Structural characterization of the α-N-acetylglucosaminidase,a key enzyme in the pathogenesis of Sanfilippo syndrome B

Mucopolysaccharidosis III B (MPS III-B) is a rare lysosomal storage disorder caused by deficiencies in Alpha-N-acetylglucosaminidase (NAGLU) for which there is currently no cure, and present treatment is largely supportive. Understanding the structure of NAGLU may allow for identification of novel therapeutic targets for MPS III-B. Here we describe the first crystal structure of human NAGLU, determined to a resolution of 2.3?Å. The crystal structure reveals a novel homotrimeric configuration, maintained primarily by hydrophobic and electrostatic interactions via domain II of three contiguous domains from the N- to C-terminus. The active site cleft is located between domains II and III. Catalytic glutamate residues, E316 and E446, are located at the top of the (α/β)₈ barrel structure in domain II. We utilized the three-dimensional structure of NAGLU to map several MPS III-B mutations, and hypothesize their functional consequences. Revealing atomic level structural information about this critical lysosomal enzyme paves the way for the design of novel therapeutics to target the underlying causes of MPS III-B.     

Purification and Characterization of Thermophilic Xylanase Isolated from the Xerophytic-<Emphasis Type="Italic">Cereus pterogonus</Emphasis> sp.

A thermo stable xylanase was purified and characterized from the cladodes of Cereus pterogonus plant species. The enzyme was purified to homogeneity by ammonium sulfate (80%) fractionation, ion exchange and size exclusion chromatography. The enzyme showed a final specific activity of 216.2 U/mg and the molecular mass of the protein was 80 KDa. The optimum pH and temperature for xylanase activity were 5.0 and 80 °C, respectively,. With oat spelt xylan as a substrate the enzyme yielded a Km value of 2.24 mg/mL and a Vmax of 5.8 μmol min⁻¹ mg⁻¹. In the presence of metal ions (1 mM) such as Co²⁺,Mn²⁺, Ni²⁺, Ca²⁺ and Fe³⁺ the activity of the enzyme increased, where as strong inhibition of the enzyme activity was observed with the use of Hg²⁺, Cd²⁺, Cu²⁺, while partial inhibition was noted with Zn²⁺ and Mg²⁺. The substrate specificity of the xylanase yielded maximum activity with oat spelt xylan.     

Neuronal Networks during Burst Suppression as Revealed by Source Analysis

Introduction

Burst-suppression (BS) is an electroencephalography (EEG) pattern consisting of alternant periods of slow waves of high amplitude (burst) and periods of so called flat EEG (suppression). It is generally associated with coma of various etiologies (hypoxia, drug-related intoxication, hypothermia, and childhood encephalopathies, but also anesthesia). Animal studies suggest that both the cortex and the thalamus are involved in the generation of BS. However, very little is known about mechanisms of BS in humans. The aim of this study was to identify the neuronal network underlying both burst and suppression phases using source reconstruction and analysis of functional and effective connectivity in EEG.

Material/Methods

Dynamic imaging of coherent sources (DICS) was applied to EEG segments of 13 neonates and infants with burst and suppression EEG pattern. The brain area with the strongest power in the analyzed frequency (1–4 Hz) range was defined as the reference region. DICS was used to compute the coherence between this reference region and the entire brain. The renormalized partial directed coherence (RPDC) was used to describe the informational flow between the identified sources.

Results/Conclusion

Delta activity during the burst phases was associated with coherent sources in the thalamus and brainstem as well as bilateral sources in cortical regions mainly frontal and parietal, whereas suppression phases were associated with coherent sources only in cortical regions. Results of the RPDC analyses showed an upwards informational flow from the brainstem towards the thalamus and from the thalamus to cortical regions, which was absent during the suppression phases. These findings may support the theory that a “cortical deafferentiation” between the cortex and sub-cortical structures exists especially in suppression phases compared to burst phases in burst suppression EEGs. Such a deafferentiation may play a role in the poor neurological outcome of children with these encephalopathies.     

Therapeutic efficacy of natural dipeptide carnosine against human cervical carcinoma cells

Natural substances have been attracted several researchers in the recent years, because of its potential antioxidant, anti‐inflammatory and anti‐cancer properties. We have investigated the effect of carnosine on cell viability, apoptosis, DNA damage, reactive oxygen species (ROS) and caspase 3 enzyme expression in human cervical carcinoma and Madin‐Darby Kidney Cells (MDCK) cells . Carnosine inhibited cancer cell growth up to 23%. ROS level was increased up to 30 and 31% in MDCK and HeLa cells respectively. Tunnel assay showed 42 and 14% of positive apoptotic cells in cancer and normal cells respectively. The alteration in mitochondrial and nuclear morphology was determined. The extended lace‐like network of normal mitochondria found in control cells. Carnosine treatment significantly altered the mitochondrial morphology of normal cervical carcinoma cell. Mitochondria were condensed clump structures in carnosine treated cancer cells. Carnosine reduced the number of colonies of cervical carcinoma cells. Caspase 3 expression was corresponded to the appearance of immunofluorescence in the cytoplasm. Caspase 3 expression was gradually increased in cervical carcinoma cells. In Silico, docking study was performed to recognize the binding activity of carnosine against a subunit of the caspase 3 , and carnosine was able to bind to the drug binding pocket of caspase 3. The glide energy is ?5.2 kcal/mol, suggesting the high binding affinity of carnosine to caspase 3. Taking all these data together, the natural dipeptide L‐carnosine could be a suitable antiproliferative agent in cervical carcinoma cells. Copyright © 2016 John Wiley & Sons, Ltd.     

EEG-MEG Integration Enhances the Characterization of Functional and Effective Connectivity in the Resting State Network

At the sensor level many aspects, such as spectral power, functional and effective connectivity as well as relative-power-ratio ratio (RPR) and spatial resolution have been comprehensively investigated through both electroencephalography (EEG) and magnetoencephalography (MEG). Despite this, differences between both modalities have not yet been systematically studied by direct comparison. It remains an open question as to whether the integration of EEG and MEG data would improve the information obtained from the above mentioned parameters. Here, EEG (64-channel system) and MEG (275 sensor system) were recorded simultaneously in conditions with eyes open (EO) and eyes closed (EC) in 29 healthy adults. Spectral power, functional and effective connectivity, RPR, and spatial resolution were analyzed at five different frequency bands (delta, theta, alpha, beta and gamma). Networks of functional and effective connectivity were described using a spatial filter approach called the dynamic imaging of coherent sources (DICS) followed by the renormalized partial directed coherence (RPDC). Absolute mean power at the sensor level was significantly higher in EEG than in MEG data in both EO and EC conditions. At the source level, there was a trend towards a better performance of the combined EEG+MEG analysis compared with separate EEG or MEG analyses for the source mean power, functional correlation, effective connectivity for both EO and EC. The network of coherent sources and the spatial resolution were similar for both the EEG and MEG data if they were analyzed separately. Results indicate that the combined approach has several advantages over the separate analyses of both EEG and MEG. Moreover, by a direct comparison of EEG and MEG, EEG was characterized by significantly higher values in all measured parameters in both sensor and source level. All the above conclusions are specific to the resting state task and the specific analysis used in this study to have general conclusion multi-center studies would be helpful.     

Effects of stress hormone cortisol on the mRNA expression of myogenenin,MyoD, Myf5, PAX3 and PAX7

The present investigation was carried out to evaluate the effect of stress hormone cortisol on the myogenic markers in the C2C12 cells co-cultured with 3T3-L1 preadipocytes. Co-culturing was achieved by transwell inserts with a 0.4 μm porous membrane. C2C12 and 3T3-L1 cells were grown independently on the transwell plates. After differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates for co-culturing. 10 μg/μl of cortisol was added to the medium. After 72 h of treatment, C2C12 cells which were in the lower well were harvested for analysis. RT-PCR analysis of myogenic markers such as of myogenin, MyoD, Myf5, PAX3 and PAX7 showed a significant reduction in the mRNA expression of these myogenic markers. In addition, cortisol increased calpain activity, which led to accelerated protein degradation, which in turn reduced the myogenic rate. In conclusion, cortisol treatment reduced mRNA expression of myogenic markers in the co-cultured C2C12 cells, which is quite distinct from one dimensional mono-cultured C2C12 cells.     

Changes in proteolytic enzymes mRNAs and proteins relevant for meat quality during myogenesis and hypoxia of primary bovine satellite cells

The current study was conducted to evaluate the functions of μ-calpain (CAPN1), calpastatin, HSPs (heat shock proteins), and caspases during myogenesis and cell death induced by sodium azide (NaN(3)) hypoxia. The cell samples were divided into three groups: satellite cells formed at confluent monolayer (stage 1), stage 1 cells fusion into myotubes on d eight post-differentiation (stage 2), and stage 2 cells treated with 1 mM NaN(3) for 24 h (stage 3). Real-time RT-PCR showed that stage 2 cells had increased CAPN1, calpastatin, caspase 7, and CARD9 (Caspase activation and recruitment domain 9) mRNA expressions compared to stage 1 cells (*p < 0.05). By Western blotting caspase 3, caspase 7, caspase 8, and caspase 9 protein levels increased in cells at stage 2 compared to cells at stage 1 (*p < 0.05). Real-time RT-PCR showed that stage 3 cells had increased CAPN1, calpastatin, caspase 7, HSP70 (70 kDA heat shock proteins), and HSP90 (90 kDA heat shock proteins-alpha) and decreased CARD9 mRNA expression compared to stage 2 cells (*p < 0.05). Stage 3 samples had increase caspase 7 and caspase 12 activities compared to stage 2 samples, and by Western blotting protein levels of both HSP70 and HSP90 expressions, increased significantly under hypoxia condition (*p < 0.05). Here, we conclude that CAPN1, calpastatin, caspase 3, caspase 7, caspase 8, and CARD9 have important roles for satellite cell myogenesis; and that caspase 7, 12, HSP70, and HSP90 are involved in the process of apoptotic cell death under hypoxia conditions and we speculate that these proteins may be involved in early postmortem proteolysis and meat tenderization.     

Dynamic Imaging of Coherent Sources Reveals Different Network Connectivity Underlying the Generation and Perpetuation of Epileptic Seizures

The concept of focal epilepsies includes a seizure origin in brain regions with hyper synchronous activity (epileptogenic zone and seizure onset zone) and a complex epileptic network of different brain areas involved in the generation, propagation, and modulation of seizures. The purpose of this work was to study functional and effective connectivity between regions involved in networks of epileptic seizures. The beginning and middle part of focal seizures from ictal surface EEG data were analyzed using dynamic imaging of coherent sources (DICS), an inverse solution in the frequency domain which describes neuronal networks and coherences of oscillatory brain activities. The information flow (effective connectivity) between coherent sources was investigated using the renormalized partial directed coherence (RPDC) method. In 8/11 patients, the first and second source of epileptic activity as found by DICS were concordant with the operative resection site; these patients became seizure free after epilepsy surgery. In the remaining 3 patients, the results of DICS / RPDC calculations and the resection site were discordant; these patients had a poorer post-operative outcome. The first sources as found by DICS were located predominantly in cortical structures; subsequent sources included some subcortical structures: thalamus, Nucl. Subthalamicus and cerebellum. DICS seems to be a powerful tool to define the seizure onset zone and the epileptic networks involved. Seizure generation seems to be related to the propagation of epileptic activity from the primary source in the seizure onset zone, and maintenance of seizures is attributed to the perpetuation of epileptic activity between nodes in the epileptic network. Despite of these promising results, this proof of principle study needs further confirmation prior to the use of the described methods in the clinical praxis.     

Ameliorative potential of montelukast on ischemia-reperfusion injury induced vasculitic neuropathic pain in rat

AimsIschemia -reperfusion (I/R) event in vascular and nervous system has been documented to rising ischemic and vasculitic neuropathic pain, clinically resembles the complex regional pain syndrome (CRPS). The present study evaluated the effect of montelukast, a cysteinyl leukotriene receptor (Cys-LTC₄ and Cys-LTD₄) antagonist on ischemia -reperfusion (I/R) induced vasculitic neuropathic pain in rats.Main MethodsBehavioral parameters were assessed at different time intervals (i.e. 0, 1, 7, 14 and 21st day) and biochemical analysis in sciatic nerve tissue samples were also performed along with histopathological studies.Key FindingsBehavioral pain assessment has shown increase in paw and tail withdrawal threshold in montelukast treated groups against thermal and mechanical stimuli as compared to I/R control group. We observed a decrease in the total calcium, thiobarbituric acid reactive substance (TBARS) and myeloperoxidase (MPO) activity levels, whereas there is rise in reduced glutathione level in montelukast treated groups as compared to I/R control group. However, significant behavioral and biochemical results were observed only in medium and high dose of treated groups which were comparable to normal control group. Moreover, histopathological study has revealed the reduction of I/R induced neuronal edema and axonal degeneration due to montelukast.SignificanceMontelukast has ameliorated I/R induced vasculitic neuropathic pain, these effects may be due to inhibition of lipid peroxidation, reduction of oxidative stress, release of inflammatory mediators and neuroprotective actions. Hence, it could be used as a novel therapeutic agent for the management of vasculitic inflammation related neuropathic pain.