Cell‐free production of a therapeutic protein: Expression,purification, and characterization of recombinant streptokinase using a CHO lysate

The use of cell‐free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell‐free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell‐free system using instrumented mini‐bioreactors for highly reproducible protein production. We achieved recombinant protein production (～600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell‐free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag‐free self‐cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose‐level production of therapeutic proteins at the point‐of‐care.     

Characterizing Brain Structures and Remodeling after TBI Based on Information Content,Diffusion Entropy

Background

To overcome the limitations of conventional diffusion tensor magnetic resonance imaging resulting from the assumption of a Gaussian diffusion model for characterizing voxels containing multiple axonal orientations, Shannon''s entropy was employed to evaluate white matter structure in human brain and in brain remodeling after traumatic brain injury (TBI) in a rat.

Methods

Thirteen healthy subjects were investigated using a Q-ball based DTI data sampling scheme. FA and entropy values were measured in white matter bundles, white matter fiber crossing areas, different gray matter (GM) regions and cerebrospinal fluid (CSF). Axonal densities'' from the same regions of interest (ROIs) were evaluated in Bielschowsky and Luxol fast blue stained autopsy (n = 30) brain sections by light microscopy. As a case demonstration, a Wistar rat subjected to TBI and treated with bone marrow stromal cells (MSC) 1 week after TBI was employed to illustrate the superior ability of entropy over FA in detecting reorganized crossing axonal bundles as confirmed by histological analysis with Bielschowsky and Luxol fast blue staining.

Results

Unlike FA, entropy was less affected by axonal orientation and more affected by axonal density. A significant agreement (r = 0.91) was detected between entropy values from in vivo human brain and histologically measured axonal density from post mortum from the same brain structures. The MSC treated TBI rat demonstrated that the entropy approach is superior to FA in detecting axonal remodeling after injury. Compared with FA, entropy detected new axonal remodeling regions with crossing axons, confirmed with immunohistological staining.

Conclusions

Entropy measurement is more effective in distinguishing axonal remodeling after injury, when compared with FA. Entropy is also more sensitive to axonal density than axonal orientation, and thus may provide a more accurate reflection of axonal changes that occur in neurological injury and disease.     

Combination effect of exercise training and eugenol supplementation on the hippocampus apoptosis induced by chlorpyrifos

The aim of this study was to investigate the combination effect of exercise training and eugenol supplementation on the hippocampus apoptosis induced by CPF. 64 adult male albino rats were randomly selected and devided into eight groups of eight including: control, exercise (EXE), chlorpyrifos (CPF), Control?+?Oil (Co?+?Oil), Control?+?DMSO (Co?+?DMSO), chlorpyrifos?+?eugenol (CPF?+?Sup), chlorpyrifos?+?exercise (CPF?+?Exe) and, chlorpyrifos?+?exercise?+?eugenol (CPF?+?Exe?+?Eu). Four experimental groups received intraperitoneal injection (5 days a week) of 3.0 mg/kg body weight CPF in DMSO for 6 consecutive weeks. The exercise groups performed aerobic 5 days per week over 4 weeks. Eugenol were administered by gavage. Finally, the animals were sacrificed using CO₂ gas (a half of the rats were anesthetized with ketamine and xylazine and then perfused) to evaluate hippocampus histology and parameters. The results of this study showed that CPF injection significantly decreased BDNF, AChE and ATP in CA1 area of the hippocampus (p ? 0.05). Also, CA1 apoptosis by tunnel assay, it was found that CPF receiving groups with different dosage, showed a significant increase compared to other groups, which was confirmed by increasing cytochrome C and procaspase-3 in CPF groups (p ? 0.05). The result of this study show that 4 weeks of exercise training and eugenol supplementation does not improve the destructive effects of CPF in CA1 area of the hippocampus. As a result, it is recommended that future studies longer periods for treatment with exercise and eugenol supplementation.

     

Diffusion of MRI and CT Contrast Agents in Articular Cartilage under Static Compression

Cartilage has a limited capacity for self-repair and focal damage can eventually lead to complete degradation of the tissue. Early diagnosis of degenerative changes in cartilage is therefore essential. Contrast agent-based computed tomography and magnetic resonance imaging provide promising tools for this purpose. However, the common assumption in clinical applications that contrast agents reach steady-state distributions within the tissue has been of questionable validity. Characterization of nonequilibrium diffusion of contrast agents rather than their equilibrium distributions may therefore be more effective for image-based cartilage assessment. Transport of contrast agent through the extracellular matrix of cartilage can be affected by tissue compression due to matrix structural and compositional changes including reduced pore size and fluid content. We therefore investigate the effects of static compression on diffusion of three common contrast agents: sodium iodide, sodium diatrizoate, and gadolinium diethylenetriamine-pentaacid (Gd-DTPA). Results showed that static compression was associated with significant decreases in diffusivities for sodium iodide and Gd-DTPA, with similar (but not significant) trends for sodium diatrizoate. Molecular mass of contrast agents affected diffusivities as the smallest one tested, sodium iodide, showed higher diffusivity than sodium diatrizoate and Gd-DTPA. Compression-associated cartilage matrix alterations such as glycosaminoglycan and fluid contents were found to correspond with variations in contrast agent diffusivities. Although decreased diffusivity was significantly correlated with increasing glycosaminoglycan content for sodium iodide and Gd-DTPA only, diffusivity significantly increased for all contrast agents by increasing fluid fraction. Because compounds based on iodine and gadolinium are commonly used for computed tomography and magnetic resonance imaging, present findings can be valuable for more accurate image-based assessment of variations in cartilage composition associated with focal injuries.     

Clinical grade cultivation of human Schwann cell, by the using of human autologous serum instead of fetal bovine serum and without growth factors

Clinical grade cultivation of human schwann cell by the utilization of human autologous serum instead of fetal bovine serum, and also avoiding any growth factors, can increase safety level of this procedure in cases of clinical cell transplantation. The aim of this study was demonstration of the feasibility of clinical grade schwann cell cultivation. In this experimental study after obtaining consent from close relatives we harvested 10 sural nerves from brain death donors and then cultured in 10 seperated culture media plus autologous serum. We also prepared autologous serum from donor??s whole blood. Then cultured cells were evaluated by S100 antibody staining for both morphology and purity. Cell purity range was from 97% to 99% (mean?=?98.11?±?0.782%). Mean of the cell count was 14,055.56?±?2,480.479 per micro liter. There was not significant correlation between cell purity and either the culture period or the age of donors (P?>?0.05). The spearman correlation coefficient for the cell purity with the period or the age of donors was 0.21 and 0.09, respectively. We demonstrated the feasibility of clinical grade schwann cell cultivation by the using of human autologous serum instead of fetal bovine serum and also without the using of growth factors. We also recommended all cell preparation facilities to adhere to the GMP and other similar quality disciplines especially in the preparation of clinically-used cell products.     

Synthesis of 2-deoxy-2-fluoro and 1,2-ene derivatives of the naturally occurring glycosidase inhibitor, salacinol, and their inhibitory activities against recombinant human maltase glucoamylase

2-Deoxy-2-fluorosalacinol and a 1,2-ene derivative of the naturally occurring glycosidase inhibitor salacinol were synthesized for structure activity studies with human maltase glucoamylase (MGA). 2-Deoxy-2-fluorosalacinol was synthesized through the coupling reaction of 2-deoxy-2-fluoro-3,5-di-O-p-methoxybenzyl-1,4-anhydro-4-thio-D-arabinitol with 2,4-O-benzylidene-l-erythritol-1,3-cyclic sulfate in hexafluoroisopropanol (HFIP) containing 0.3 equiv of K(2)CO(3). Excess of K(2)CO(3) resulted in the elimination of HF from the coupled product, and the formation of an alkene derivative of salacinol. Nucleophilic attack of the 1,4-anhydro-4-thio-D-arabinitol moiety on the cyclic sulfate did not proceed in the absence of K(2)CO(3). No reaction was observed in acetonitrile containing K(2)CO(3). The target compounds were obtained by deprotection with TFA. The 2-deoxy-1-ene derivative of salacinol and 2-deoxy-2-fluorosalacinol inhibited recombinant human maltase glucoamylase, one of the key intestinal enzymes involved in the breakdown of glucose, with an IC(50) value of 150 microM and a K(i) value of 6+/-1 microM, respectively.     

Memantine inhibits and reverses the Alzheimer type abnormal hyperphosphorylation of tau and associated neurodegeneration

Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, reduces the clinical deterioration in moderate-to-severe Alzheimer disease (AD) for which other treatments are not available. The activity of protein phosphatase (PP)-2A is compromised in AD brain and is believed to be a cause of the abnormal hyperphosphorylation of tau and the consequent neurofibrillary degeneration. Here we show that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation and accumulation of tau in organotypic culture of rat hippocampal slices. Such restorative effects of memantine were not detected either with 5,7-dichlorokynurenic acid or with D(-)-2-amino-5-phosphopentanoic acid, NMDA receptor antagonists active at the glycine binding site and at the glutamate binding site, respectively. These findings show (1) that memantine inhibits and reverses the PP-2A inhibition-induced abnormal hyperphosphorylation of tau/neurofibrillary degeneration and (2) that this drug might be useful for the treatment of AD and related tauopathies.     

Homer modulates NFAT-dependent signaling during muscle differentiation

While changes in intracellular calcium are well known to influence muscle contraction through excitation contraction coupling, little is understood of the calcium signaling events regulating gene expression through the calcineurin/NFAT pathway in muscle. Here, we demonstrate that Ca(+2) released via the inositol trisphosphate receptor (IP3R) increases nuclear entry of NFAT in undifferentiated skeletal myoblasts, but the IP3R Ca(+2) pool in differentiated myotubes promotes nuclear exit of NFAT despite a comparable quantitative change in [Ca(+2)]i. In contrast, Ca(+2) released via ryanodine receptors (RYR) increases NFAT nuclear entry in myotubes. The scaffolding protein Homer, known to interact with both IP3R and RYR, is expressed as part of the myogenic differentiation program and enhances NFAT-dependent signaling by increasing RYR Ca(+2) release. These results demonstrate that differentiated skeletal myotubes employ discrete pools of intracellular calcium to restrain (IP3R pool) or activate (RYR pool) NFAT-dependent signaling, in a manner distinct from undifferentiated myoblasts. The selective expression of Homer proteins contributes to these differentiation-dependent features of calcium signaling.