Methylene Blue Protects Astrocytes against Glucose Oxygen Deprivation by Improving Cellular Respiration

Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration.     

Role of cysteine residues in 4-oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1

4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K(m)-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl(2); and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.     

Anti-Human VEGF Repebody Effectively Suppresses Choroidal Neovascularization and Vascular Leakage

Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness among people over the age of 60. Vascular endothelial growth factor (VEGF) plays a major role in pathological angiogenesis in AMD. Herein, we present the development of an anti- human VEGF repebody, which is a small-sized protein binder consisting of leucine-rich repeat (LRR) modules. The anti-VEGF repebody selected through a phage-display was shown to have a high affinity and specificity for human VEGF. We demonstrate that this repebody effectively inhibits in vitro angiogenic cellular processes, such as proliferation and migration, by blocking the VEGF-mediated signaling pathway. The repebody was also shown to have a strong suppression effect on choroidal neovascularization (CNV) and vascular leakage in vivo. Our results indicate that the anti-VEGF repebody has a therapeutic potential for treating neovascular AMD as well as other VEGF-involved diseases including diabetic retinopathy and metastatic cancers.     

Effects of Individual and Coexisting Diabetes and Cardiomyopathy on Diastolic Function in Rats (Rattus norvegicus domestica)

The goal of this study was to evaluate diastolic intraventricular pressure gradients (IVPG) and 2-dimensional tissue tracking (2DTT) patterns during diabetes and cardiomyopathy. Rats (n = 60) were induced to become diabetic (DM group, n = 15) by using streptozotocin, to become cardiomyopathic (CM group, n = 15) by using isoproterenol, and to become both diabetic and cardiomyopathic (DMCM group, n = 15); control rats (CT group, n = 15) were injected with saline. Two months after induction, all rats underwent conventional echocardiography, IVPG, and 2DTT and then were euthanized for microscopic examination of cardiac fibrosis. Compared with the controls, all 3 treated groups showed diastolic dysfunction and delayed cardiac relaxation. DMCM rats showed the most pronounced cardiac abnormalities. In addition, CM and DMCM groups had showed decreased middle IVPG, whereas DMCM rats had decreased midapical IVPG. Although the overall IVPG of the CM group was normal, the middle segment was significantly decreased. 2DTT results showed that the DMCM group had a delay in relaxation compared with other groups. IVPG and 2DTT can be used to overcome the limitation of conventional echocardiographic methods and reveal diastolic dysfunction. DM worsened diastolic function during cardiac disease.

Diabetes mellitus (DM) is a chronic disease identified by hyperglycemia.¹ DM and associated insulin abnormalities result in hypertension, cardiovascular disease, and multiple organ dysfunction.¹⁸ Diabetic cardiomyopathy is defined as abnormal myocardial structure and performance in the absence of other cardiac risk factors, such as coronary artery disease, hypertension, and significant valvular disease, in patients with DM.³ A previous study showed that diastolic dysfunction could result from DM; and cardiac fibrosis in the interstitial and perivascular areas was a hallmark of long-term DM.²³ Cardiac fibrosis in DM rats results from myocardial infarction.Cardiomyopathy can be induced in rats by using isoproterenol.¹⁷ Cardiomyopathy refers to the cardiac disorder in which myocardial abnormalities result from myocyte injury. Cardiomyopathy has several etiologies, including chronic hypertension, valvular abnormality, and toxin.¹⁵ Both DM and cardiomyopathy are fibrotic heart diseases, but their pathogenesis differs. Moreover, DM and CM cause fibrosis in different heart locations.The diastolic function of the left ventricle (LV) can be defined as the pull of blood into the LV under low filling pressure. Two groups have described the pressure differences that pull the blood through the mitral valve into the ventricle during diastole.^6,16 The difference of pressure between the LV and aorta during systole and the importance of regional pressure differences within the ventricle during relaxation has recently gained attention.^9,29 The diastolic intraventricular pressure gradient (IVPG) in the LV is closely related to LV relaxation. Deterioration of the IVPG is associated with abnormal LV blood flow patterns.²2D tissue tracking (2DTT) echocardiography is a promising technique that quantifies myocardial deformation by tracking the ultrasonographic speckle pattern of the cardiac cycle.¹³ Because of the importance of measuring diastolic function, we used conventional echocardiography, IVPG and 2DTT to evaluate the diastolic impairment without altering systolic function. We studied the relationship between mild (DM rats) and moderate (CM rats) cardiac fibrosis due to myocardial injury and infarction together with the severe fibrosis that develops due to the combination of DM and cardiomyopathy.Streptozotocin and isoproterenol were used to induce DM and cardiomyopathy, respectively. Both cause cardiac fibrosis, but the pathogenesis and affected location are different. Our study was designed to evaluate diastolic IVPG and 2DTT as diastolic indices. Our goal was to delineate IVPG and 2DTT patterns in DM, CM, and very severe fibrotic heart disease induced by combining DM and cardiomyopathy.     

Risk factors for radiographic progression in psoriatic arthritis: subanalysis of the randomized controlled trial ADEPT

Introduction  

To identify independent predictors of radiographic progression in psoriatic arthritis (PsA) for patients treated with adalimumab or placebo in the Adalimumab Effectiveness in PsA Trial (ADEPT).     

Effect of ammonium and nitrate on current generation using dual-cathode microbial fuel cells

These studies were conducted to determine the effects of various concentrations of ammonium and nitrate on current generation using dual-cathode microbial fuel cells (MFCs). Current generation was not affected by ammonium up to 51.8+/-0.0 mg/l, whereas 103.5+/-0.0 mg/l ammonium chloride reduced the current slightly. On the other hand, when 60.0+/-0.0 and 123.3+/-0.1 mg/l nitrate were supplied, the current was decreased from 10.23+/-0.07 mA to 3.20+/-0.24 and 0.20+/-0.01 mA, respectively. Nitrate did not seem to serve as a fuel for current generation in these studies. At this time, COD and nitrate removal were increased except at 123+/-0.1 mg NO(3)(-)/l. These results show that proper management of ammonium and nitrate is very important for increasing the current in a microbial fuel cell.     

Molecular dynamics of synthetic leucine-serine ion channels in a phospholipid membrane

Molecular dynamics calculations were carried out on models of two synthetic leucine-serine ion channels: a tetrameric bundle with sequence (LSLLLSL)(3)NH(2) and a hexameric bundle with sequence (LSSLLSL)(3)NH(2). Each protein bundle is inserted in a palmitoyloleoylphosphatidylcholine bilayer membrane and solvated by simple point charge water molecules inside the pore and at both mouths. Both systems appear to be stable in the absence of an electric field during the 4 ns of molecular dynamics simulation. The water motion in the narrow pore of the four-helix bundle is highly restricted and may provide suitable conditions for proton transfer via a water wire mechanism. In the wider hexameric pore, the water diffuses much more slowly than in bulk but is still mobile. This, along with the dimensions of the pore, supports the observation that this peptide is selective for monovalent cations. Reasonable agreement of predicted conductances with experimentally determined values lends support to the validity of the simulations.     

In vitro amplification of misfolded prion protein using lysate of cultured cells

Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrP(C)). PrP(Sc) was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrP(C) in cell lysate was a critical factor to drive efficient PrP(Sc) amplification, our results demonstrate that cell lysate in which PrP(C) is present abundantly serves as an excellent substrate source for PMCA.     

Effects of SGI-1027 on Formation and Elimination of PrPSc in Prion-Infected Cells

Molecular Biology - Recently, SGI-1027, a well-known inhibitor of DNA-methyl transferases (DNMTs), was reported to effectively reduce formation of pathogenic PrPSc in prion-infected cells. Herein,...