Coomassie Brilliant Blue Method Research Articles

Determination of cyclic adenosine phosphate and protein content in dormant chlamydospore and nondormant chlamydospore of Arthrobotrys flagrans.

Arthrobotrys flagrans, a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and nondormant chlamydospore of A. flagrans to investigate the differences in cyclic adenosine monophosphate (cAMP) and protein content between the two types of spores. cAMP and soluble proteins were extracted from the nondormant chlamydospore and dormant chlamydospore of two isolates of A. flagrans. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in nondormant spores (p < 0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of nondormant spores (p < 0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of nondormant spores, but the difference was not significant (p > 0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus A. flagrans.

Effects of Ginsenoside Rg1 on the Biological Activity of Human Periodontal Ligament Cells.

, Objective. To investigate the effect of ginsenoside Rg1 on the biological activity of primary cultured human periodontal ligament cells (PDLC). Methods. The effects of ginsenoside Rg1 on the proliferation activity, protein synthesis, and alkaline phosphatase (ALP) activity of primary cultured human periodontal ligament cells were investigated by thiazole blue (MTT) colorimetric method, Coomassie brilliant blue method, and enzyme kinetics method. The effect of ginsenoside Rg1 on cell cycle was detected by flow cytometry, and the cells were labeled with calcium ion-sensitive fluorescent probe Fluo3/AM, and the effect of ginsenoside Rg1 on intracellular free calcium concentration was detected by laser scanning confocal microscope. Results. Compared with the control group, the experimental groups of ginsenoside Rg1 at various concentrations could significantly promote cell proliferation, and the effect time was the longest in the concentration range of 0.01-0.05 μmol/L;, Rg1 0.01umol/L and 0.05umol/L. The protein content in the 72-hour cell culture medium of the μmol/L group was significantly higher than that of the control group; the ALP activity in the 72-hour cell culture medium of the Rg1 0.01 μmol/L, 0.05 μmol/L, and 0.1 μmol/L groups was significantly higher than that of the control group; FCM assay showed that after 0.1 μmol/L Rg1 for 48 hours, compared with the control group, the proportion of cells in the early stage of DNA synthesis (G1%) of PDLC was significantly reduced, while the proportion of cells in the DNA synthesis stage (S%) and the value of cell proliferation index PrI (S + G2M)% were significantly increased; Rg1 increased intracellular calcium in PDLC cells at first and then decreased and finally maintained at a slightly higher resting calcium level than before drug addition. Conclusion. Ginsenoside Rg1 can increase the proliferation activity, protein synthesis, and alkaline phosphatase activity of periodontal ligament cells within a certain concentration range; Rg1 reduces the cells in G1 phase and increases cells in S phase of periodontal ligament fibroblasts. Change the concentration of free calcium ions in cells and promote more cells to enter a proliferative state.

Modification in Silver Staining Procedure for Enhanced Protein Staining.

Silver staining is an excellent technique for detecting proteins that are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein silver staining technology has higher sensitivity and is suitable for the detection of low-concentration proteins compared to other staining techniques including the Coomassie brilliant blue detection method. The present study was conducted to enhance the detection ability of the protein staining method. Herein, we modified the recipe of silver staining, a very reproducible method, by adding AMP, PVP, Tween-80, and xylene to enhance the detection ability of protein staining. Furthermore, the particle size and potentiometer were used to detect the particle size and potential difference of the silver ions in the prepared dyeing materials, and then, the morphology, transparency, and size of the dyed silver particles in different dyeing solutions were studied using a transmission electron microscopy (TEM). The obtained results revealed that the use of 0.5% of AMP, PVP, Tween-80, and xylene improved the staining ability of protein silver staining, compared with the original method. Furthermore, 0.5% AMP, 0.5% PVP, 0.5% Tween-80 reagents significantly influenced the morphology, size, potential, and dispersion of silver ions. These results suggested a new idea for further improving the detection ability of protein silver staining.

Effect and mechanism of Jingqi Yukui Capsules on gastric ulcer mucosa healing quality: based on network pharmacology and animal experiment

This study aims to identify the active components and the mechanism of Jingqi Yukui Capsules(JQYK) in the treatment of gastric ulcer based on network pharmacology, and verify some key targets and signaling pathways through animal experiment. To be specific, first, the active components and targets of JQYK were retrieved from a Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM) and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the targets of gastric ulcer from GeneCards and Online Mendelian Inheritance in Man(OMIM) with the search term &quot;gastric ulcer&quot;. The common targets of the two were the potential targets of the prescription for the treatment of the di-sease. Then, protein-protein interaction(PPI) network of key targets were constructed based on STRING and Cytoscape 3.7.2, followed by Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment by matescape database and pathway visualization by Omicshare. For the animal experiment, the improved method of Okabe was used to induce gastric ulcer in rats, and the model rats were classified into the model group, JQYK high-dose(JQYK-H), medium-dose(JQYK-M), and low-dose(JQYK-L) groups, Anweiyang Capsules(WYA) group, and Rabeprazole Sodium Enteric Capsules(RBPZ) group. Normal rats were included in the blank group. Rats in the blank group and model group were given distilled water and those in the administration groups received corresponding drugs. Then gastric ulcer healing in rats was observed. The changes of the gastric histomorphology in rats were evaluated based on hematoxylin-eosin(HE) staining, and the content of inducible nitric oxide synthase(iNOS) in rat gastric tissue was detected with Coomassie brilliant blue method. The mRNA and protein levels of some proteins in rat gastric tissue were determined by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot(WB) to further validate some key targets and signaling pathways. A total of 206 active components and 535 targets of JQYK, 1 305 targets of gastric ulcer, and 166 common targets of the disease and the drug were yielded. According to PPI analysis and KEGG pathway enrichment analysis, multiple key targets, such as interleukin-6(IL-6), tumor necrosis factor(TNF), mitogen-activated protein kinase 1(MAPK1), MAPK3, and MAPK14, as well as nuclear factor kappa-B(NF-κB) signaling pathway, IL-17 signaling pathway, and leukocyte transendothelial migration in the top 20 key signaling pathways were closely related to inflammation. The key protein p38 MAPK and NF-κB signaling pathway were selected for further verification by animal experiment. The gastric ulcer in the JQYK-H group recovered nearly to the level in the blank group, with significant decrease in the content of iNOS in rat gastric tissue and significant reduction in the mRNA and phosphorylation levels of p38 MAPK and the mRNA and protein levels of NF-κB p65 in rat gastric tissue. The results indicated that JQYK can inhibit the phosphorylation of the key protein p38 MAPK and the expression of NF-κB p65 in the NF-κB signaling pathway, thereby exerting the anti-inflammatory effect and effectively improving the quality of gastric ulcer healing in rats. Thus, the animal experiment result verifies some predictions of network pharmacology.

Tripterygium glycoside ameliorates kidney injury in diabetic rats by regulating the PI3K/Akt signaling pathway

To discuss protective effect of Tripterygium Glycoside (TG) on renal injury in diabetic rats and preliminary exploration of its mechanism. The rats were divided into Normal, Model, Low, Middle and High groups. Measuring FBG, TG, TC, HDL-C, BUN and Scr concentration by ELISA assay; using coomassie brilliant blue method to measure 24 h Urine protein. Measuring Kidney index; using HE staining to observation renal histomorphological changes; The ultrastructural changes of renal tissue were observed by transmission electron microscope; TUNEL staining was used to detect the apoptosis of rat renal cells; using WB assay to evaluate PTEN, PI3K, Akt, Bcl-2, Bax and caspase-3 expression. Compared with Normal group, FBG, TG and TC concentrations significantly increased and HDL-C concentration significantly decreased (P < 0.001); BUN, Scr and 24 h Urine protein significantly up-regulated (P < 0.001); Kidney weight and KI significantly down-regulated; Kidney injury score and apoptosis cell number significantly increased (P < 0.001) with PTEN, Bcl-2 protein downregulation and PI3K, Akt, Bax and caspase-3 protein expression significantly up-regulation in Model group (P < 0.001). With TG supplement, the kidney injury significantly improved. TG improved diabetic induced kidney injury via PI3K/Akt pathway in vivo.

Study on self-assembly of novel hydrophilic silane containing PEG segments and its resistance to protein adsorption

A new type of hydrophilic silane containing PEG (polyethylene glycol) segments was produced in this study. Membranes were produced through self-assembly. Hydrophilic silane was characterized by FTIR, 1H-NMR; The chemical composition and surface morphology of the self-assembled membrane surface were characterized by AFM, CA, SEM; The material’s resistance to protein adsorption was studied by UV-Vis and coomassie brilliant blue method [1]. The results showed that the novel hydrophilic silane containing PEG segments can be successfully assembled on silicon substrates; self-assembled membranes and blank silicon substrates in comparison, protein adsorption decreased from 274.8 μg/cm2 to 145.7 μg/cm2. This study has important reference significance for the preparation of anti-protein adsorption and bacterial adhension materials.

Comparison of dye-based proteinuria estimation methods with immunoturbidimetric microalbuminuria estimation, in screening for diabetic

We compared the performance of Coomassie Brilliant Blue (CBB) and Ponceau S dyes based proteinuria estimation methods with immunoturbidimetry microalbumin estimation to develop new cut-offs for proteinuria, which would help us detect diabetic nephropathy patients with better sensitivity using the above mentioned dye based methods. These proteinuria testing methods are easy to adapt in smaller laboratories typically associated with peripheral government health centers. Urine sample was collected from 158 diabetic individuals and tested simultaneously for Urine Albumin Creatinine ratio (UACR) using immunoturbidimetry and Urine Protein Creatinine Ratio (UPCR) using Ponceau S method and CBB method. The Ponceau S method gives good correlation with immunoturbidimetry, for screening of diabetic nephropathy, at UPCR cut-off of 50mg/g creatinine. The CBB method gives higher false positives. The overall agreement of Ponceau S and CBB with immunotubidimetry is maximum at cut off 50mg/g, i.e. 91% and 84% respectively. In conclusion, proteinuria estimation with Ponceau S method with UPCR >50mg/g creatinine, may be used for routine screening of diabetic patients to diagnose microalbuminuria. Keywords: Diabetic nephropathy screening, Proteinuria, Microalbuminuria, Ponceau S.

Hot Water Extraction of Crude Polysaccharide from Codonopsis pilosula and Determination of the Rheological Properties

The extraction condition of crude polysaccharides from Codonopsis pilosula by hot water reflux method was studied and the results analyzed by kinetic model. Then, the extraction rate and maximum extraction temperature were determined. furthermore, the thermal and pH stability and the rheological properties of the crude polysaccharide were also studied. crude Codonopsis pilosula polysaccharide (CCPP) under different conditions was extracted by hot water reflux method, the content was determined by phenol-sulfuric acid and DNS method, and the kinetic model of the extraction process was established. The viscosity was determined under different solute concentration, salt concentration, rotation speed, pH, and temperature. The thermal stability and pH stability were determined; the protein content and sulfate ion content were determined by the coomassie brilliant blue method and barium sulfate turbidimetry method, respectively. The yield of CCPP by the hot water reflux method was 25.72%. At 50 oC, the maximum extraction concentration, the rate constant and the linear relationship between the rate constant and the time were obtained at different ratios of material to liquid. The extraction process of this experiment coincided with Fick`s second law, R2 was greater than 0.88 and with the increase of extraction time, the dissolution rate increased, but with the increase of the ratio of material to liquid, the dissolution rate was decreased. CCPP showed a loss of polysaccharide fluidity as temperature increased and was affected by pH change. The apparent viscosity was affected by a change in solute concentration, temperature, pH, rotation speed and salt and sucrose concentrations.

Effects of arbuscular mycorrhizal fungi on the repair capacity of perennial ryegrass (Lolium perenne L.) in the uranium-containing soils

This research inclines to study the ramification on arbuscular mycorrhizal fungi (AMF), including Glomusclaroideum (G. claroideum) and G. mosseae strains as well as the repair process of perennial ryegrass (Lolium perenne L.) in the uranium-containing soils. Three times at 120°C for an hour was required to disinfect the soil mixture. Spraying the uranium (U3O8) into the soil mixture and mixing until the ingredients were thoroughly blended, serving the purpose of obtaining the uranium-containing soil mixture with 5 mg/kg U3O8. A week later, AMF was inoculated into every flowerpot and chamber facility. Seeds of perennial ryegrass were cultivated with thirty seeds in each flowerpot or each indoor equipment in a fortnight’s time. Perennial ryegrass plants tended to be harvested after sowing and meeting the conditions of 60 days. Three substances are detected by three different detection means(Photosynthetic pigments, soluble proteins, and malondialdehyde correspond to ethanol extraction method, Coomassie brilliant blue method, and thiobarbituric acid method, respectively). Additionally, testing included activities of alkaline phosphatase and succinate dehydrogenase as well. Moreover, Hitachi H-7650 transmission electron microscope performed the function of observing cellular and subcellular distributions of uranium in plant epidermal cells. Photosynthetic pigment standards of perennial ryegrass are subject to AMF, which not only has a tendency to enhance the soluble protein levels in perennial ryegrass, but also gains the possibility of holding up the uranium-induced improvement of malondialdehyde levels in perennial ryegrass. G. mosseaes is properly more efficacious than G. claroideum in the attenuation of uranium damages in cell structures of perennial ryegrass. Our results demonstrate that AMF is capable of developing the capacity of perennial ryegrass on repairing the uranium-related soil contamination.

Determination of lignin concentration in bleaching effluent via Coomassie brilliant blue G-250 method

Kraft pulp and alkali peroxide mechanical pulp were prepared with aspen and wheat straw and bleached with chlorine dioxide, an alkali extraction strengthened by hydrogen peroxide, hydrogen peroxide, or peroxyacetic acid. Eight different bleaching effluents were obtained, and a Coomassie brilliant blue G-250 method was applied to determine the lignin content in each of the bleaching effluents. The visible light spectra indicated that the alkali-soluble lignin and the alkali bleaching effluent could increase the absorbance of Coomassie brilliant blue G-250 at 640 nm, but the acidic soluble lignin and the acidic bleaching effluent did not increase absorbance. Thus, the Coomassie brilliant blue G-250 method applied to alkali bleaching effluents but did not apply to acidic bleaching effluents. An analysis of the pH, particle size, zeta potential, and visible light spectra determination showed the Coomassie brilliant blue G-250 absorbed on lignin particles, which caused the absorbance increase at 640 nm. With the actual measured value of the lignin content obtained via the Klason lignin method, the relative error of the results obtained via the Coomassie brilliant blue G-250 method was less than or equal to 3.8%.

Repair capacity of perennial ryegrass (Lolium perenne L.) based on arbuscular mycorrhizal fungi on the in uranium contaminated soil

This study is to research effects of arbuscular mycorrhizal fungi (AMF) on the repair capacity of perennial ryegrass (Lolium perenne L.) in the uranium-containing soils. Perennial ryegrass plants were harvested 60 days after sowing in soils. Photosynthetic pigments, soluble proteins, and malondialdehyde were determined by ethanol extraction method, Coomassie brilliant blue method, and thiobarbituric acid method, respectively. Activities of alkaline phosphatase and succinate dehydrogenase were also determined. And, cellular and subcellular distributions of uranium in plant epidermal cells were observed with Hitachi H-7650 transmission electron microscope. AMF influents photosynthetic pigment levels of perennial ryegrass. AMF increases the soluble protein levels in perennial ryegrass and represses the uranium-induced increase of malondialdehyde levels in perennial ryegrass. It suggested that AMF can effectively increase the ability of perennial ryegrass on repairing the uranium-related soil contamination.

Effects of glutamine on skeletal muscle membrane repair in severely burned mice and the functional mechanism

Objective: To observe how glutamine affect the skeletal muscle membrane repair in severely burned mice through promoting the mitsugumin 53 (MG53) dimerization in skeletal muscle and to explore its functional mechanism. Methods: (1) Animal experiments. A total of 179 BALB/c male mice aged 6 to 8 weeks were divided into sham injury group (n=43), burn group (n=73) and burn+ glutamine group (n=63) according to the random number table (the same grouping method below). Mice in sham injury group were sham injured on the back, and mice in burn group and burn+ glutamine group were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Mice in burn+ glutamine group were intragastrically administered with glutamine (1 mg/kg), and the other two groups were given the same amount of amino acid solution once per day for 14 days. On post burn hour 12, 10 mice from burn group were taken for preparation of burn serum, which is used in the following cell experiments. Blood samples were collected from the hearts to prepare serum from 10 mice in sham injury group immediately after burn and from 10 mice in burn group and burn+ glutamine group on post burn day (PBD) 5, 10, and 14, respectively. And then the whole gastrocnemius muscle was harvested after the mice were sacrificed. On PBD 10, the whole flexor brevis digitorum was harvested from 6 mice in the 3 groups respectively after the mice were sacrificed. On PBD 5, 10, and 14, the whole gastrocnemius muscle tissue was harvested from another 9 mice in the 3 groups respectively after the mice were sacrificed. The mass of the whole gastrocnemius muscle of mice was weighed. The total protein content of gastrocnemius muscle of mice was detected by coomassie brilliant blue method. The repair function of myolemma of flexor brevis digitorum of mice was detected by two-photon laser fiber membrane perforating. The serum content of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) of mice was determined with radioimmunoassay. The expressions of MG53 dimer and monomer in gastrocnemius of mice were determined with non-reductive electrophoresis-Western blotting. The protein expressions of endoplasmic reticulum stress sign proteins CCAAT/enhancer binding protein homologous protein (CHOP) and glucose-regulated protein 78 (GRP78) in gastrocnemius of mice were determined with Western blotting. (2) Cell experiments. Mice skeletal muscle precursor cells C2C12 were cultured in vitro, and cells of the second passage were selected for the experiments. The cells were divided into normal control group, burn serum group, and burn serum+ glutamine group, with 3 dishes in each group and 1×10(3) cells in each dish. Cells in normal control group were cultured with 1 mL Dulbecco's modified Eagle medium (DMEM) with fetal bovine serum of volume fraction 10%, cells in burn serum group were cultured with 1 mL DMEM with burn serum of volume fraction 10%, and cells in burn serum+ glutamine group were cultured with 1 mL DMEM with burn serum of volume fraction 10% and 4 μL glutamine with a final molar concentration of 8 mmol/L. After 24 hours of culturing, the repair function of myocyte membrane after differentiation of skeletal muscle precursor cells in mice was detected with the same method before. Another cells were grouped and cultured as before, with 3 wells in each group and 1×10(5) cells in each well. After 24 hours of culturing, the expressions of MG53 dimer and monomer and endoplasmic reticulum stress marker proteins in the cells were detected as before. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least significant difference t test, and Student Newman Keuls test. Results: Animal experiments. (1) Compared with those in sham injury group, the mass and total protein content of gastrocnemius muscle of mice in burn group were significantly decreased on PBD 5, 10, and 14 (P 0.05). Compared with 58.5±1.8 in normal control group, the expression of MG53 dimer of cells in burn serum group was significantly decreased after 24 hours of culturing (14.1±1.4, P<0.05). Compared with that in burn serum group, the expression of MG53 dimer of cells in burn serum+ glutamine group was significantly increased after 24 hours of culturing (30.9±0.6, P<0.05). Compared with those in normal control group, the protein expressions of CHOP and GRP78 of cells were significantly elevated in burn serum group after 24 hours of culturing (P<0.05). Compared with those in burn serum group, the protein expressions of CHOP and GRP78 of cells were significantly reduced in burn serum+ glutamine group after 24 hours of culturing (P<0.05). Conclusions: Glutamine can promote MG53 dimerization by alleviating endoplasmic reticulum stress in severely burned mice. Thus it can accelerate skeletal muscle membrane repair, reduce the local inflammatory reaction of skeletal muscle and consumption of skeletal muscle.

PO-071 Effects of Resistance Training of Different Loads on Gastrocnemius Protein, IGF-I and TNF-α in Aging Rats

Objective To observe the effects of different load resistance training on skeletal muscle dystrophy in aging rats by observing the expression of protein and protein of IGF-I and TNF-α in aging rats after 8 weeks of different load resistance training. The resistance training delays the theoretical basis for anti-skeletal muscle aging.&#x0D; Methods 40 aging SD rats (18 months old) were randomly divided into a quiet control group, a non-weight-bearing exercise group, 30% of the largest negative recombination, 50% of the largest negative recombination, and 70% of the largest negative recombination. The sports group carried the treadmill movement with the tail load, the slope was 35°, the running speed was 15m/min, the rest was 30s every 15s, the rest was 3min after four cycles, and the three groups were one cycle. The cycle was intermittent for 10 minutes, and the exercise frequency was the next day for 8 weeks. After 8 weeks, the rat gastrocnemius muscle was taken. The protein content of the gastrocnemius muscle was determined by Coomassie brilliant blue method. The contents of IGF-I and TNF-α in the gastrocnemius muscle were determined by enzyme-linked immunosorbent assay.&#x0D; Results After 1.8 weeks of different load resistance exercise, compared with the quiet control group, the skeletal muscle protein content of each exercise group increased, among which 50% of the largest negative recombination and 70% of the largest negative recombination was the most significant (P&lt;0.01). Compared with 70% of the largest negative recombination, the 50% maximum negative recombination content was relatively high (P&lt;0.05);&#x0D; &#x0D; After 8 weeks of different load resistance exercise, compared with the quiet control group, the content of IGF-I in the gastrocnemius of each exercise group was higher than that of the quiet control group, and there was a very significant difference (P&lt;0.01), and 50%. The highest negative recombination and 70% of the largest negative recombination content is the highest;&#x0D; After 8 weeks of different load resistance exercise, compared with the quiet control group, the TNF-α content in the gastrocnemius muscle of the rats in each resistance exercise group increased slightly, and the increase of only 50% of the largest negative recombination was statistically significant (P&lt;0.05), although there were differences between the exercise groups, there was no statistical significance (P&gt;0.05).&#x0D; &#x0D; Conclusions 50% of the maximum weight-bearing resistance training can effectively increase the skeletal muscle muscle protein content of aging SD rats, and it is related to up-regulation of IGF-I factor and down-regulation of TNF-α factor expression by resistance training.

Evaluation of the analytical variability of urine protein-to-creatinine ratio in cats.

Proteinuria quantification with the urinary protein-to-creatinine (UPC) ratio is part of the diagnostic process in feline patients suspected of chronic kidney disease (CKD). In affected cats, monitoring and substaging of the UPC according to the International Renal Interest Society (IRIS) guidelines is also necessary for appropriate patient management. No information is available about the possible effects of analytical variability on urinary proteins (UPs) and the UPC ratio in cats. This study aimed to determine whether imprecision and method-dependent differences due to the two dye-binding methods, pyrogallol red-molybdate (PRM) and Coomassie brilliant blue (CBB), could affect IRIS substaging. Urine samples were collected from proteinuric and nonproteinuric cats. Intra-assay and inter-assay repeatability were assessed with both the PRM and CBB methods. Urinary supernatants (n=120) were tested using both methods. Agreement between the methods and concordance with sample classification according to IRIS guidelines were determined. On average, the PRM method yielded a higher CV (UP 8.4±5.2%; UPC 9.5±4.8%) than the CBB method (UP 5.6±2.6%; UPC 7.2±2.6%), but similar rates of misclassification were found in samples with UPC ratios close to the IRIS cut-off. Although the two methods were correlated, the CBB method tended to yield UPs and UPC ratios that were significantly higher (P<0.0001) than that of the PRM method. The Passing-Bablok test also found constant and proportional errors between the PRM and CBB methods. Concordance in substaging samples according to IRIS was good (k coefficient=0.62). The two methods were precise, but the higher UPC ratios obtained with the CBB methods might affect the interpretation using the IRIS guidelines and clinical decisions.

Effects of Ginkgo biloba leaf extract on local renin-angiotensin system through TLR4/NF-κB pathway in cardiac myocyte.

The present study investigated the effects of Ginkgo biloba leaf extract (GBE50) on lipopolysaccharide (LPS) induced Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway and its effects on angiotensinogen (ATG) and AT1a receptor, so as to explore the mechanism of GBE50 in prevention and treatment of left ventricular remodeling. In vitro cultured neonatal rat ventricular myocytes (NRVMs) were divided into 4 groups including i) control group: DMEM medium; ii) LPS group: iii) LPS + GBE50 group; iv) LPS + caffeic acid phenethyl ester (CAPE, specific inhibitor of NF-κB) group. Nuclear translocation of NF-κB p65 was detected by immunocytochemical method after intervention for 24 h. Expression of TLR4, ATG, AT1a receptors and β-myosin heavy chain (β-MHC) mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein content of cardiomyocytes was measured by Coomassie Brilliant Blue method. Under LPS stimulation, expression level of TLR4 mRNA in NRVMs was significantly increased (P<0.01), nuclear translocation of NF-κB p65 was increased, expression levels of ATG, AT1a receptor and β-MHC mRNA and the protein content in cells were also increased significantly (P<0.01). GBE50 and CAPE significantly inhibited nuclear translocation of NF-κB p65. GBE50 and CAPE treatments also reduced the increased mRNA levels of TLR4, ATG, AT1a receptor and β-MHC and protein content in cell caused by LPS stimulation. We concluded that, GBE50 can inhibit the activation of local renin-angiotensin system by inhibiting the activation of TLR4/NF-κB and TLR4/NF-κB, signaling pathway inhibition may be one of the mechanisms of the role of Ginkgo biloba leaf extract in preventing myocardial remodeling.

Icariin improves acute kidney injury and proteinuria in a rat model of pregnancy‑induced hypertension.

Icariin has previously been demonstrated to attenuate hyperglycemia-induced renal injury, however the renoprotective effects of icariin in a rat model of pregnancy-induced hypertension (PIH) remain to be elucidated. The present study aimed to investigate the effect of icariin on PIH-induced acute kidney injury (AKI) and proteinuria. Following 18 days of icariin treatment between day 1 and day 18 of gestation, which was combined with NG-nitro-L-arginine methyl ester (L-NAME) treatment between day 12 and day 18 of gestation to induce PIH, the 24 h urine protein level, blood urea nitrogen and serum creatinine were measured by using the Coomassie Brilliant Blue method, a commercial enzymatic kit and the picric acid method, respectively. Renal tissues were collected at day 18 of gestation for hematoxylin and eosin staining and immunohistochemistry. The mRNA expression of AGT and protein expression of angiotensin II (Ang II) in the kidneys of control and PIH rats was investigated by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively, to determine the effect of icariin on components of the renin-angiotensin system. The results demonstrated that L-NAME treatment in pregnant rats resulted in significant increases in systolic blood pressure (SBP) and diastolic blood pressure, in addition to the induction of severe proteinuria. The significant increase in SBP and proteinuria in PIH rats was prevented by icariin. L-NAME-induced AKI resulted in profound renal histological alterations, including mesangial expansion and glomerular lesions. L-NAME administration exerted a marked decrease in the mRNA and protein expression levels of nephrin in the kidneys from PIH rats compared with control group. Furthermore, upregulation of circulating and renal Ang II levels in PIH rats was observed. However, icariin treatment significantly reversed the L-NAME-induced downregulation of nephrin and upregulation of circulating and renal Ang II levels in PIH rats. These results demonstrated that icariin administration improved urinary protein excretion levels and renal tissue damage in PIH rats, and the underlying mechanism was mediated in part, via upregulation of nephrin expression and downregulation of Ang II.

In vitro anticoagulant activity of different processed products of Whitmania pigra by water extraction and bionic extraction

In order to determine the scientificalness of traditionally processed Whitmania pigra, water extraction method and bionic extraction method were used respectively to extract the anticoagulating active components in W. pigra hanging dry products, talcum powder fried products and wine immersing-baked products. Activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and antithrombin activity were selected as the activity indexes to evaluate the anticoagulant activities of different processed W. pigra. Then the contents of protein in different processed W. pigra were measured by Coomassie brilliant blue method to preliminarily explain the reason of anticoagulant activity changes. When water extraction method was used, the results of APTT, PT, TT and antithrombin activity showed that the anticoagulant activities of W. pigra were decreased both in talcum powder fried products and wine immersing-baked products, and the activity order was as follows: hanging dried products> wine immersing-baked products>talcum powder fried products. This order was same as the protein content order. While when bionic extraction was used, APTT was shortened in talcum powder fried products, but all the other results indicated the anticoagulant activities of W. pigra processed products were increased, and the activity order was as follows: wine immersing-baked products>talcum powder fried products>hanging dry products. As compared with water extraction, the bionic extraction was more similar to the absorption process of W. pigra in human digestive system after oral administration and was more scientific. Therefore, the traditional processing method can not only modify the taste and smell, but also enhance the anticoagulant activity of W. pigra.

Isolation and identification of sinapine-degrading bacteria from the intestinal tract of laying hens.

This study was aimed to isolate sinapine-degrading bacteria from the intestinal tract of laying hens and to identify the predominant bacteria. Thirty-week old healthy laying hens were killed, and the chyme in the digestive tract was inoculated into modified Czapek medium containing sinapin and cultivated at 37 °C for 10 days. The optical density (OD) values of the bacterial solutions at different cultivating times were detected by a spectrophotometric method. The predominant strains were identified by 16S rRNA gene analysis. We extracted the extracellular products of the predominant strains to determine the total protein using the Coomassie brilliant blue method, and to determine the activities of some extracellular enzymes using the agar plate diffusion method. Nine strains were isolated from the lower intestinal tract of laying hens. Among the 9 strains, 5 were from the ileum, 2 were from the ceca and 2 were from the jejunum. We could not isolate any strains from the upper intestinal tract, such as the stomach and duodenum. Eight of those 9 isolated strains were gram negative and one was gram positive. Strains YD-1 and YD-2 were better than other strains in their abilities to degrade sinapine. Strains YD-1 and YD-2 were identified as Escherichia coli and Klebsiella spp., respectively, by the 16S rRNA sequence analysis. The total protein level of the extracellular products was 1.213 g/L for YD-1 and 1.990 g/L for YD-2. Both extracellular products of YD-1 and YD-2 had the activities of protease, amylase and urease. This study confirmed that the primary site of sinapine degradation is in the lower intestinal tract of laying hens. The sinapine-degrading strains are mainly gram negative. Strains YD-1 and YD-2 are predominant in degrading sinapine and they belong to E. coli and Klebsiella spp., respectively. Both extracellular products of YD-1 and YD-2 contain protease, amylase and urease. Strain YD-2 is better than strain YD-1 in its ability to degrade sinapine.

Diffusion blotting: a rapid and simple method for production of multiple blots from a single gel.

A very simple and fast method for diffusion blotting of proteins from precast SDS-PAGE gels on a solid plastic support was developed. Diffusion blotting for 3 min gives a quantitative transfer of 10 % compared to 1-h electroblotting. For each subsequent blot from the same gel a doubling of transfer time is necessary to obtain the same amount of protein onto each blot. High- and low-molecular-weight components are transferred equally efficiently when compared to electroblotting. However, both methods do give a higher total transfer of the low-molecular-weight proteins compared to the large proteins. The greatest advantage of diffusion blotting is that several blots can be made from each lane, thus enabling testing of multiple antisera on virtually identical blots. The gel remains on the plastic support, which prevents it from stretching or shrinking. This ensures identical blots and facilitates more reliable molecular weight determination. Furthermore the proteins remaining in the gel can be stained with Coomassie Brilliant Blue or other methods for exact and easy comparison with the developed blots. These advantages make diffusion blotting the method of choice when quantitative protein transfer is not required.

Hepatoprotective Effects of Yintian Granule on Experimental Liver Injury in Mice

ObjectiveYintian Granule (YTG), as a type of local preparation and applied for Chinese patent, is mainly composed of several traditional Chinese herbs used as both drug and food such as Lonicera macranthoides, Gardenia jasminoides, and Asparagus cochinchinenis, and has been reported to demonstrate the beneficial effects on human health in other researches. In this paper, the protective effects of YTG against experimental acute liver injury of mice were investigated to assess the value of this innovative Chinese herbal compound. MethodsCarbon tetrachloride (CCl4) and 50% ethanol were used respectively to induce the acute liver injury model in mice pre-administered with YTG. Lai's method was used to detect the level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, Coomassie brilliant blue method was used for the determination of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content, and hematoxylin-eosin (HE) staining was used for the observation of liver histomorphometry. ResultsYTG significantly lowered the elevated ALT and AST levels, increased the SOD activity, decreased the MDA content, and inhibited the deterioration of liver. ConclusionYTG exerts protective effected against hepatocyte damage in mice induced by CCl4 and 50% ethanol, respectively.