Protein Permeability Research Articles

MURINE MODELS OF SYSTEMIC INFLAMMATION AND DEMENTIA: HOW ARE THEY CONNECTED?

There is growing evidence for a role of systemic microbial infections in the onset and/or progression of a number of chronic neurodegenerative diseases including Alzheimer's disease (AD). While these observations further support a role of the immune system in the pathogenesis of AD, the underlying biological mechanisms by which systemic infections drive disease progression are incompletely understood. We have previously shown that a real-life systemic bacterial infection results in long-lasting changes to the blood brain barrier permeability, microglial activation and infiltration of lymphocytes into the brain of adult mice. In this study we explore the effect of systemic infection in experimental models of Alzheimer's Disease. Transgenic mice carrying hAPPswe were challenged with an attenuated bacterial strain of Salmonella typhimurium at different stages of disease (12, 16 and 20 months). Brain and serum samples were collected and analysed for amyloid load, neuroinflammation, and BBB permeability using immunohistochemistry and multiplex immune assays. Tg2576 mice showed increased amyloid load following systemic bacterial infection, regardless of the stage of the disease, whilst no amyloid was detected in wild type mice. Bacterial infection resulted in increased neuroinflammatory response in both wild type and Tg2576 mice, measured by microglial phenotype changes and pro-inflammatory cytokine levels, but this response was prolonged in Tg2576 mice, especially in 12 month old mice. Blood brain barrier permeability for plasma proteins was increased following bacterial infection in both wild type and Tg2576, but recruitment of lymphocytes was exaggerated in Tg2576. Our results suggest that environmental stimuli, such as systemic bacterial infections, will contribute to earlier onset and progression of neuropathology in the Tg2576 model of Alzheimer's disease. These observations may explain the exaggerated clinicial symptoms reported in AD patients who are exposed to systemic microbial infection.

Whey protein film properties as affected by ultraviolet treatment under alkaline conditions

The effects of UV radiation treatment on film-forming solutions adjusted to two alkaline pH values (9 or 11) on the properties (water vapour permeability, solubility, mechanical properties, colour and microstructure) of whey protein concentrate films, and on some structural characteristics of proteins (free sulphydryl groups, degree of aggregation, denaturation and secondary structure) were evaluated. UV radiation increased protein aggregation at both pH values. Ultraviolet treatment of pH 9 solutions increased free sulphydryl groups and produced films with higher solubility, tensile strength, elastic modulus, and puncture properties and lower elongation at break; UV radiation at pH 11 decreased free sulphydryl groups and films showed higher solubility and elastic modulus and lower puncture deformation and elongation at break than untreated films. The treatment also affected film colour differently depending on the pH. In conclusion, ultraviolet treatment modifies the properties of the films in different ways depending on the pH of the solutions.

Enhancement of transdermal protein delivery by photothermal effect of gold nanorods coated on polysaccharide-based hydrogel.

Transdermal protein delivery is a useful and attractive method for protein therapy and dermal vaccination. However, this delivery method is restricted by the low permeability of the stratum corneum. The purpose of this study was to develop a transdermal delivery system for enhancement of protein permeability into the skin. First, we prepared a transparent gel patch made of polysaccharides with gold nanorods on the gel surface and fluorescein isothiocyanate-modified ovalbumin (FITC-OVA) inside. Next, the gel patch was placed on mouse skin to allow contact with the coated gold nanorods, and irradiated by a continuous-wave laser. The laser irradiation heated the gold nanorods and the skin temperature increased to 43°C, resulting in enhanced translocation of FITC-OVA into the skin. These results confirmed the capability of the transdermal protein delivery system to perforate the stratum corneum and thus facilitate the passage of proteins across the skin.

The effect of netrin-1 on the retinal vascular permeability in diabetes mellitus rats

Objective To observe the effect of different concentration netrin-1 on retinal vascular permeability in diabetes mellitus (DM) rats. Methods Eighty adult Sprague-Dawley rats were randomly divided into 8 groups, 10 rats in each group, including normal control group (group A), normal+ balanced salt solution (BSS) group (group B), normal+ netrin-1 (500 μg/ml) group (group C) and DM group (50 rats in 5 subgroups). DM rats were induced by intraperitoneal injection of streptozocin. Three months after intraperitoneal injection, 10 DM rats in the control group were injected with BSS (group D). Forty DM rats were injected with 5 μl of different concentrate netrin-1, and were divided into DM+netrin-1 10 μg/ml group (group E), DM+netrin-1 50 μg/ml group (group F), DM+ netrin-1 100 μg/ml group (group G), DM+ netrin-1 500 μg/ml group (group H) according to the different concentration. Non-DM rats in group C were injected with netrin-1 500 μg/ml. The expression of occludin was determined by immunohistochemistry for protein, and by real-time fluorescence quantitative reverse transcription polymerase chain reaction for mRNA level. Retinal vascular permeability was measured by Evans blue infusion. Results The expression of occludin protein and mRNA in group D were less than group A (t=27.71, 8.59; P=0.00, 0.00). However, the retinal vascular permeability increased in group D (t=-42.72, P=0.00). The expression of occluding protein, occludin mRNA and retinal vascular permeability showed significant differences between group D, E, F, G and H (F=146.31, 16.54, 67.77; P=0.00, 0.00, 0.00). Compared the group B with group C, there was no significant differences between the expression of occludin protein, occludin mRNA and the retinal vascular permeability (t=-1.13, 0.93, 1.04; P=0.27, 0.36, 0.31). The concentrate of netrin-1 showed a significant positive correlation to the expression level of occludin and occludin mRNA (r=0.73, 0.81; P=0.00, 0.00), but negative correlation to the vascular permeability (r=-0.61, P=0.00). Conclusion Netrin-1 can reduce the DM rats' retinal vascular permeability, which depended on the concentration of netrin-1. Key words: Diabetic retinopathy/physiopathology; Blood retinal barrier/physiopathology; Nerve growth factors; Animal experimentation

Inhibition of PKR ameliorates lipopolysaccharide-induced acute lung injury by suppressing NF-κB pathway in mice

Acute lung injury (ALI) is characterized by dramatic lung inflammation and alveolar epithelial cell death. Although protein kinase R (PKR) (double-stranded RNA-activated serine/threonine kinase) has been implicated in inflammatory response to bacterial cell wall components, whether it plays roles in lipopolysaccharide (LPS)-induced ALI remains unclear. This study was aimed to reveal whether and how PKR was involved in LPS-induced ALI pathology and the potential effects of its specific inhibitor, C16 (C13H8N4OS). During the experiment, mice received C16 (100 or 500 ug/kg) intraperitoneally 1 h before intratracheal LPS instillation. Then, whole lung lavage was collected for analysis of total protein levels and proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. The lungs were tested for Western blot, transferase-mediated dUTP nick-end labeling (TUNEL) stain and immunohistochemistry. Results showed that PKR phosphorylation increased significantly after LPS instillation. Furthermore, PKR specific inhibition attenuated LPS-induced lung injury (hematoxylin and eosin stain), reduced lung protein permeability (total protein levels in whole lung lavage) and suppressed proinflammatory cytokines (TNF-α, IL-1β and IL-6) and lung apoptosis (TUNEL stain and caspase3 activation). Moreover, mechanism-study showed that C16 significantly suppressed I kappa B kinase (IKK)/I kappa B alpha (IκBα)/NF-κB signaling pathway after LPS challenge. These findings suggested that PKR inhibition ameliorated LPS-induced lung inflammation and apoptosis in mice by suppressing NF-κB signaling pathway.

Contribution of thrombin-reactive brain pericytes to blood-brain barrier dysfunction in an in vivo mouse model of obesity-associated diabetes and an in vitro rat model.

Diabetic complications are characterized by the dysfunction of pericytes located around microvascular endothelial cells. The blood–brain barrier (BBB) exhibits hyperpermeability with progression of diabetes. Therefore, brain pericytes at the BBB may be involved in diabetic complications of the central nervous system (CNS). We hypothesized that brain pericytes respond to increased brain thrombin levels in diabetes, leading to BBB dysfunction and diabetic CNS complications. Mice were fed a high-fat diet (HFD) for 2 or 8 weeks to induce obesity. Transport of i.v.-administered sodium fluorescein and 125I-thrombin across the BBB were measured. We evaluated brain endothelial permeability and expression of tight junction proteins in the presence of thrombin–treated brain pericytes using a BBB model of co-cultured rat brain endothelial cells and pericytes. Mice fed a HFD for 8 weeks showed both increased weight gain and impaired glucose tolerance. In parallel, the brain influx rate of sodium fluorescein was significantly greater than that in mice fed a normal diet. HFD feeding inhibited the decline in brain thrombin levels occurring during 6 weeks of feeding. In the HFD fed mice, plasma thrombin levels were significantly increased, by up to 22%. 125I-thrombin was transported across the BBB in normal mice after i.v. injection, with uptake further enhanced by co-injection of unlabeled thrombin. Thrombin-treated brain pericytes increased brain endothelial permeability and caused decreased expression of zona occludens-1 (ZO-1) and occludin and morphological disorganization of ZO-1. Thrombin also increased mRNA expression of interleukin-1β and 6 and tumor necrosis factor-α in brain pericytes. Thrombin can be transported from circulating blood through the BBB, maintaining constant levels in the brain, where it can stimulate pericytes to induce BBB dysfunction. Thus, the brain pericyte–thrombin interaction may play a key role in causing BBB dysfunction in obesity-associated diabetes and represent a therapeutic target for its CNS complications.

ATRP-grown protein-polymer conjugates containing phenylpiperazine selectively enhance transepithelial protein transport

Despite its patient-friendliness, the oral route is not yet a viable strategy for the delivery of biomacromolecular therapeutics. This is, in part, due to the large size of proteins, which greatly limits their absorption across the intestinal epithelium. Although chemical permeation enhancers can improve macromolecular transport, their positive impact is often accompanied by toxicity. One element potentially contributing to this toxicity is the lack of co-localization of the enhancer with the protein drug, which can result in non-specific permeation of the intestine as well as enhancer overdosing in some areas due to non-uniform distribution. To circumvent these issues, this study describes a new way of increasing protein permeability via a polymer conjugation process that co-localizes permeation enhancer with the protein. Based on previous reports demonstrating the utility of 1-phenylpiperazine as an intestinal permeation enhancer, we synthesized protein-polymer conjugates with a phenylpiperazine-containing polymer using polymer-based protein engineering. A novel phenylpiperazine acrylamide monomer was synthesized and chain extended using atom transfer radical polymerization from the model protein bovine serum albumin (BSA). At non-cytotoxic doses, the protein-polymer conjugates induced a dose dependent reduction in the trans-epithelial electrical resistance of Caco-2 monolayers and an impressive ~30-fold increase in BSA permeability. Furthermore, this permeability increase was selective, as the permeability of the small molecule calcein co-incubated with the protein-polymer conjugate increased only 5-fold. Together, these data represent an important first step in the development of protein polymer conjugates that facilitate selective protein transport across membranes that are typically impermeable to macromolecules.

Su1515 - Plecanatide and Dolcanatide, Guanylate Cyclase-C Agonists, Attenuate Paracellular Permeability and Enhance Normal Localization of Tight Junctional Proteins to Maintain Intestinal Barrier Function

Su1515 - Plecanatide and Dolcanatide, Guanylate Cyclase-C Agonists, Attenuate Paracellular Permeability and Enhance Normal Localization of Tight Junctional Proteins to Maintain Intestinal Barrier Function

Pyrrolidine dithiocarbamate administered during ex-vivo lung perfusion promotes rehabilitation of injured donor rat lungs obtained after prolonged warm ischemia.

Damaged lung grafts obtained after circulatory death (DCD lungs) and warm ischemia may be at high risk of reperfusion injury after transplantation. Such lungs could be pharmacologically reconditioned using ex-vivo lung perfusion (EVLP). Since acute inflammation related to the activation of nuclear factor kappaB (NF-κB) is instrumental in lung reperfusion injury, we hypothesized that DCD lungs might be treated during EVLP by pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. Rat lungs exposed to 1h warm ischemia and 2 h cold ischemia were subjected to EVLP during 4h, in absence (CTRL group, N = 6) or in presence of PDTC (2.5g/L, PDTC group, N = 6). Static pulmonary compliance (SPC), peak airway pressure (PAWP), pulmonary vascular resistance (PVR), and oxygenation capacity were determined during EVLP. After EVLP, we measured the weight gain of the heart-lung block (edema), and the concentration of LDH (cell damage), proteins (permeability edema) and of the cytokines IL-6, TNF-α and CINC-1 in bronchoalveolar lavage (BAL), and we evaluated NF-κB activation by the degree of phosphorylation and degradation of its inhibitor IκBα in lung tissue. In CTRL, we found significant NF-κB activation, lung edema, and a massive release of LDH, proteins and cytokines. SPC significantly decreased, PAWP and PVR increased, while oxygenation tended to decrease. Treatment with PDTC during EVLP inhibited NF-κB activation, did not influence LDH release, but markedly reduced lung edema and protein concentration in BAL, suppressed TNFα and IL-6 release, and abrogated the changes in SPC, PAWP and PVR, with unchanged oxygenation. In conclusion, suppression of innate immune activation during EVLP using the NF-κB inhibitor PDTC promotes significant improvement of damaged rat DCD lungs. Future studies will determine if such rehabilitated lungs are suitable for in vivo transplantation.

Intestinal fatty acid-binding protein and gut permeability responses to exercise

PurposeIntestinal cell damage due to physiological stressors (e.g. heat, oxidative, hypoperfusion/ischaemic) may contribute to increased intestinal permeability. The aim of this study was to assess changes in plasma intestinal fatty acid-binding protein (I-FABP) in response to exercise (with bovine colostrum supplementation, Col, positive control) and compare this to intestinal barrier integrity/permeability (5 h urinary lactulose/rhamnose ratio, L/R).MethodsIn a double-blind, placebo-controlled, crossover design, 18 males completed two experimental arms (14 days of 20 g/day supplementation with Col or placebo, Plac). For each arm participants performed two baseline (resting) intestinal permeability assessments (L/R) pre-supplementation and one post-exercise following supplementation. Blood samples were collected pre- and post-exercise to determine I-FABP concentration.ResultsTwo-way repeated measures ANOVA revealed an arm × time interaction for L/R and I-FABP (P < 0.001). Post hoc analyses showed urinary L/R increased post-exercise in Plac (273% of pre, P < 0.001) and Col (148% of pre, P < 0.001) with post-exercise values significantly lower with Col (P < 0.001). Plasma I-FABP increased post-exercise in Plac (191% of pre-exercise, P = 0.002) but not in the Col arm (107%, P = 0.862) with post-exercise values significantly lower with Col (P = 0.013). Correlations between the increase in I-FABP and L/R were evident for visit one (P = 0.044) but not visit two (P = 0.200) although overall plots/patterns do appear similar for each.ConclusionThese findings suggest that exercise-induced intestinal cellular damage/injury is partly implicated in changes in permeability but other factors must also contribute.

Raman Spectroscopy Reveals Selective Interactions of Cytochrome c with Cardiolipin That Correlate with Membrane Permeability

Permeabilization of the outer mitochondrial membrane is an integral step in apoptosis. The resulting release of pro-apoptotic signaling proteins leads to cell destruction through activation of the cysteine-aspartic protease (caspase) cascade. However, the mechanism of outer mitochondrial membrane (OMM) permeabilization remains unclear. It was recently shown that cytochrome c can induce pore formation in cardiolipin-containing phospholipid membranes, leading to large dextran and protein permeability. In this work, the interaction of cytochrome c with cardiolipin-containing phospholipid vesicles, serving as models of the OMM, is investigated to probe cytochrome c-induced permeability. Lipid vesicles having either a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixed-DPPC/cardiolipin membrane and containing a membrane-impermeable Raman tracer 3-nitrobenzenesulfonate (3-NBS) were optically trapped, translated into a solution containing cytochrome c, and monitored for 3-NBS leakage. Cytochrome-correlated leakage was observed only in cardiolipin-containing vesicles. Structural changes observed in the Raman spectra during permeabilization indicated acyl chain disordering along with decreased intensity of the cardiolipin cis-double-bond stretching modes. When the vesicle-associated cytochrome c Raman spectrum is compared with a spectrum in buffer, heme-resonance bands are absent, indicating loss of Met-80 coordination. To verify selective interactions of cytochrome c with cardiolipin, these experiments were repeated where the DPPC acyl chains were deuterated (D62-DPPC), allowing spectral resolution of the DPPC acyl chain response from that of cardiolipin. Interestingly, D62-DPPC acyl chains were unaffected by cytochrome c accumulation, while cardiolipin showed major changes in acyl chain structure. These results suggest that cytochrome-induced permeabilization proceeds through selective interaction of cytochrome c with cardiolipin, resulting in protein unfolding, where the unfolded form interacts with cardiolipin acyl chains within the bilayer to induce permeability.

Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable Gas Vesicle Nanoparticles.

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant protein organelles produced by the extremophilic microbe Halobacterium sp. NRC-1 and are being developed as bioengineerable and biocompatible antigen and drug-delivery systems (DDS). Dynamic light scattering measurements of purified GVNP suspensions showed a mean diameter of 245 nm. In vitro diffusion studies using Yucatan miniature pig skin showed GVNP permeation to be enhanced after MN-treatment compared to untreated skin. GVNPs were found to be nontoxic to mammalian cells (human kidney and rat mycocardial myoblasts). These findings support the use of GVNPs as DDS for intradermal/transdermal permeation of protein- and peptide-based drugs.

Reduction of Lactococcus lactis phage contamination in whey by means of membrane filtration: Impact of phage morphology and of bacterial host cells functioning as “phage fishing tool”

For dairy bacteriophages, the impact of phage morphology and whey components on phage retention efficiencies in filtration processes is not known. Therefore, the influence of (i) five differently shaped Lactococcus lactis phages as well as (ii) differently treated bacterial host cells added to native whey were analysed via cross-flow membrane filtration experiments with polyethersulfone membranes. Filtration efficiency with respect to phage retention and whey protein permeation was investigated. Compared with untreated native whey, phage retention was significantly higher (up to 4.8 log units) in the experiments using whey that was spiked with mechanically disrupted lactococcal host cells. However, the retention of phages commonly found in the dairy environment (i.e., members of the 936, c2, and P335 phage groups) did not correlate with the phage morphology. Results of this model study clearly demonstrate that phage titres can be reduced in whey (containing a sufficient protein concentration) by flat sheet membrane filtration processes.

Re-Validation of New Develop Highly Sensitive, Simple LCMS/MS Method for the Estimation of Rohitukine and its Application in ADME/Pre-Clinical Pharmacokinetics

The purpose of the research was to develop a simple, rapid, accurate, reproducible and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for determination of Rohitukine, a chromone alkaloid in plasma. The chromatographic separation was achieved with high resolution RP18e Chromolith column (100 × 4.6 mm, 2 μm) employing a isocratic composition of organic solvent acetonitrile with 0.1% (v/v) formic acid (80:20, %v/v) at a flow rate of 0.5 mL/min. Triple quadrupole mass spectrometry with positive electrospray ionization (ESI) technique operating in multiple reaction monitoring used to estimate MS/MS ion transitions like 306.05>245.10 and 306.05>231.05 for Rohitukine and 330.30>97.0 for IS. Simple single step protein precipitate method was used for sample preparation. The method was validated for specificity, linearity, accuracy, precision, recovery, matrix effect and stability as per FDA guidelines. Linearity of the analyte was acquired throughout the concentration range from 0.1 ng/mL to 1000 ng/mL in mice plasma. Pharmacokinetic study was performed on female BALB/c mice through oral (20 mg/kg) and intravenous (2 mg/kg) route where the oral bioavailability of Rohitukine obtained was 84%. The bioanalytical method was successfully used for determination of plasma protein binding study, permeability and microsomal stability in mouse, rat and human liver microsomes.

Flow and aggregation of rod-like proteins in slit and cylindrical pores coated with polymer brushes: an insight from dissipative particle dynamics.

We use a meso-scale dissipative particle dynamics method to simulate the flow and aggregation of rod-like protein solutions through pores grafted with a solvent-sensitive polymer brush. The coated pores can control protein permeability and aggregation by a stretch-to-collapse conformational transition of the brush polymers in response to changes in the solvent quality. The protein solutions mimic aqueous glycoprotein solutions and proteins are represented as rod-like objects formed by coarse-grain beads. The model further employs two types of beads to represent the existence of cystein-like terminal groups in real glycoproteins and mimic the aggregation of real glycoproteins in aqueous solutions. We vary the solvent quality with respect to the brush chains and study the flow and aggregation of rod-like proteins in the slit and cylindrical pores as the brush polymers undergo the stretch-to-collapse transition. The results show that stretched brush chains close the pore, hamper proteins' flow and promote proteins' aggregation. The collapsed brush chains open the pores for proteins' flow and suppress their aggregation. Therefore, we observe more than a ten-fold reduction in the permeation rate of proteins in both pore geometries. Finally, due to pore confinement, larger proteins' aggregates are formed in the slit pore than in the cylindrical pore, while more pronounced orientation of proteins in the flow direction is seen in the cylindrical pore than in the slit pore.

Design of Stable α-Helical Peptides and Thermostable Proteins in Biotechnology and Biomedicine

α-Helices are the most frequently occurring elements of the secondary structure in water-soluble globular proteins. Their increased conformational stability is among the main reasons for the high thermal stability of proteins in thermophilic bacteria. In addition, α-helices are often involved in protein interactions with other proteins, nucleic acids, and the lipids of cell membranes. That is why the highly stable α-helical peptides used as highly active and specific inhibitors of protein-protein and other interactions have recently found more applications in medicine. Several different approaches have been developed in recent years to improve the conformational stability of α-helical peptides and thermostable proteins, which will be discussed in this review. We also discuss the methods for improving the permeability of peptides and proteins across cellular membranes and their resistance to intracellular protease activity. Special attention is given to the SEQOPT method (http://mml.spbstu.ru/services/seqopt/), which is used to design conformationally stable short α-helices.

Protein Kinase Cζ Inhibitor Promotes Resolution of Bleomycin-Induced Acute Lung Injury.

Protein kinase Cζ (PKCζ) is highly expressed in the lung, where it plays several regulating roles in the pathogenesis of acute lung injury (ALI). Proliferation and differentiation of integrin β4+ distal lung epithelial progenitor cells seem to play a key role in proper lung regeneration. We investigated the effects of a myristoylated PKCζ inhibitor (PKCζi) in a murine model of bleomycin-induced ALI. After intratracheal injury, we treated mice three times a week with PKCζi or its vehicle, DMSO. We found that mice injured with bleomycin and then treated with PKCζi for one week showed decreased activation of PKCζ, improved lung compliance, and decreased lung protein permeability compared to injured mice treated with DMSO. Mice treated continuously with PKCζi for 6 weeks showed reduced evidence of lung fibrosis by computed tomographic images, decreased lung collagen deposition, and decreased active transforming growth factor-β in the bronchoalveolar lavage fluid. In addition, we found an increased number of lung β4+ cells compared to DMSO at Week 6. Therefore, we grew isolated integrin β4+ lung progenitor cells in the presence of PKCζi or DMSO and found that β4+ cells treated with PKCζi proliferated more in vitro compared to DMSO. We conclude that the use of a PKCζi promotes resolution of lung fibrosis in a bleomycin ALI model and increases the number of β4+ progenitor cells with regenerative potential in the lung.

On Dioxygen and Substrate Access to Soluble Methane Monooxygenases: An all-Atom Molecular Dynamics Investigation in Water Solution.

In a preliminary exploration of the dummy model for diiron proteins, random-acceleration molecular dynamics (RAMD) revealed that a pure four-helix bundle structure, like hemerythrin, constitutes an efficient cage for dioxygen (O2 ), which can only leave from defined, albeit very broad, gates. However, this well ordered structure does not constitute an archetype on which to compare O2 permeation of other diiron proteins, like the complex of soluble methane monooxygenase hydroxylase with the regulatory protein (sMMOH-MMOB). The reason is that with this complex, unlike hemerythrin, the four helices of the four-helix bundle are heavily bent, and RAMD showed that most traps for O2 lie outside them. It was also observed that, in spite of a nearly identical van der Waals radius for O2 and the natural substrate CH4 , the latter behaves under RAMD as a bulkier molecule than O2 , requiring a higher external force to be brought out of sMMOH-MMOB along trajectories of viable length. All that determined with sMMOH-MMOB multiple gates and multiple pathways to each of them through several binding pockets, for both O2 and CH4 . Of the two equally preferred pathways for O2 , at right angle with one another, one proved to be in accordance with the Xe-atom mapping for sMMOH. In contrast, none of the pathways identified for CH4 proved to be in accordance with such mapping, CH4 looking for more open avenues instead.

Effect of tight junction protein Occludin expression and permeability in blood-brain barrier of rats after heat stress

Effect of tight junction protein Occludin expression and permeability in blood-brain barrier of rats after heat stress

Biodegradation and detoxification of chloronitroaromatic pollutant by Cupriavidus

Current study reports isolation of Cupriavidus strain a3 which can utilize 2-chloro-4-nitrophenol (C4NP) as sole source of carbon and nitrogen, leading to its detoxification. Degradation process was initiated by release of nitrite ion resulting in the formation of 2-chlorohydroquinone as intermediate. The nitrite releasing activity was also evident in the cell free protein extract. Different parameters for 2C4NP biodegradation were optimized. The degradation pattern followed Haldane substrate inhibition model with maximum specific degradation rate (qmax) of 0.13/h, half saturation constant (Ks) of 0.05mM, and 2C4NP inhibition constant (Ki) of 0.64mM. The isolate was successfully applied to remediation of 2C4NP-contaminated soil in microcosm study. 2-Dimensional protein electrophoresis analysis showed that growth of the isolate in the presence of 2C4NP resulted in modification of membrane permeability and induction of signal transduction protein. In our knowledge, this is the first study reporting degradation and detoxification of 2C4NP by Cupriavidus.