Mitofusin-2 mediates cannabidiol-induced neuroprotection against cerebral ischemia in rats.

Cannabidiol (CBD) reportedly exerts protective effects against many psychiatric disorders and neurodegenerative diseases, but the mechanisms are poorly understood. In this study, we explored the molecular mechanism of CBD against cerebral ischemia. HT-22 cells or primary cortical neurons were subjected to oxygen-glucose deprivation insult followed by reoxygenation (OGD/R). In both HT-22 cells and primary cortical neurons, CBD pretreatment (0.1, 0.3, 1 µM) dose-dependently attenuated OGD/R-induced cell death and mitochondrial dysfunction, ameliorated OGD/R-induced endoplasmic reticulum (ER) stress, and increased the mitofusin-2 (MFN2) protein level in HT-22 cells and primary cortical neurons. Knockdown of MFN2 abolished the protective effects of CBD. CBD pretreatment also suppressed OGD/R-induced binding of Parkin to MFN2 and subsequent ubiquitination of MFN2. Overexpression of Parkin blocked the effects of CBD in reducing MFN2 ubiquitination and reduced cell viability, whereas overexpressing MFN2 abolished Parkin's detrimental effects. In vivo experiments were conducted on male rats subjected to middle cerebral artery occlusion (MCAO) insult, and administration of CBD (2.5, 5 mg · kg-1, i.p.) dose-dependently reduced the infarct volume and ER stress in the brains. Moreover, the level of MFN2 within the ischemic penumbra of rats was increased by CBD treatment, while the binding of Parkin to MFN2 and the ubiquitination of MFN2 was decreased. Finally, short hairpin RNA against MFN2 reversed CBD's protective effects. Together, these results demonstrate that CBD protects brain neurons against cerebral ischemia by reducing MFN2 degradation via disrupting Parkin's binding to MFN2, indicating that MFN2 is a potential target for the treatment of cerebral ischemia.

Ferroptosis mediated by the interaction between Mfn2 and IREα promotes arsenic-induced nonalcoholic steatohepatitis.

Exposure to arsenic is a risk factor for nonalcoholic steatohepatitis (NASH). Ferroptosis is a form of regulated cell death defined by the accumulation of lipid peroxidation. In the current study, we observed the occurrence of ferroptosis in arsenic-induced NASH by assessing ferroptosis related hallmarks. In vitro, we found that ferrostatin-1 effectively attenuated the executing of ferroptosis and NASH. Simultaneously, the expression of ACSL4 (acyl-CoA synthetase long-chain family member 4) was upregulated in rat's liver and L-02 cells exposed to arsenic. While, suppression of ACSL4 with rosiglitazone or ACSL4 siRNA remarkably alleviated arsenic-induced NASH and ferroptosis through diminishing 5-hydroxyeicosatetraenoic acid (5-HETE) content. Additionally, Mitofusin 2 (Mfn2), a physical tether between endoplasmic reticulum and mitochondria, has rarely been explored in the ferroptosis. Using Mfn2 siRNA or inositol-requiring enzyme 1 alpha (IRE1α) inhibitor, we found NASH and ferroptosis were obviously mitigated through reducing 5-HETE content. Importantly, Co-IP assay indicated that Mfn2 could interact with IRE1α and promoted the production of 5-HETE, ultimately led to ferroptosis and NASH. Collectively, our data showed that ferroptosis is involved in arsenic-induced NASH. These data provide insightful viewpoints into the mechanism of arsenic-induced NASH.

Melatonin Attenuates Calcium Deposition from Vascular Smooth Muscle Cells by Activating Mitochondrial Fusion and Mitophagy via an AMPK/OPA1 Signaling Pathway.

Mitochondrial fusion/mitophagy plays a role in cardiovascular calcification. Melatonin has been shown to protect against cardiovascular disease. This study sought to explore whether melatonin attenuates vascular calcification by regulating mitochondrial fusion/mitophagy via the AMP-activated protein kinase/optic atrophy 1 (AMPK/OPA1) signaling pathway. The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualized by Alizarin Red S staining, while calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure expression of runt-related transcription factor 2 (Runx2), mitofusin 2 (Mfn2), mito-light chain 3 (mito-LC3) II, and cleaved caspase 3. Melatonin markedly reduced calcium deposition and ALP activity. Runx2 and cleaved caspase 3 were downregulated in response to melatonin, whereas Mfn2 and mito-LC3II were enhanced and accompanied by decreased mitochondrial superoxide levels. Melatonin also maintained mitochondrial function and promoted mitochondrial fusion/mitophagy via the OPA1 pathway. However, OPA1 deletion abolished the protective effects of melatonin on VSMC calcification. Melatonin treatment significantly increased p-AMPK and OPA1 protein expression, whereas treatment with compound C ablated the observed benefits of melatonin treatment. Collectively, our results demonstrate that melatonin protects VSMCs against calcification by promoting mitochondrial fusion/mitophagy via the AMPK/OPA1 pathway.

The expression of glucose transporters and mitochondrial division and fusion proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity.

Purpose: Evidence has shown that propofol may cause widespread apoptotic neurodegeneration. Hypoxic preconditioning has been demonstrated to provide neuroprotection and brain recovery from both acute and chronic neurodegeneration in several cellular and animal models. However, the mechanism has not been well elucidated. Therefore, the present study was designed to investigate the expression of glucose transporters (GLUT1 and GLUT3) and mitochondrial division and fusion (Drp1 and Mfn2) proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity.Methods: Propofol (100 mg/kg) was given to 7-day-old Sprague-Dawley rats; in some rats, hypoxic preconditioning was administered before intraperitoneal propofol injection by subjecting rats to five cycles of 10 min of hypoxia (8% O2) and 10 min of normoxia (21% O2). Then, the rats were allowed to breathe room air for 2 h. Neuronal mitochondrial morphology was observed by transmission electron microscopy. ATP content was detected using an ATP assay kit. The expression levels of GLUT1, GLUT3, pDrp1, Drp1 and Mfn2 were detected by Western blot, and the expression levels of GLUT1 and GLUT3 were further examined by immunohistochemistry.Results: Propofol damaged mitochondria, and decreased ATP content and GLUT3 and pDrp1 protein expression. However, our results suggested that hypoxic preconditioning could attenuate propofol neurotoxicity by reducing mitochondrial damage and increasing ATP content and pDrp1, GLUT1 and GLUT3 protein expression.Conclusion: Hypoxic preconditioning reduced propofol-induced damage in the hippocampus of neonatal rats by attenuating the increase in mitochondrial division and decrease in GLUT3 expression.

Mitoquinone attenuates blood-brain barrier disruption through Nrf2/PHB2/OPA1 pathway after subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE: Mitochondrial dysfunction is involved in the mechanism of early brain injury (EBI) following subarachnoid hemorrhage (SAH). Blood-brain barrier disruption is a devastating outcome in the early stage of SAH. In this study, we aimed to investigate the role of a mitochondria-related drug Mitoquinone (MitoQ) in blood-brain barrier disruption after SAH in rats. METHODS: A total of 181 male Sprague-Dawley SAH rats with the endovascular perforation model were utilized. Intraperitoneal MitoQ was given 1 h (h) post-SAH. Cerebroventricular ML385, an inhibitor of NF-E2-related factor 2 (Nrf2) and Small interfering ribonucleic acid (siRNA) for Prohibitin 2 (PHB2) were injected respectively 24 h and 48 h before SAH. Neurological function evaluation was performed before sacrifice. SAH grade was measured during the sacrifice of each animal. Brain water content was performed at 24 h. Co-immunoprecipitation was used to demonstrate the relationship of proteins Nrf2 and PHB2. Mitochondrial and cytoplasmic fractions were gathered using mitochondria isolation kits. Pathway related proteins were investigated with Western blot and immunofluorescence staining. Transmission electron microscopy was performed for mitochondrial morphology. RESULTS: Expression of Nrf2 levels peaked at the 3 h time point following SAH and then decreased to normal levels at 24 h, while PHB2 and Optic Atrophy 1 (OPA1) decreased at 24 h and 72 h after SAH compared with the Sham group. MitoQ treatment attenuated neurological deficits and brain edema, thereby resulting in a decreased expression of Albumin, while an increase of Nrf2, PHB2, OPA1 and Claudin-5 proteins compared with SAH + vehicle group. With co-immunoprecipitation, Nrf2 and PHB2 were further demonstrated to show their interaction. And MitoQ administration lead to more binding of the two proteins. ML385 abolished the effects of MitoQ on neurobehavior and protein levels post-SAH. Similarly, PHB2 siRNA reversed the neuroprotection of MitoQ administration with the decreased expression of PHB2 and OPA1 after SAH. Further, MitoQ treatment improved mitochondrial morphology after SAH with an increase of PHB2 and OPA1 in mitochondrial extraction. CONCLUSIONS: MitoQ attenuates blood-brain barrier disruption via Nrf2/PHB2/OPA1 pathway after SAH in rats. MitoQ may serve as a potential therapeutic strategy for SAH patients.

Baicalein Targets GTPase-Mediated Autophagy to Eliminate Liver Tumor-Initiating Stem Cell-Like Cells Resistant to mTORC1 Inhibition.

Drug resistance is a major problem in the treatment of liver cancer. Mammalian Target of Rapamycin 1 (mTORC1) inhibitors have been tested for the treatment of liver cancer based on hyperactive mTOR in this malignancy. However, their clinical trials showed poor outcome, most likely due to their ability to upregulate CD133 and promote chemoresistance. The CD133+ tumor-initiating stem cell-like cells (TICs) isolated from mouse and human liver tumors are chemoresistant, and identification of an approach to abrogate this resistance is desired. In search of a compound that rescinds resistance of TICs to mTORC1 inhibition and improves chemotherapy, we identified baicalein (BC), which selectively chemosensitizes TICs and the human hepatocellular carcinoma (HCC) cell line Huh7 cells but not mouse and human primary hepatocytes. Nanobead pull-down and mass-spectrometric analysis, biochemical binding assay, and three-dimensional computational modeling studies reveal BC's ability to competitively inhibit guanosine triphosphate binding of SAR1B guanosine triphosphatase, which is essential for autophagy. Indeed, BC suppresses autophagy induced by an mTORC1 inhibitor and synergizes cell death caused by mTORC1 inhibition in TIC and Huh7 spheroid formation and in the patient-derived xenograft model of HCC. The BC-induced chemosensitization is rescued by SAR1B expression and phenocopied by SAR1B knockdown in cancer cells treated with a mTORC1 inhibitor. Conclusion: These results identify SAR1B as a target in liver TICs and HCC cells resistant to mTORC1 inhibition.

Aqua-soluble DDQ reduces the levels of Drp1 and Aß and inhibits abnormal interactions between Aß and Drp1 and protects Alzheimer's disease neurons from Aß- and Drp1-induced mitochondrial and synaptic toxicities.

The purpose of our study was to develop a therapeutic target that can reduce Aß and Drp1 levels, and also can inhibit abnormal interactions between Aß and Drp1 in AD neurons. To achieve this objective, we designed various compounds and their 3-dimensional molecular structures were introduced into Aß and Drp1 complex and identified their inhibitory properties against Aß-Drp1 interaction. Among all, DDQ was selected for further investigation because of 1) its best docking score and 2) its binding capability at interacting sites of Drp1 and Aß complex. We synthesized DDQ using retro-synthesis and analyzed its structure spectrally. Using biochemical, molecular biology, immunostaining and transmission electron microscopy (TEM) methods, we studied DDQ's beneficial effects in AD neurons. We measured the levels of Aß and Drp1, Aß and Drp1 interaction, mRNA and protein levels of mitochondrial dynamics, biogenesis and synaptic genes, mitochondrial function and cell viability and mitochondrial number in DDQ-treated and untreated AD neurons. Our qRT-PCR and immunoblotting analysis revealed that reduced levels of mitochondrial fission and increased fusion, biogenesis and synaptic genes in DDQ-treated AD neurons. Our immunoblotting and immunostaining analyses revealed that Aß and Drp1 levels were reduced in DDQ-treated AD neurons. Interaction between Aß and Drp1 is reduced in DDQ-treated AD neurons. Aß42 levels were significantly reduced in DDQ-treated mutant APPSwe/Ind cells. Mitochondrial number is significantly reduced and mitochondrial length is significantly increased. Mitochondrial function and cell viability were maintained in AD neurons treated with DDQ. These observations indicate that DDQ reduces excessive mitochondrial fragmentation, enhances fusion, biogenesis and synaptic activity and reduces Aß42 levels and protects AD neurons against Aß-induced mitochondrial and synaptic toxicities.

Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

Arabidopsis thaliana mitochondrial EF-G1 functions in two different translation steps.

Translation elongation factor G (EF-G) in bacteria catalyses the translocation of transfer RNA on ribosomes in the elongation step as well as dissociation of post-termination state ribosomes into two subunits in the recycling step. In contrast, the dual functions of EF-G are exclusively divided into two different paralogues in human mitochondria, named EF-G1mt for translocation and EF-G2mt for ribosomal dissociation. Many of the two eukaryotic EF-G paralogues are phylogenetically associated with EF-G1mt and EF-G2mt groups. However, plant paralogues are associated with EF-G1mt and plastid EF-G, not with EF-G2mt. In this study, we phylogenetically and biochemically characterized Arabidopsis thaliana EF-G1mt (AtEF-G1mt) to clarify the factor responsible for the dissociation of ribosomes in plant mitochondria. We showed that eukaryotic EF-G1mts form one monophyletic group separated from bacterial EF-G and are classified into five sister groups. AtEF-G1mt is classified into a different group from its human counterpart. We also demonstrated that AtEF-G1mt catalyses both translocation and ribosomal dissociation, unlike in humans. Meanwhile, AtEF-G1mt is resistant to fusidic acid, an inhibitor of bacterial EF-G. Here, we propose that the functional division is not necessarily conserved among mitochondriate eukaryotes and also that EF-G1mt in organisms lacking EF-G2mt functions in two steps, similar to conventional bacterial EF-G.

Narciclasine as well as other Amaryllidaceae isocarbostyrils are promising GTP-ase targeting agents against brain cancers.

The anticancer activity of Amaryllidaceae isocarbostyrils is well documented. At pharmacological concentrations, that is, approximately 1 µM in vitro and approximately 10 mg/kg in vivo, narciclasine displays marked proapoptotic and cytotoxic activity, as does pancratistatin, and significant in vivo anticancer effects in various experimental models, but it is also associated with severe toxic side effects. At physiological doses, that is, approximately 50 nM in vitro and approximately 1 mg/kg in vivo, narciclasine is not cytotoxic but cytostatic and displays marked anticancer activity in vivo in experimental models of brain cancer (including gliomas and brain metastases), but it is not associated with toxic side effects. The cytostatic activity of narciclasine involves the impairment of actin cytoskeleton organization by targeting GTPases, including RhoA and the elongation factor eEF1A. We have demonstrated that chronic treatments of narciclasine (1 mg/kg) significantly increased the survival of immunodeficient mice orthotopically xenografted with highly invasive human glioblastomas and apoptosis-resistant brain metastases, including melanoma- and non-small-cell-lung cancer- (NSCLC) related brain metastases. Thus, narciclasine is a potentially promising agent for the treatment of primary brain cancers and various brain metastases. To date, efforts to develop synthetic analogs with anticancer properties superior to those of narciclasine have failed; thus, research efforts are now focused on narciclasine prodrugs.

3-Nitropropionic acid induces autophagy by forming mitochondrial permeability transition pores rather than activating the mitochondrial fission pathway.

BACKGROUND AND PURPOSE: Huntington's disease is a neurodegenerative process associated with mitochondrial alterations. Inhibitors of the electron-transport channel complex II, such as 3-nitropropionic acid (3NP), are used to study the molecular and cellular pathways involved in this disease. We studied the effect of 3NP on mitochondrial morphology and its involvement in macrophagy. EXPERIMENTAL APPROACH: Pharmacological and biochemical methods were used to characterize the effects of 3NP on autophagy and mitochondrial morphology. SH-SY5Y cells were transfected with GFP-LC3, GFP-Drp1 or GFP-Bax to ascertain their role and intracellular localization after 3NP treatment using confocal microscopy. KEY RESULTS: Untreated SH-SY5Y cells presented a long, tubular and filamentous net of mitochondria. After 3NP (5 mM) treatment, mitochondria became shorter and rounder. 3NP induced formation of mitochondrial permeability transition pores, both in cell cultures and in isolated liver mitochondria, and this process was inhibited by cyclosporin A. Participation of the mitochondrial fission pathway was excluded because 3NP did not induce translocation of the dynamin-related protein 1 (Drp1) to the mitochondria. The Drp1 inhibitor Mdivi-1 did not affect the observed changes in mitochondrial morphology. Finally, scavengers of reactive oxygen species failed to prevent mitochondrial alterations, while cyclosporin A, but not Mdivi-1, prevented the generation of ROS. CONCLUSIONS AND IMPLICATIONS: There was a direct correlation between formation of mitochondrial permeability transition pores and autophagy induced by 3NP treatment. Activation of autophagy preceded the apoptotic process and was mediated, at least partly, by formation of reactive oxygen species and mitochondrial permeability transition pores.

Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1.

Mutations in the parkin gene cause early-onset, autosomal recessive Parkinson's disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non-degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly-ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.

Comparison of NMR structural and dynamics features of the urea and guanidine-denatured states of GED.

Denatured states of proteins, the starting points as well as the intermediates of folding in vivo, play important roles in biological function. In this context, we describe here urea unfolding and characterization of the denatured state of GTPase effector domain (GED) of dynamin created by 9.7 M urea. These are compared with similar data for guanidine induced denaturation reported earlier. The unfolding characteristics in the two cases, as measured by the optical probes, are significantly different, urea unfolding proceeding via an intermediate. The structural and motional characteristics, determined by NMR, of the two denatured states are also strikingly different. The urea-denatured state shows a combination of alpha- and beta-preferences in contrast to the entirely beta-preferences in the guanidine-denatured state. Higher (15)N transverse relaxation rates suggest higher folding propensities in the urea-denatured state. The implications of these to GED folding are discussed.

Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition.

The statins, hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower serum cholesterol, exhibit myriad clinical benefits, including enhanced vascular integrity. One potential mechanism underlying increased endothelial cell (EC) barrier function is inhibition of geranylgeranylation, a covalent modification enabling translocation of the small GTPases Rho and Rac to the cell membrane. While RhoA inhibition attenuates actin stress fiber formation and promotes EC barrier function, Rac1 inhibition at the cell membrane potentially prevents activation of NADPH oxidase and subsequent generation of superoxides known to induce barrier disruption. We examined the relative regulatory effects of simvastatin on RhoA, Rac1, and NADPH oxidase activities in the context of human pulmonary artery EC barrier protection. Confluent EC treated with simvastatin demonstrated significantly decreased thrombin-induced FITC-dextran permeability, a reflection of vascular integrity, which was linked temporally to simvastatin-mediated actin cytoskeletal rearrangement. Compared with Rho inhibition alone (Y-27632), simvastatin afforded additional protection against thrombin-mediated barrier dysfunction and attenuated LPS-induced EC permeability and superoxide generation. Statin-mediated inhibition of both Rac translocation to the cell membrane and superoxide production were attenuated by geranylgeranyl pyrophosphate (GGPP), indicating that these effects are due to geranylgeranylation inhibition. Finally, thrombin-induced EC permeability was modestly attenuated by reduced Rac1 expression (small interfering RNA), whereas these effects were made more pronounced by simvastatin pretreatment. Together, these data suggest EC barrier protection by simvastatin is due to dual inhibitory effects on RhoA and Rac1 as well as the attenuation of superoxide generation by EC NADPH oxidase and contribute to the molecular mechanistic understanding of the modulation of EC barrier properties by simvastatin.

Anti-cancer therapy: targeting the mevalonate pathway.

The mevalonate pathway has become an important target for anti-cancer therapy. Manipulation of this pathway results in alteration of malignant cell growth and survival in cell culture and animal models, with promising potential for application in human cancers. Mevalonate is synthesized from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). Mevalonate is further metabolized to farnesyl pyrophosphate (FPP), which is the precursor for sterols. In addition, the farnesyl moiety from FPP is utilized for post-translational modification of proteins including small GTPases, such as Ras and Ras related proteins, which play a role in malignant transformation of cells. FPP is a precursor for geranylgeranyl pyrophosphate (GGPP), which is similarly involved in post-translational modification of proteins. There has been intense interest in manipulating the pathway through HMG-CoA reductase inhibition. More recently, the focus has been on manipulating the pathway by post-translational modification of key regulatory proteins through farnesyl prenyl transferase (FPTase) or geranylgeranyl prenyl transferase (GGPTase) inhibition. This review focuses on the mevalonate pathway and the application of rational drug therapies to manipulate this pathway. Included in the review are a summary of agents demonstrating success in preclinical investigations such as; farnesyl transferase inhibitors, geranylgeranyl transferase inhibitors, dual inhibitors, statins, bisphosphonates, histone deacetylase inhibitors and other compounds. While these agents have shown preclinical success, translation to success in clinical trials has been more difficult. These clinical trials are reviewed along with evaluation of some of the potential problems with these agents in their clinical application.

Signal transduction through Ras-GTPase and Ca2+/ calmodulin-dependent protein kinase II contributes to development of diabetes-induced renal vascular dysfunction.

This study examined the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Ras-GTPase in the development of abnormal reactivity to vasoactive agents in the renal artery of diabetic rats. The vasoconstrictor response induced by norepinephrine (NE), endothelin-1 (ET-1) or angiotensin II (Ang II) was significantly increased whereas vasodilator response to carbachol, histamine or sodium nitroprusside (SNP) was not altered in the renal artery segments of the streptozotocin (STZ)-diabetic rats. Chronic intraperitoneal administration of KN-93 (5 mg/kg/ alt diem), an inhibitor of CaMKII or FPTIII (1.5 mg/kg/ alt diem), an inhibitor of Ras-GTPase, produced significant normalization of the altered agonist-induced vasoconstrictor responses without affecting blood glucose levels. All the inhibitors were administered for four weeks starting from day one of diabetes induction. Inhibition of Ras-GTPase or CaMKII did not affect the agonist-induced vasoconstrictor and vasodilator responses in the non-diabetic control animals. These data suggest that inhibition of signal transduction involving CaMKII and Ras-GTPase can prevent development of diabetes-induced abnormal vascular reactivity in the renal artery.

Brassinosteroids stimulate plant tropisms through modulation of polar auxin transport in Brassica and Arabidopsis.

Brassinosteroids (BRs) are important plant growth regulators in multiple developmental processes. Previous studies have indicated that BR treatment enhanced auxin-related responses, but the underlying mechanisms remain unknown. Using (14)C-labeled indole-3-acetic acid and Arabidopsis thaliana plants harboring an auxin-responsive reporter construct, we show that the BR brassinolide (BL) stimulates polar auxin transport capacities and modifies the distribution of endogenous auxin. In plants treated with BL or defective in BR biosynthesis or signaling, the transcription of PIN genes, which facilitate functional auxin transport in plants, was differentially regulated. In addition, BL enhanced plant tropistic responses by promoting the accumulation of the PIN2 protein from the root tip to the elongation zone and stimulating the expression and dispersed localization of ROP2 during tropistic responses. Constitutive overexpression of ROP2 results in enhanced polar accumulation of PIN2 protein in the root elongation region and increased gravitropism, which is significantly affected by latrunculin B, an inhibitor of F-actin assembly. The ROP2 dominant negative mutants (35S-ROP2-DA/DN) show delayed tropistic responses, and this delay cannot be reversed by BL addition, strongly supporting the idea that ROP2 modulates the functional localization of PIN2 through regulation of the assembly/reassembly of F-actins, thereby mediating the BR effects on polar auxin transport and tropistic responses.

A retromerlike complex is a novel Rab7 effector that is involved in the transport of the virulence factor cysteine protease in the enteric protozoan parasite Entamoeba histolytica.

Vesicular trafficking plays an important role in a virulence mechanism of the enteric protozoan parasite Entamoeba histolytica as secreted and lysosomal cysteine protease (CP) contributes to both cytolysis of tissues and degradation of internalized host cells. Despite the primary importance of intracellular sorting in pathogenesis, the molecular mechanism of CP trafficking remains largely unknown. In this report we demonstrate that transport of CP is regulated through a specific interaction of Rab7A small GTPase (EhRab7A) with the retromerlike complex. The amoebic retromerlike complex composed of Vps26, Vps29, and Vps35 was identified as EhRab7A-binding proteins. The amoebic retromerlike complex specifically bound to GTP-EhRab7A, but not GDP-EhRab7A through the direct binding via the carboxy terminus of EhVps26. In erythrophagocytosis the retromerlike complex was recruited to prephagosomal vacuoles, the unique preparatory vacuole of digestive enzymes, and later to phagosomes. This dynamism was indistinguishable from that of EhRab7A, and consistent with the premise that the retromerlike complex is involved in the retrograde transport of putative hydrolase receptor(s) from preparatory vacuoles and phagosomes to the Golgi apparatus. EhRab7A overexpression caused enlargement of lysosomes and decrease of the cellular CP activity. The reduced CP activity was restored by the coexpression of EhVps26, implying that the EhRab7A-mediated transport of CP to phagosomes is regulated by the retromerlike complex.

Preclinical evidence for the effect of bisphosphonates and cytotoxic drugs on tumor cell invasion.

Bisphosphonates (BPs) are stable pyrophosphate analogs currently used in the treatment of patients with metastatic bone disease, known to affect bone resorption by reducing osteoclast activity. Use of these drugs in adjuvant therapy is currently under investigation following reports of an effect of BPs on tumor cell apoptosis in preclinical models. Recent evidence has suggested that BPs might also affect tumor cell invasion in vitro, and the component processes of adhesion, migration and degradation, through mechanisms including inhibition of prenylation of intracellular small GTPases such as Ras and Rho. The effects potentially may be enhanced through co-administration with chemotherapy agents, as both synergistic and additive effects have been described in vitro. This review discusses the preclinical evidence for the potential use of BPs and cytotoxic drugs for inhibiting tumor cell invasion, a key process in cancer progression.

The small GTPase Rac is involved in clustering of hippocampal neurons and fasciculation of their neurites.

In hippocampal neurons cultured from brains of newborn rats, the glutamate receptor agonist N-methyl-D-aspartate induced the clustering of neuronal perikarya and the fasciculation of neurites. In addition, N-methyl-D-aspartate activated the small GTPase Rac1. Other stimuli of Rac activity, such as the Rho kinase inhibitors Y-27632, H-1152, and H89, as well as the cytotoxic necrotizing factor-1 from Escherichia coli, also caused neuronal clustering and neurite bundling. In neurons transiently transfected with dominant negative Rac1N17 neither N-methyl-D-aspartate nor Y-27632 induced clustering and fasciculation. In addition, the PI3-kinase inhibitors wortmannin and LY-294002 prevented these effects, as did a dominant negative form of p110PI3-Kgamma. Time-lapse microscopy showed that lethal toxin from Clostridium sordellii, which inhibits Rac, and wortmannin blocked the neuronal migration induced by Y-27632. In contrast, only lethal toxin reversed the clustering and fasciculation induced by pre-treatment with Y-27632. This effect of the toxin may be due to inactivation of Ras, since FTI-277, which prevents the farnesylation of Ras and thereby inactivates the GTPase, also dissolved the preformed clusters. We suggest that active Rac and a PI3-kinase synergistically induce neuronal migration, whereas a Ras isoform is responsible for the lasting attachment of neurons necessary for clustering and neurite fasciculation.