Mitochondrial dysfunction and Purkinje cell loss in autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a childhood-onset neurological disease resulting from mutations in the SACS gene encoding sacsin, a 4,579-aa protein of unknown function. Originally identified as a founder disease in Québec, ARSACS is now recognized worldwide. Prominent features include pyramidal spasticity and cerebellar ataxia, but the underlying pathology and pathophysiological mechanisms are unknown. We have generated an animal model for ARSACS, sacsin knockout mice, that display age-dependent neurodegeneration of cerebellar Purkinje cells. To explore the pathophysiological basis for this observation, we examined the cell biological properties of sacsin. We show that sacsin localizes to mitochondria in non-neuronal cells and primary neurons and that it interacts with dynamin-related protein 1, which participates in mitochondrial fission. Fibroblasts from ARSACS patients show a hyperfused mitochondrial network, consistent with defects in mitochondrial fission. Sacsin knockdown leads to an overly interconnected and functionally impaired mitochondrial network, and mitochondria accumulate in the soma and proximal dendrites of sacsin knockdown neurons. Disruption of mitochondrial transport into dendrites has been shown to lead to abnormal dendritic morphology, and we observe striking alterations in the organization of dendritic fields in the cerebellum of knockout mice that precedes Purkinje cell death. Our data identifies mitochondrial dysfunction/mislocalization as the likely cellular basis for ARSACS and indicates a role for sacsin in regulation of mitochondrial dynamics.

High NRBP1 expression in prostate cancer is linked with poor clinical outcomes and increased cancer cell growth

We recently established the rationale that NRBP1 (nuclear receptor binding protein 1) has a potential growth-promoting role in cell biology. NRBP1 interacts directly with TSC-22, a potential tumor suppressor gene that is differently expressed in prostate cancer. Consequently, we analyzed the role of NRBP1 expression in prostate cancer cell lines and its expression on prostate cancer tissue microarrays (TMA).

Measuring Correlation Between HIV Sequence Variation and Disease Stage of HIV-Infected Individuals

With a virus such as Human Immunodeficiency Virus (HIV) that has infected millions of people worldwide, and with many unaware that they are infected, it becomes vital to understand how the virus works and how it functions at the molecular level. Because there currently is no vaccine and no way to eradicate the virus from an infected person, any information about how the virus interacts with its host greatly increases the chances of understanding how HIV works and brings scientists one step closer to being able to combat such a destructive virus. Thousands of HIV viruses have been sequenced and are available in many online databases for public use. Attributes that are linked to each sequence include the viral load within the host and how sick the patient is currently. Being able to predict the stage of infection for someone is a valuable resource, as it could potentially aid in treatment options and proper medication use. Our approach of analyzing region-specific amino acid composition for select genes has been able to predict patient disease state up to an accuracy of 85.4%. Moreover, we output a set of classification rules based on the sequence that may prove useful for diagnosing the expected clinical outcome of the infected patient.

The Impact of Foreign Direct Investment on Economic Growth

Over the past four decades, the number of democracies in the world has increased exponentially. This project considers how democracy and FDI affect economic growth as well as whether the impact of FDI depends on the level of democracy in a country. Thus, I explore two major research questions: 1) Whether increased FDI speeds up economic growth, controlling for political regime type, urbanization and other developmental indicators; and 2) Whether an increase in political freedom helps or hinders economic growth, and specifically whether the impact of FDI varies depending on the political regime in the recipient country. To examine these questions, this paper used data from 150 countries over a period between 1980 and 2010 and utilized several models, testing variables such as institutions, agglomerations, urbanization, FDI and type of political regime, among others, for their impact on economic growth. I found that FDI does have a positive impact on economic growth, and that this impact is often magnified when it interacts with other relevant factors. I also found that, after controlling for other variables, FDI inflows do not have a different impact on economic growth in autocracies than they do in democracies. This may be partially explained by autocratic outliers such as China and the OPEC states, which have recently experienced rapid export-led growth. This suggests that factors such as education could have a greater impact on a country¿s economic growth than does its political system.

Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes

During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α). Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα) phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB). The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis). Our data suggest that remifentanil increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration.

Repetitive TMS over the human oculomotor cortex: comparison of 1-Hz and theta burst stimulation

The aim of the study was to compare the effect duration of two different protocols of repetitive transcranial magnetic stimulation (rTMS) on saccade triggering. In four experiments, two regions (right frontal eye field (FEF) and vertex) were stimulated using a 1-Hz and a theta burst protocol (three 30Hz pulses repeated at intervals of 100ms). The same number of TMS pulses (600 pulses) was applied with stimulation strength of 80% of the resting motor threshold for hand muscles. Following stimulation the subjects repeatedly performed an oculomotor task using a modified overlap paradigm, and saccade latencies were measured over a period of 60min. The results show that both 1-Hz and theta burst stimulation had inhibitory effects on saccade triggering when applied over the FEF, but not over the vertex. One-hertz rTMS significantly increased saccade latencies over a period of about 8min. After theta burst rTMS, this effect lasted up to 30min. Furthermore, the decay of rTMS effects was protocol-specific: After 1-Hz stimulation, saccade latencies returned to a baseline level much faster than after theta burst stimulation. We speculate that these time course differences represent distinct physiological mechanisms of how TMS interacts with brain function.

Expression of FGFRL1, a novel fibroblast growth factor receptor, during embryonic development

FGFRL1 is a novel member of the fibroblast growth factor receptor (FGFR) family. To investigate its expression during mammalian embryonic development, we have used the mouse system. Expression of Fgfrl1 is very low in mouse embryos of day 6 but steadily increases until birth. As demonstrated by in situ hybridization of 16-day-old embryos, the Fgfrl1 mRNA occurs in cartilaginous structures such as the primordia of bones and the permanent cartilage of the trachea, the ribs and the nose. In addition, some muscle types, including the muscles of the tongue and the diaphragm, express Fgfrl1 at relatively high level. In contrast, the heart and the skeletal muscles of the limbs, as well as many other organs (brain, lung, liver, kidney, gut) express Fgfrl1 only at basal level. It is conceivable that Fgfrl1 interacts with other Fgfrs, which are expressed in cartilage and muscle, to modulate FGF signaling.

Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase

Eph receptor tyrosine kinases are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Caveolae are flask-shaped invaginations of the cell membrane; their major structural protein, caveolin-1, has been shown to regulate signaling molecules localized in these micro-domains. The interaction of caveolin-1 with several of these proteins is mediated by the binding of its scaffolding domain to a region containing hydrophobic amino acids within these proteins. The presence of such a motif within the EphB1 kinase domain prompted us to investigate the caveolar localization and regulation of EphB1 by caveolin-1. We report that EphB1 receptors are localized in caveolae, and directly interact with caveolin-1 upon ligand stimulation. This interaction, as well as EphB1-mediated activation of extracellular-signal-regulated kinase (ERK), was abrogated by overexpression of a caveolin-1 mutant lacking a functional scaffolding domain. Interaction between Ephs and caveolin-1 is not restricted to the B-subclass of receptors, since we show that EphA2 also interacts with caveolin-1. Furthermore, we demonstrate that the caveolin-binding motif within the kinase domain of EphB1 is primordial for its correct membrane targeting. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of EphB1.

Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl-2 and Bcl-xL genes

OBJECTIVE: To determine whether a specifically designed bispecific (Bcl-2/Bcl-xL) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl-2 and Bcl-xL are both anti-apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer. MATERIALS AND METHODS: Inhibition of Bcl-2 and Bcl-xL expression by the bispecific ASO was evaluated using real-time reverse transcription-polymerase chain reaction and Western blotting, while growth inhibition and induction of apoptosis were analysed by a crystal violet assay, flow cytometry and Western blotting of apoptosis-relevant proteins. The effect of combined treatment with bispecific ASO and chemotherapy or small-interference RNA (siRNA) targeting the clusterin gene was also investigated. RESULTS: Bispecific ASO reduced Bcl-2 and Bcl-xL expression in LNCaP cells in a dose-dependent manner. There was cell growth inhibition, increases in the sub-G0-G1 fraction, and cleavage of caspase-3 and poly(ADP-Ribose) polymerase proteins in LNCaP cells after bispecific ASO treatment. Interestingly, Bcl-2/Bcl-xL bispecific ASO treatment also resulted in the down-regulation of Mcl-1 and up-regulation of Bax. The sensitivity of LNCaP cells to mitoxantrone, docetaxel or paclitaxel was significantly increased, reducing the 50% inhibitory concentration by 45%, 80% or 90%, respectively. Furthermore, the apoptotic induction by Bcl-2/Bcl-xL bispecific ASO was synergistically enhanced by siRNA-mediated inhibition of clusterin, a cytoprotective chaperone that interacts with and inhibits activated Bax. CONCLUSIONS: These findings support the concept of the targeted suppression of Bcl-2 anti-apoptotic family members using multitarget inhibition strategies for prostate cancer, through the effective induction of apoptosis.

The role of stress hormones in the relationship between resting blood pressure and coagulation activity

BACKGROUND: Systemic hypertension confers a hypercoagulable state. We hypothesized that resting mean blood pressure (MBP) interacts with stress hormones in predicting coagulation activity at rest and with acute mental stress. METHODS: We measured plasma clotting factor VII activity (FVII:C), FVIII:C, fibrinogen, D-dimer, epinephrine and norepinephrine, and saliva cortisol in 42 otherwise healthy normotensive and hypertensive medication-free men (mean age 43 +/- 14 years) at rest, immediately after stress, and twice during 60 min of recovery from stress. RESULTS: At rest, the MBP-by-epinephrine interaction predicted FVII:C (beta = -0.33, P < 0.04) and D-dimer (beta = 0.26, P < 0.05), and the MBP-by-cortisol interaction predicted D-dimer (beta = 0.43, P = 0.001), all independent of age and body mass index (BMI). Resting norepinephrine predicted fibrinogen (beta = 0.42, P < 0.01) and D-dimer (beta = 0.37, P < 0.03), both independent of MBP. MBP predicted FVIII:C change from rest to immediately post-stress independent of epinephrine (beta = -0.37, P < 0.03) and norepinephrine (beta = -0.38, P < 0.02). Cortisol change predicted FVIII:C change (beta = -0.30, P < 0.05) independent of age, BMI and MBP. Integrated norepinephrine change from rest to recovery (area under the curve, AUC) predicted D-dimer AUC (beta = 0.34, P = 0.04) independent of MBP. The MBP-by-epinephrine AUC interaction predicted FVII:C AUC (beta = 0.28) and fibrinogen AUC (beta = -0.30), and the MBP-by-norepinephrine AUC interaction predicted FVIII:C AUC (beta = -0.28), all with borderline significance (Ps < 0.09) and independent of age and BMI. CONCLUSIONS: MBP significantly altered the association between stress hormones and coagulation activity at rest and, with borderline significance, across the entire stress and recovery interval. Independent of MBP, catecholamines were associated with procoagulant effects and cortisol reactivity dampened the acute procoagulant stress response.

F90927: a new member in the class of cardioactive steroids

F90927 is a newly developed cardioactive drug with a steroid-like structure. It acts directly and agonistically on the cardiac L-type Ca2+ channel by shifting its voltage-dependent activation toward more negative potentials. This leads to an increased influx of Ca2+ and, therefore, to a stronger contraction; however, no arrhythmias occur. Calcium current stimulation can already be observed at nanomolar concentrations, but higher concentrations of F90927 elevate intracellular Ca2+ concentration, causing a reduction of the myocardial compliance and an increased diastolic blood pressure. Vessels also react to F90927 and contract in its presence. Binding of F90927 with the L-type Ca2+ channel presumably occurs in the vicinity of the transmembrane domains III and IV of the alpha1 subunit. F90927 exhibits no use dependence and interacts with Ca2+ channel inhibitors of all three known classes of channel modulators (dihydropyridines, phenylalkylamines, and benzothiazepines), suggesting that it is a member of a new class of Ca2+ channel modulators. Due to its adverse effects on blood pressure and vessel contraction, F90927 is not an ideal drug candidate. It has, however, some unique properties, which makes it a promising tool to study the function of the L-type Ca2+ channel.

Messenger RNA levels and binding sites of muscarinic acetylcholine receptors in gastrointestinal muscle layers from healthy dairy cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M(1)to M(5) in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [(3)H]-QNB (1-quinuclidinyl-[phenyl-4-(3)H]-benzilate) binding by M antagonists [atropine (M(1 - 5)), pirenzepine (M(1)), methoctramine (M(2)), 4-DAMP (M(3)), and tropicamide (M(4))] was used to identify receptors at the functional level. Maximal binding (B(max)) was determined through saturation binding with atropine as a competitor. The mRNA levels of M(1), M(2), M(3), and M(5) represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M(4) was undetectable. The mRNA levels of M(2) and of M(3) in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [(3)H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [(3)H]-QNB binding. The [(3)H]-QNB binding was dose-dependent and saturable. B(max) in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. B(max) and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.

Regulation of Id1 expression by SRC: implications for targeting of the bone morphogenetic protein pathway in cancer

Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)-induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1.

Angiotensinergic neurons in sympathetic coeliac ganglia innervating rat and human mesenteric resistance blood vessels

In contrast to the current belief that angiotensin II (Ang II) interacts with the sympathetic nervous system only as a circulating hormone, we document here the existence of endogenous Ang II in the neurons of rat and human sympathetic coeliac ganglia and their angiotensinergic innervation with mesenteric resistance blood vessels. Angiotensinogen - and angiotensin converting enzyme-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia, while renin mRNA was untraceable. Cathepsin D, a protease responsible for cleavage beneath other substrates also angiotensinogen to angiotensin I, was successfully detected in rat coeliac ganglia indicating the possibility of existence of alternative pathways. Angiotensinogen mRNA was also detected by in situ hybridization in the cytoplasm of neurons of rat coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia as well as with mesenteric resistance blood vessels. By using confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally with the mesenteric resistance blood vessels.

Posttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3' untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation.