Kinetic characterization and identification of the acylation and glycosylation sites of recombinant human gamma-glutamyltranspeptidase

Gamma-glutamyltranspeptidase (GGT) is a heterodimeric enzyme important for glutathione homeostasis control. It has also been implicated in many physiological disorders, including Parkinson's disease, apoptosis inhibition, and diabetes. In the first step of its ping-pong mechanism it binds glutathione, its in vivo substrate, and releases cysteinylglycine upon formation of an acyl-enzyme intermediate. This intermediate can then react with water to release glutamate as a hydrolysis product or with an amino acid or dipeptide to form a transpeptidation product. Further detailed study of the mechanism underlying these reactions is hindered at least for some GGTs by the low quantities of protein available after a multistep purification from tissue. In the present work the gene for human GGT was cloned into the pPICZalphaA vector and transformed into Pichia pastoris to express as a 68 kDa His-tagged protein. The optimized expression and secretion of this enzyme in 1 L of culture and subsequent purification by immobilized metal affinity chromatography yielded 1.6 mg of purified enzyme having a specific activity of 237 U/mg. Kinetic parameters for the transpeptidation reaction between glutathione and glycylglycine were determined by mass spectrometry, giving a kcat of 13.4 x 10(3) min-1 and apparent KM values of 1.11 mM for glutathione and 8.1 mM for glycylglycine. The GGT-mediated hydrolysis of glutathione was also studied, providing a kcat of 53 min-1 and a KM value of 7.3 microM for glutathione. Incubation of the enzyme with a mechanism-based inhibitor, enzymatic digest, and mass spectrometric analysis provided the first unambiguous identification of Thr381 as the active site nucleophile of human gamma-glutamyltranspeptidase, and confirmed four of the seven N-linked glycosylation sites. These structural and kinetic data are discussed with respect to a homology model generated to facilitate visualization.     

MT1-MMP downregulation via the PI3K/Akt signaling pathway is required for the mechanical stretching-inhibited invasion of bone-marrow-derived mesenchymal stem cells

Mobilization from the bone marrow and the migration of bone-marrow-derived mesenchymal stem cells (BMSCs) through the peripheral circulation to injured tissue sites are regulated by multiple mechanical and chemical factors. We previously demonstrated that mechanical stretching promotes the migration but inhibits the invasion of BMSCs. However, the involved mechanisms, especially the mechanism of stretching-inhibited BMSC invasion, have not been thoroughly elucidated to date. In this study, we found that mechanical stretching with a 10% amplitude at a 1-Hz frequency for 8 hr significantly reduces BMSC invasion and downregulates the expression of membrane type-1 matrix metalloproteinases (MT1-MMP) at both the messenger RNA and protein levels. The overexpression of MT1-MMP restores mechanical stretching-reduced BMSC invasion. Moreover, phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation in BMSCs was found to be inactivated by mechanical stretching. Pharmacological inhibitors of PI3K/Akt signaling (LY294002 or A443654) reduced the expression of MT1-MMP and impaired BMSC invasion. In addition, the upregulation of Akt phosphorylation by a pharmacological activator (SC79) increased MT1-MMP expression and suppressed mechanical stretching-reduced BMSC invasion. Taken together, our results suggest that mechanical stretching inhibits BMSC invasion by downregulating MT1-MMP expression by suppressing the PI3K/Akt signaling pathway.     

The convenient use of fluorescamine for spectrofluorimetric determination of midodrine hydrochloride in pure form and its tablets formulation: Application to content uniformity testing

A novel sensitive and simple spectrofluorimetric method was developed then validated for determination of midodrine in both its authentic pure form and its tablets. This method is based on the reaction between midodrine's aliphatic primary amine moiety with fluorescamine reagent, using borate buffer at pH 7.8 and yielding a highly fluorescent product whose fluorescence intensity was measured at 462 nm after excitation at 388 nm. This method represents the first attempt for determination of midodrine spectrofluorimetrically. A calibration curve was constructed showing that the linear range was 0.2–3.0 μg/ml. The limit of detection and limit of quantitation values were 0.06 and 0.19 μg/ml respectively. The correlation coefficient (r) and the determination coefficient (r²) values were 0.9992 and 0.9984 respectively. The proposed method was validated according to ICH guidelines and successfully applied for determination of midodrine in its tablets with an overall % recovery of 99.56 ± 0.95. Finally, the presented method was adapted to study the content uniformity test according to United States Pharmacopeia guidelines.     

A Facile Thermal-Treatment Route to Synthesize ZnO Nanosheets and Effect of Calcination Temperature

A facile thermal-treatment route was successfully used to synthesize ZnO nanosheets. Morphological, structural, and optical properties of obtained nanoparticles at different calcination temperatures were studied using various techniques. The FTIR, XRD, EDX, SEM and TEM images confirmed the formation of ZnO nanosheets through calcination in the temperature between 500 to 650°C. The SEM images showed a morphological structure of ZnO nanosheets, which inclined to crumble at higher calcination temperatures. The XRD and FTIR spectra revealed that the samples were amorphous at 30°C but transformed into a crystalline structure during calcination process. The average particle size and degree of crystallinity increased with increasing calcination temperature. The estimated average particle sizes from TEM images were about 23 and 38 nm for the lowest and highest calcination temperature i.e. 500 and 650°C, respectively. The optical properties were determined by UV–Vis reflection spectrophotometer and showed a decrease in the band gap with increasing calcination temperature.     

The Effects of Comprehensive Warm-Up Programs on Proprioception,Static and Dynamic Balance on Male Soccer Players

Purpose

The study investigated the effects of FIFA 11+ and HarmoKnee, both being popular warm-up programs, on proprioception, and on the static and dynamic balance of professional male soccer players.

Methods

Under 21 year-old soccer players (n = 36) were divided randomly into 11+, HarmoKnee and control groups. The programs were performed for 2 months (24 sessions). Proprioception was measured bilaterally at 30°, 45° and 60° knee flexion using the Biodex Isokinetic Dynamometer. Static and dynamic balances were evaluated using the stork stand test and Star Excursion Balance Test (SEBT), respectively.

Results

The proprioception error of dominant leg significantly decreased from pre- to post-test by 2.8% and 1.7% in the 11+ group at 45° and 60° knee flexion, compared to 3% and 2.1% in the HarmoKnee group. The largest joint positioning error was in the non-dominant leg at 30° knee flexion (mean error value = 5.047), (p<0.05). The static balance with the eyes opened increased in the 11+ by 10.9% and in the HarmoKnee by 6.1% (p<0.05). The static balance with eyes closed significantly increased in the 11+ by 12.4% and in the HarmoKnee by 17.6%. The results indicated that static balance was significantly higher in eyes opened compared to eyes closed (p = 0.000). Significant improvements in SEBT in the 11+ (12.4%) and HarmoKnee (17.6%) groups were also found.

Conclusion

Both the 11+ and HarmoKnee programs were proven to be useful warm-up protocols in improving proprioception at 45° and 60° knee flexion as well as static and dynamic balance in professional male soccer players. Data from this research may be helpful in encouraging coaches or trainers to implement the two warm-up programs in their soccer teams.     

The protection of selenium on adriamycin-induced mitochondrial damage in rat

Although adriamycin (ADR) exhibits high anti-tumor efficacy in vitro, its clinical use in cancer chemotherapy is limited due to its high renal toxicity. This study investigated the mechanism of ADR nephropathy and the protective effect of selenium on ADR-induced kidney damage by analyzing of the relationship between selenium and mitochondria. Rats were divided into four groups. The first group was injected with saline i.p. for 21 days, the second group received the 4 mg/kg i.p. ADR every alternate day for 8 days, the third group received the 50 μg/kg i.p. Se for 21 days, and the fourth group received the Se. ADR co-administration i.p. blood pressures were assessed, the mitochondrial membrane potential (MMP) was assessed, and the adenosine triphosphate (ATP) levels were determined. The total antioxidant (TAS) and oxidant status (TOS) in cytosol, the mitochondria of kidney cells, and plasma were measured. Mitochondrial TAS decreased and TOS increased in the ADR group compared to the Se group. ADR-treated rats showed significantly lower MMP than did the control and Se groups. MMP was significantly restored in the Se + ADR group through selenium treatment compared to the ADR group (p < 0.01). In the ADR group, a reduction in ATP content was seen compared to the control and Se groups (p < 0.01). ATP level was significantly restored through treatment with selenium in the Se + ADR group compared to the ADR group (p < 0.01). We concluded that selenium is effective in vivo against ADR-induced kidney damage via the restoration of TAS and TOS, which prevented mitochondrial damage.     

High Fill Factor Polymer Solar Cells Incorporating a Low Temperature Solution Processed WO3 Hole Extraction Layer

We demonstrate solution‐processed tungsten trioxide (WO₃) incorporated as hole extraction layer (HEL) in polymer solar cells (PSCs) with active layers comprising either poly(3‐hexylthiophene) (P3HT) or poly[(4,4'‐bis(2‐ethylhexyl)dithieno[3,2‐b:2′,3′‐d]silole)‐2,6‐diyl‐alt‐(4,7‐bis(2‐thienyl)‐2,1,3‐benzothiadiazole)‐5,50‐diyl] (Si‐PCPDTBT) mixed with a fullerene derivative. The WO₃ layers are deposited from an alcohol‐based, surfactant‐free nanoparticle solution. A short, low‐temperature (80 °C) annealing is sufficient to result in fully functional films without the need for an oxygen‐plasma treatment. This allows the application of the WO₃ buffer layer in normal as well as inverted architecture solar cells. Normal architecture devices based on WO₃ HELs show comparable performance to the PEDOT:PSS reference devices with slightly better fill factors and open circuit voltages. Very high shunt resistances (over 1 MΩ cm²) and excellent diode rectification underline the charge selectivity of the solution‐processed WO₃ layers.