The Effect Of Celastrol, A Triterpene With Antitumorigenic Activity, On Conformational And Functional Aspects Of The Human 90kda Heat Shock Protein Hsp90α, A Chaperone Implicated In The Stabilization Of The Tumor Phenotype.

Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive. In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol. Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α. To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α.

Changes In Liver Cell Dna Methylation Status In Diabetic Mice Affect Its Ft-ir Characteristics.

Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1) and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

Tadalafil-induced Improvement In Left Ventricular Diastolic Function In Resistant Hypertension.

Left ventricular hypertrophy and diastolic dysfunction (LVDD) remain highly frequent markers of cardiac damage and risk of progression to symptomatic heart failure, especially in resistant hypertension (RHTN). We have previously demonstrated that administration of sildenafil in hypertensive rats improves LVDD, restoring phosphodiesterase type 5 (PDE-5) inhibition in cardiac myocytes. We hypothesized that the long-acting PDE-5 inhibitor tadalafil may be clinically useful in improving LVDD in RHTN independently of blood pressure (BP) reduction. A single blinded, placebo-controlled, crossover study enrolled 19 patients with both RHTN and LVDD. Firstly, subjects received tadalafil (20 mg) for 14 days and after a 2-week washout period, they received placebo orally for 14 days. Patients were evaluated by office BP and ambulatory BP monitoring (ABPM), endothelial function (FMD), echocardiography, plasma brain natriuretic peptide (BNP-32), cyclic guanosine monophosphate (cGMP) and nitrite levels. No significant differences were detected in BP measurements. Remarkably, at least four echocardiographic parameters related with diastolic function improved accompanied by decrease in BNP-32 in tadalafil use. Although increasing cGMP, tadalafil did not change endothelial function or nitrites. There were no changes in those parameters after placebo. The current findings suggest that tadalafil improves LV relaxation through direct effects PDE-5-mediated in the cardiomyocytes with potential benefit as an adjunct to treat symptomatic subjects with LVDD such as RHTN patients.

Enhanced Glucose-induced Intracellular Signaling Promotes Insulin Hypersecretion: Pancreatic Beta-cell Functional Adaptations In A Model Of Genetic Obesity And Prediabetes.

Obesity is associated with insulin resistance and is known to be a risk factor for type-2 diabetes. In obese individuals, pancreatic beta-cells try to compensate for the increased insulin demand in order to maintain euglycemia. Most studies have reported that this adaptation is due to morphological changes. However, the involvement of beta-cell functional adaptations in this process needs to be clarified. For this purpose, we evaluated different key steps in the glucose-stimulated insulin secretion (GSIS) in intact islets from female ob/ob obese mice and lean controls. Obese mice showed increased body weight, insulin resistance, hyperinsulinemia, glucose intolerance and fed hyperglycemia. Islets from ob/ob mice exhibited increased glucose-induced mitochondrial activity, reflected by enhanced NAD(P)H production and mitochondrial membrane potential hyperpolarization. Perforated patch-clamp examination of beta-cells within intact islets revealed several alterations in the electrical activity such as increased firing frequency and higher sensitivity to low glucose concentrations. A higher intracellular Ca(2+) mobilization in response to glucose was also found in ob/ob islets. Additionally, they displayed a change in the oscillatory pattern and Ca(2+) signals at low glucose levels. Capacitance experiments in intact islets revealed increased exocytosis in individual ob/ob beta-cells. All these up-regulated processes led to increased GSIS. In contrast, we found a lack of beta-cell Ca(2+) signal coupling, which could be a manifestation of early defects that lead to beta-cell malfunction in the progression to diabetes. These findings indicate that beta-cell functional adaptations are an important process in the compensatory response to obesity.

The Psychedelic State Induced By Ayahuasca Modulates The Activity And Connectivity Of The Default Mode Network.

The experiences induced by psychedelics share a wide variety of subjective features, related to the complex changes in perception and cognition induced by this class of drugs. A remarkable increase in introspection is at the core of these altered states of consciousness. Self-oriented mental activity has been consistently linked to the Default Mode Network (DMN), a set of brain regions more active during rest than during the execution of a goal-directed task. Here we used fMRI technique to inspect the DMN during the psychedelic state induced by Ayahuasca in ten experienced subjects. Ayahuasca is a potion traditionally used by Amazonian Amerindians composed by a mixture of compounds that increase monoaminergic transmission. In particular, we examined whether Ayahuasca changes the activity and connectivity of the DMN and the connection between the DMN and the task-positive network (TPN). Ayahuasca caused a significant decrease in activity through most parts of the DMN, including its most consistent hubs: the Posterior Cingulate Cortex (PCC)/Precuneus and the medial Prefrontal Cortex (mPFC). Functional connectivity within the PCC/Precuneus decreased after Ayahuasca intake. No significant change was observed in the DMN-TPN orthogonality. Altogether, our results support the notion that the altered state of consciousness induced by Ayahuasca, like those induced by psilocybin (another serotonergic psychedelic), meditation and sleep, is linked to the modulation of the activity and the connectivity of the DMN.

Stress-induced neuroinflammation: mechanisms and new pharmacological targets

Stress is triggered by numerous unexpected environmental, social or pathological stimuli occurring during the life of animals, including humans, which determine changes in all of their systems. Although acute stress is essential for survival, chronic, long-lasting stress can be detrimental. In this review, we present data supporting the hypothesis that stress-related events are characterized by modifications of oxidative/nitrosative pathways in the brain in response to the activation of inflammatory mediators. Recent findings indicate a key role for nitric oxide (NO) and an excess of pro-oxidants in various brain areas as responsible for both neuronal functional impairment and structural damage. Similarly, cyclooxygenase-2 (COX-2), another known source of oxidants, may account for stress-induced brain damage. Interestingly, some of the COX-2-derived mediators, such as the prostaglandin 15d-PGJ2 and its peroxisome proliferator-activated nuclear receptor PPARγ, are activated in the brain in response to stress, constituting a possible endogenous anti-inflammatory mechanism of defense against excessive inflammation. The stress-induced activation of both biochemical pathways depends on the activation of the N-methyl-D-aspartate (NMDA) glutamate receptor and on the activation of the transcription factor nuclear factor kappa B (NFκB). In the case of inducible NO synthase (iNOS), release of the cytokine TNF-α also accounts for its expression. Different pharmacological strategies directed towards different sites in iNOS or COX-2 pathways have been shown to be neuroprotective in stress-induced brain damage: NMDA receptor blockers, inhibitors of TNF-α activation and release, inhibitors of NFκB, specific inhibitors of iNOS and COX-2 activities and PPARγ agonists. This article reviews recent contributions to this area addressing possible new pharmacological targets for the treatment of stress-induced neuropsychiatric disorders.

Simvastatin-induced cardiac autonomic control improvement in fructose-fed female rats

OBJECTIVE: Because autonomic dysfunction has been found to lead to cardiometabolic disorders and because studies have reported that simvastatin treatment has neuroprotective effects, the objective of the present study was to investigate the effects of simvastatin treatment on cardiovascular and autonomic changes in fructose-fed female rats. METHODS: Female Wistar rats were divided into three groups: controls (n=8), fructose (n=8), and fructose+ simvastatin (n=8). Fructose overload was induced by supplementing the drinking water with fructose (100 mg/L, 18 wks). Simvastatin treatment (5 mg/kg/day for 2 wks) was performed by gavage. The arterial pressure was recorded using a data acquisition system. Autonomic control was evaluated by pharmacological blockade. RESULTS: Fructose overload induced an increase in the fasting blood glucose and triglyceride levels and insulin resistance. The constant rate of glucose disappearance during the insulin intolerance test was reduced in the fructose group (3.4+ 0.32%/min) relative to that in the control group (4.4+ 0.29%/min). Fructose+simvastatin rats exhibited increased insulin sensitivity (5.4+0.66%/min). The fructose and fructose+simvastatin groups demonstrated an increase in the mean arterial pressure compared with controls rats (fructose: 124+2 mmHg and fructose+simvastatin: 126 + 3 mmHg vs. controls: 112 + 2 mmHg). The sympathetic effect was enhanced in the fructose group (73 + 7 bpm) compared with that in the control (48 + 7 bpm) and fructose+simvastatin groups (31+8 bpm). The vagal effect was increased in fructose+simvastatin animals (84 + 7 bpm) compared with that in control (49 + 9 bpm) and fructose animals (46+5 bpm). CONCLUSION: Simvastatin treatment improved insulin sensitivity and cardiac autonomic control in an experimental model of metabolic syndrome in female rats. These effects were independent of the improvements in the classical plasma lipid profile and of reductions in arterial pressure. These results support the hypothesis that statins reduce the cardiometabolic risk in females with metabolic syndrome.

Clinical evaluation and induced corneal vascularization study by native and anionic collagen membranes in rabbits corneas

PURPOSE: To evaluate the corneal vascularization (CV) and the clinical aspects induced by interlamellar graft with native (NCM) and anionic (ACM) collagen membranes in rabbits corneas. METHODS: An interlamellar graft with a 0.25 x 0.25 cm NCM (group 1) or ACM (group 2) fragment was performed in the right eye (treated eye). In the left eye, an estromal tunnel was done (control eye). Sixteen rabbits were used, and they were subdivided into two experimental groups of eight animals each. The clinical evaluation was performed at the 1st, 3rd, 7th, 15th and 30th postoperative days. Corneal vascularization analysis was performed after 30 days by the Images Analizator System Leica Qwin-550®. RESULTS: After 7 days, corneal vascularization was observed at about 2.25 ± 0.71 mm (NCM) and at about 1.0 ± 1.69 mm (ACM), respectively, from the limbus in direction to the central cornea. After 15 days, CV increased in both groups (5.25 ± 1.03 mm - NCM; 2.0 ± 2.39 mm - ACM) and then progressively decreased until day 30 (2.25 ± 2.10 mm - NCM; 0.75 ± 2.12 mm - ACM). The statistical analysis indicated that the averages of the distances from the limb vessels to the grafts observed after 7 and 15 days had not differed statistically (p=0.17), and after 15 and 30 postoperative days had a tendency to differ statistically (p=0.09). The control eyes did not present any changes. CONCLUSION: The interlamellar graft with native and anionic collagen membranes induced corneal vascularization when applied to rabbit corneas, but anionic collagen membrane induced a smaller corneal vascularization when compared to native collagen membrane. Although further studies are required, the results found in this study demonstrated the usefulness of interlamellar graft with native and anionic collagen membranes in keratoplasties. These membranes consists in one more graft option for the surgical treatment of corneal repair in rabbits and others animals, when other forms of medical and surgical treatment are not effective.

Defects in vesicle core induced by Escherichia coli dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate during the fourth step of the de novo pyrimidine synthesis pathway. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies in that pathway for nucleotide synthesis. Moreover, as certain parasites lack salvage enzymes, relying solely on the de novo pathway, DHODH inhibition has turned out as an efficient way to block pyrimidine biosynthesis. Escherichia coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used electronic spin resonance (ESR) to study the interaction of EcDHODH with vesicles of 1,2-dioleoyl-sn-glycero-phosphatidylcholine/detergent. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions of phospholipid derivatives. Two-component ESR spectra are obtained for labels 5- and 1 0-phosphatidylcholine in presence of EcDHODH, whereas other probes show a single-component spectrum. The appearance of an additional spectral component with features related to fast-motion regime of the probe is attributed to the formation of a defect-like structure in the membrane hydrophobic region. This is probably the mechanism used by the protein to capture quinones used as electron acceptors during catalysis. The use of specific spectral simulation routines allows us to characterize the ESR spectra in terms of changes in polarity and mobility around the spin-labeled phospholipids. We believe this is the first report of direct evidences concerning the binding of class 2 DHODH to membrane systems.

Loxosceles gaucho Venom-Induced Acute Kidney Injury - In Vivo and In Vitro Studies

Background: Accidents caused by Loxosceles spider may cause severe systemic reactions, including acute kidney injury (AKI). There are few experimental studies assessing Loxosceles venom effects on kidney function in vivo. Methodology/Principal Findings: In order to test Loxosceles gaucho venom (LV) nephrotoxicity and to assess some of the possible mechanisms of renal injury, rats were studied up to 60 minutes after LV 0.24 mg/kg or saline IV injection (control). LV caused a sharp and significant drop in glomerular filtration rate, renal blood flow and urinary output and increased renal vascular resistance, without changing blood pressure. Venom infusion increased significantly serum creatine kinase and aspartate aminotransferase. In the LV group renal histology analysis found acute epithelial tubular cells degenerative changes, presence of cell debris and detached epithelial cells in tubular lumen without glomerular or vascular changes. Immunohistochemistry disclosed renal deposition of myoglobin and hemoglobin. LV did not cause injury to a suspension of fresh proximal tubules isolated from rats. Conclusions/Significance: Loxosceles gaucho venom injection caused early AKI, which occurred without blood pressure variation. Changes in glomerular function occurred likely due to renal vasoconstriction and rhabdomyolysis. Direct nephrotoxicity could not be demonstrated in vitro. The development of a consistent model of Loxosceles venom-induced AKI and a better understanding of the mechanisms involved in the renal injury may allow more efficient ways to prevent or attenuate the systemic injury after Loxosceles bite.

Minor and Unsystematic Cortical Topographic Changes of Attention Correlates between Modalities

In this study we analyzed the topography of induced cortical oscillations in 20 healthy individuals performing simple attention tasks. We were interested in qualitatively replicating our recent findings on the localization of attention-induced beta bands during a visual task [1], and verifying whether significant topographic changes would follow the change of attention to the auditory modality. We computed corrected latency averaging of each induced frequency bands, and modeled their generators by current density reconstruction with Lp-norm minimization. We quantified topographic similarity between conditions by an analysis of correlations, whereas the inter-modality significant differences in attention correlates were illustrated in each individual case. We replicated the qualitative result of highly idiosyncratic topography of attention-related activity to individuals, manifested both in the beta bands, and previously studied slow potential distributions [2]. Visual inspection of both scalp potentials and distribution of cortical currents showed minor changes in attention-related bands with respect to modality, as compared to the theta and delta bands, known to be major contributors to the sensory-related potentials. Quantitative results agreed with visual inspection, supporting to the conclusion that attention-related activity does not change much between modalities, and whatever individual changes do occur, they are not systematic in cortical localization across subjects. We discuss our results, combined with results from other studies that present individual data, with respect to the function of cortical association areas.

Alstonine as an Antipsychotic: Effects on Brain Amines and Metabolic Changes

Managing schizophrenia has never been a trivial matter. Furthermore, while classical antipsychotics induce extrapyramidal side effects and hyperprolactinaemia, atypical antipsychotics lead to diabetes, hyperlipidaemia, and weight gain. Moreover, even with newer drugs, a sizable proportion of patients do not show significant improvement. Alstonine is an indole alkaloid identified as the major component of a plant-based remedy used in Nigeria to treat the mentally ill. Alstonine presents a clear antipsychotic profile in rodents, apparently with differential effects in distinct dopaminergic pathways. The aim of this study was to complement the antipsychotic profile of alstonine, verifying its effects on brain amines in mouse frontal cortex and striatum. Additionally, we examined if alstonine induces some hormonal and metabolic changes common to antipsychotics. HPLC data reveal that alstonine increases serotonergic transmission and increases intraneuronal dopamine catabolism. In relation to possible side effects, preliminary data suggest that alstonine does not affect prolactin levels, does not induce gains in body weight, but prevents the expected fasting-induced decrease in glucose levels. Overall, this study reinforces the proposal that alstonine is a potential innovative antipsychotic, and that a comprehensive understanding of its neurochemical basis may open new avenues to developing newer antipsychotic medications.

Serotonin receptors are involved in the spinal mediation of descending facilitation of surgical incision-induced increase of Fos-like immunoreactivity in rats

Background: Descending pronociceptive pathways may be implicated in states of persistent pain. Paw skin incision is a well-established postoperative pain model that causes behavioral nociceptive responses and enhanced excitability of spinal dorsal horn neurons. The number of spinal c-Fos positive neurons of rats treated intrathecally with serotonin, noradrenaline or acetylcholine antagonists where evaluated to study the descending pathways activated by a surgical paw incision. Results: The number of c-Fos positive neurons in laminae I/II ipsilateral, lamina V bilateral to the incised paw, and in lamina X significantly increased after the incision. These changes: remained unchanged in phenoxybenzamine-treated rats; were increased in the contralateral lamina V of atropine-treated rats; were inhibited in the ipsilateral lamina I/II by 5-HT(1/2B/2C) (methysergide), 5-HT(2A) (ketanserin) or 5-HT(1/2A/2C/5/6/7) (methiothepin) receptors antagonists, in the ipsilateral lamina V by methysergide or methiothepin, in the contralateral lamina V by all the serotonergic antagonists and in the lamina X by LY 278,584, ketanserin or methiothepin. Conclusions: We conclude: (1) muscarinic cholinergic mechanisms reduce incision-induced response of spinal neurons inputs from the contralateral paw; (2) 5-HT(1/2A/2C/3) receptors-mediate mechanisms increase the activity of descending pathways that facilitates the response of spinal neurons to noxious inputs from the contralateral paw; (3) 5-HT(1/2A/2C) and 5-HT(1/2C) receptors increases the descending facilitation mechanisms induced by incision in the ipsilateral paw; (4) 5-HT(2A/3) receptors contribute to descending pronociceptive pathways conveyed by lamina X spinal neurons; (5) alpha-adrenergic receptors are unlikely to participate in the incision-induced facilitation of the spinal neurons.

Leucine supplementation improves adiponectin and total cholesterol concentrations despite the lack of changes in adiposity or glucose homeostasis in rats previously exposed to a high-fat diet

Background: Studies suggest that leucine supplementation (LS) has a therapeutic potential to prevent obesity and to promote glucose homeostasis. Furthermore, regular physical exercise is a widely accepted strategy for body weight maintenance and also for the prevention of obesity. The aim of this study was to determine the effect of chronic LS alone or combined with endurance training (ET) as potential approaches for reversing the insulin resistance and obesity induced by a high-fat diet (HFD) in rats. Methods: Forty-seven rats were randomly divided into two groups. Animals were fed a control diet-low fat (n = 10) or HFD (n = 37). After 15 weeks on HFD, all rats received the control diet-low fat and were randomly divided according to treatment: reference (REF), LS, ET, and LS+ET (n = 7-8 rats per group). After 6 weeks of treatment, the animals were sacrificed and body composition, fat cell volume, and serum concentrations of total cholesterol, HDL-cholesterol, triacylglycerol, glucose, adiponectin, leptin and tumor necrosis factor-alpha (TNF-alpha) were analyzed. Results: At the end of the sixth week of treatment, there was no significant difference in body weight between the REF, LS, ET and LS+ET groups. However, ET increased lean body mass in rats (P = 0.019). In addition, ET was more effective than LS in reducing adiposity (P = 0.019), serum insulin (P = 0.022) and TNF-alpha (P = 0.044). Conversely, LS increased serum adiponectin (P = 0.021) levels and reduced serum total cholesterol concentration (P = 0.042). Conclusions: The results showed that LS had no beneficial effects on insulin sensitivity or adiposity in previously obese rats. On the other hand, LS was effective in increasing adiponectin levels and in reducing total cholesterol concentration.

Giant Vesicles under Oxidative Stress Induced by a Membrane-Anchored Photosensitizer

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid head-group. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.