade. The downregulation of IRAK1 by miR-K9 resulted in the decreased stimulation of NF-κB activity in endothelial cells treated with IL-1α and in B cells treated with a TLR7/8 agonist. Interestingly, miR-K9 had a greater effect on NF-κB activity than did a small interfering RNA (siRNA) targeting IRAK1 despite the more efficient downregulation of IRAK1 expression with the siRNA. We hypothesized that KSHV miRNAs may also be regulating a second component of the TLR/IL-1R signaling cascade, resulting in a stronger phenotype. Reanalysis of the array data set identified myeloid differentiation primary response protein 88 (MYD88) as an additional potential target. 3′UTR luciferase reporter assays and Western blot analysis confirmed the targeting of MYD88 by miR-K5. The presence of miR-K9 and miR-K5 inhibited the production of IL-6 and IL-8 upon the IL-1α stimulation of endothelial cells. These results demonstrate KSHV-encoded miRNAs regulating the TLR/IL-1R signaling cascade at two distinct points and suggest the importance of these pathways during viral infection.     

Antihyperglycemic effect of iridoid glucoside, isolated from the leaves of Vitex negundo in streptozotocin-induced diabetic rats with special reference to glycoprotein components

The aim of present study was to isolate an iridoid glucoside from the leaves of Vitex negundo and evaluates its effects on dearrangement in plasma and tissues glycoprotein components in streptozotocin-induced diabetic rats. The levels of blood glucose, plasma and tissues glycoproteins such as hexose, hexosamine, fucose and sialic acid were significantly increased whereas plasma insulin levels were significantly decreased in diabetic rats. On oral administration of iridoid glucoside at a concentration of 50 mg/kg b.w. once daily to diabetic rats for the period of 30 days, reversed the above-mentioned hyperglycemia-induced biochemical changes to near normal levels. The anti-hyperglycemic effect of iridoid glucoside was comparable with glibenclamide, a known hypoglycemic drug. Based on the results obtained from the present study, it may be concluded that iridoid glucoside possesses significant productive effect on glycoprotein metabolism in addition to its antidiabetic effect.     

Actin-capping protein promotes microtubule stability by antagonizing the actin activity of mDia1

In migrating fibroblasts, RhoA and its effector mDia1 regulate the selective stabilization of microtubules (MTs) polarized in the direction of migration. The conserved formin homology 2 domain of mDia1 is involved both in actin polymerization and MT stabilization, and the relationship between these two activities is unknown. We found that latrunculin A (LatA) and jasplakinolide, actin drugs that release mDia1 from actin filament barbed ends, stimulated stable MT formation in serum-starved fibroblasts and caused a redistribution of mDia1 onto MTs. Knockdown of mDia1 by small interfering RNA (siRNA) prevented stable MT induction by LatA, whereas blocking upstream Rho or integrin signaling had no effect. In search of physiological regulators of mDia1, we found that actin-capping protein induced stable MTs in an mDia1-dependent manner and inhibited the translocation of mDia on the ends of growing actin filaments. Knockdown of capping protein by siRNA reduced stable MT levels in proliferating cells and in starved cells stimulated with lysophosphatidic acid. These results show that actin-capping protein is a novel regulator of MT stability that functions by antagonizing mDia1 activity toward actin filaments and suggest a novel form of actin–MT cross-talk in which a single factor acts sequentially on actin and MTs.     

FE65 as a link between VLDLR and APP to regulate their trafficking and processing

Background

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinson??s disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains.

Results

In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1?/? cells recapitulate the respiratory defect in isolated mitochondria from PINK1?/? mouse brains, suggesting that these PINK1?/? cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1?/? cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1?/? cells, and this genotypic difference between PINK1?/? and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1?/? cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1?/? and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1?/? compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1.

Conclusions

Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1?/? cells.     

Brassica juncea chitinase BjCHI1 inhibits growth of fungal phytopathogens and agglutinates Gram-negative bacteria

Brassica juncea BjCHI1 is a plant chitinase with two chitin-binding domains. Its expression, induced in response to wounding, methyl jasmonate treatment, Aspergillus niger infection, and caterpillar Pieris rapae feeding, suggests that it plays a role in defence. In this study, to investigate the potential of using BjCHI1 in agriculture, Pichia-expressed BjCHI1 and its deletion derivatives that lack one or both chitin-binding domains were tested against phytopathogenic fungi and bacteria. Transplastomic tobacco expressing BjCHI1 was also generated and its extracts assessed. In radial growth-inhibition assays, BjCHI1 and its derivative with one chitin-binding domain showed anti-fungal activities against phytopathogens, Colletotrichum truncatum, C. acutatum, Botrytis cinerea, and Ascochyta rabiei. BjCHI1 also inhibited spore germination of C. truncatum. Furthermore, BjCHI1, but not its derivatives lacking one or both domains, inhibited the growth of Gram-negative bacteria (Escherichia coli, Ralstonia solanacearum, Pseudomonas aeruginosa) more effectively than Gram-positive bacteria (Micrococcus luteus and Bacillus megaterium), indicating that the duplicated chitin-binding domain, uncommon in chitinases, is essential for bacterial agglutination. Galactose, glucose, and lactose relieved agglutination, suggesting that BjCHI1 interacts with the carbohydrate components of the Gram-negative bacterial cell wall. Retention of chitinase and bacterial agglutination activities in transplastomic tobacco extracts implicates that BjCHI1 is potentially useful against both fungal and bacterial phytopathogens in agriculture.