Volume Of Phosphate Buffer Research Articles

Effect of rapamycin on the tumor growth of bearing cancer mice of RM-1 and mechansim

Objective To investigate the effect of rapamycin (RAPA) on the tumor growth of bearing cancer mice of RM-1 and the underlying mechanism. Methods In cellular level, RM-1 cells were treated with different concentrations of RAPA. The expression of p62 and cysteinyl aspartate-specific protease (Caspase)-3 proteins was detected by Western blotting. In Animal level, 30 bearing cancer mice of RM-1 were randomly divided into model group, 5 mg/kg RAPA group, 10 mg/kg RAPA group and 20 mg/kg RAPA group. Mice in 5 mg/kg RAPA, 10 mg/kg RAPA and 20 mg/kg RAPA groups were given intraperitoneal injection of RAPA with the doses of 5, 10 and 20 mg/kg respectively. Mice in mode group were given the same volume of phosphate buffer (PBS). Tumor growth and survival rate of four groups were observed after administration for 30 days. Caspase-3 mRNA and protein levels were analyzed by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting respectively. Cell apoptosis index in tumor tissue was analyzed by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining method. Results As compared with the control group (1.89±0.17), the levels of p62 in the cells treated with 125 nmol/L, 250 nmol/L and 500 nmol/L (0.91±0.19, 0.68±0.12, 0.31±0.10) decreased significantly (t=2.019, 3.491, 4.910, P<0.05). As compared with the control group (0.21±0.09), the expression levels of Caspase-3 protein in cells treated with 125 nmol/L, 250 nmol/L and 500 nmol/L were significantly increased (0.41±0.14, 0.87±0.18, 2.01±0.21) (t=1.989, 2.582, 5.019, P<0.05). As compared with the model group, RAPA of 5, 10 and 20 mg/kg significantly slowed down the growth of tumors in mice, and the survival rate was significantly higher than that in the model group in a dose-dependent manner. As compared with the model group, the levels of Caspase-3 and p62 proteins in tumor tissues of mice in 5 mg/kg RAPA, 10 mg/kg RAPA and 20 mg/kg RAPA groups increased significantly, and those of p62 protein decreased significantly. Casepase-3 and p62 proteins in 5 mg/kg RAPA, 10 mg/kg RAPA and 20 mg/kg RAPA groups were dose-dependently correlated with RAPA concentrations. As compared the model group (5.01±1.12), the apoptotic levels of tumor cells in 3 mg/kg RAPA, 6 mg/kg RAPA and 9 mg/kg RAPA groups [(20.14±4.12), (51.23±8.99), (78.43±10.32)] increased significantly (t=3.109, 5.192, 7.102, P<0.05). Conclusion RAPA can induce excessive autophagy of tumor cells, and excessive autophagy can result in tumor cell apoptosis to achieve anti-tumor effects. Key words: Rapamycin; Prostate cancer; Autophagy; Apoptosis

Effects of rat mesenchymal stem cell-derived exosomes on rat experimental autoimmune uveitis

Objective To observe the effects of exosomes derived from rat mesenchymal stem cells(MSC-exosomes) on the rat experimental autoimmune uveitis (EAU) model. Methods Twelve Lewis rats were randomly divided into experimental group and control group by random number table, with 6 rats in each group. Rats in the experimental group were established with EAU model, 100 μl of MSC-exosomes (50 μg) were periocular injected on the 9th day after modeling while the control rats were injected with the same volume of phosphate buffer. At different time points after modeling, the retinal structure was observed by hematoxylin and eosin (HE) staining, and the clinical and pathological manifestations were evaluated. T cells from the two groups were analyzed by flow cytometry. Immunohistochemical staining was used to observe the expression of macrophage surface marker CD68. The effect of MSC-exosomes on T cells was measured by lymphocyte proliferation assays. And flow cytometry was used to detect Th1, Th17 and regulatory T cells Variety. Electroretinogram (ERG) was used to evaluate the retinal function. Data were compared between the two groups using the t test. Results HE staining showed that the retina structure of the experimental group was more complete than that of the control group on the 15th day after modeling. Immunohistochemical staining showed that the positive expression of CD68 in the experimental group was significantly less than that in the control group. On the 15th day after modeling, the retinal pathological score of the experimental group was lower than that of the control group. On the 9th to 13th day after modeling, compared to the control group, the average clinical scores of the retina in the experimental group were lower, and the difference was statistically significant(t=3.665, 3.21, 3.181, 4.121, 3.227; P 0.05). Conclusions MSC- exosomes can reduce the clinical and pathological manifestations of EAU, protect retinal function, reduce ocular macrophage infiltration, down-regulate the proportion of inflammatory cells in the eye, and inhibit T cell proliferation. Key words: Mesenchymal stem cells; Exosomes; Uveitis/therapy; Animal experimentation

Effects of bone marrow mesenchymal stem cells on blood glucose concentration in diabetic rats

Objective To investigate the effects of different concentrations of bone marrow mesenchymal stem cells (BMSCs) on blood glucose concentration in diabetic rats. Methods SD rats’ bone marrow was isolated, purified and cultured to obtain BMSCs. The expression of surface markers CD34, CD44, CD45 and CD90 was detected. The models of diabetic rats were randomly divided into control group, the low concentration group, high concentration group and normal feeding group. Low concentration group and high concentration group were injected with 1×105 and 5×106 BMSCs respectively, and the normal group and the control group were injected with equal volume of phosphate buffer (PBS). The dynamic changes of blood glucose concentrations at 1st, 2nd and 3rd week after injection were statistically analyzed. Results BMSCs were proved by immunofluorescence with CD34 (-), CD44 (+ ), CD45 (-) and CD90 (+ ). After treatment with BMSCs, blood glucose had no significant change in the first two weeks, but decreased by 16.1% at 3rd week in the low concentration group. As compared with the control group, blood glucose concentration was significantly decreased, and decreased by 42.0% at 3rd week in the high concentration group. There was no obvious change in blood glucose concentration before and after treatment in the normal and control groups. Conclusion High concentration (5×106 cells) BMSCs can control and regulate the blood glucose concentration in diabetic rats. Key words: Bone marrow mesenchymal stem cells; Diabets; Rat; Blood glucose concentration

Protective mechanism of rapamycin on D-amino galactoose/lipopolysaccharide-induced acute liver injury in mice

Objective To investigate the protective mechanism of rapamycin (Rapa) on D-amino galactoose (Gal-N)/lipopolysaccharide (LPS)-induced acute liver injury in mice. Methods The acute liver injury model was established by intraperitoneal injection of Gal-N (700 mg/kg) and LPS (10 g/kg) in 45 C57/B6 mice and the animals were randomly divided into model group, low dose Rapa group, middle dose Rapa group and high dose Rapa group. At the same time, 15 mice were selected as control group. The mice in low dose Rapa group, medium dose Rapa group and high dose Rapa group were injected with 5, 10 and 20 mg/kg Rapa at 20 min after modeling respectively. The mice in control group and model group were intraperitoneally injected with equal volume of phosphate buffer (PBS). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels of serum were determined. The tumor necrosis factor-α (TNF-α) levels were analyzed by enzyme linked immunosorbent assay (ELISA) Kit. The pathological changes in liver tissues of mice were observed by hematoxylin and eosin (HE) staining. Autophagy substrates microtubule-associated protein 1 light chain 3 (LC3) and p62 were analyzed by Western blotting. Results Compared with AST and ALT of control group [(30.41±5.23) U/L and (110.23±12.02)], serum AST and ALT levels in model group, low, middle and high dose group [((489.39±35.65) U/L and (310.24±41.25) U/L); (385.21±30.59) U/L and (278.96±35.68) U/L; (279.94±35.14) U/L and (214.37±34.12); (159.24±30.12) U/L and (168.63±28.32) U/L]significantly increased [ (t=8.120, t=6.384); (t=7.013, t=5.132); (t=5.198, t=4.318); (t=3.957, t=3.048); P=0.000]. Compared with the model group, the serum AST and ALT levels in the low, middle and high dose groups significantly reduced [ (t=3.012, t=3.419); (t=3.901, t=4.012); (t=4.820, t=5.018), P=0.000]. Compared with TNF-alpha in the control group [(150.44±23.19) ng/L], the level of TNF- α in the model group, low, middle and high dose group [(509.44±49.32), (401.43±38.09), (298.44±29.41), (218.29±24.21) ng/L] significantly increased (t=6.109, t=5.184, t=4.109, t=3.921, P=0.000). Compared with the model group, the TNF-α levels in the low, middle and high dose groups significantly reduced (t=2.194, t=3.331, t=5.016, P=0.000). HE staining showed that the liver of model group mice was seriously damaged, the liver red blood cells diffused, the intercellular space of liver cells increased, the vacuoles deformed and the hepatic cords arranged irregularly. With the increase of rapamycin dose, the pathological damage degree of liver in mice was dose-dependent. Compared with the control group, autophagy in the model group was inhibited, and the autophagy level obviously enhanced after rapamycin treatment. Conclusion Rapa can induce autophagy and decrease TNF-α by inhibiting the activity of mammalian target of Rapa kinase, thus protecting the liver. Key words: Rapamycin; Acute liver injury; Mammalian target of rapamycin; Autophagy; Inflammatory factors

Design of experiments and principal component analysis as approaches for enhancing performance of gas-diffusional air-breathing bilirubin oxidase cathode

Two statistical methods, design of experiments (DOE) and principal component analysis (PCA) are employed to investigate and improve performance of air-breathing gas-diffusional enzymatic electrodes. DOE is utilized as a tool for systematic organization and evaluation of various factors affecting the performance of the composite system. Based on the results from the DOE, an improved cathode is constructed. The current density generated utilizing the improved cathode (755 ± 39 μA cm−2 at 0.3 V vs. Ag/AgCl) is 2–5 times higher than the highest current density previously achieved. Three major factors contributing to the cathode performance are identified: the amount of enzyme, the volume of phosphate buffer used to immobilize the enzyme, and the thickness of the gas-diffusion layer (GDL). PCA is applied as an independent confirmation tool to support conclusions made by DOE and to visualize the contribution of factors in individual cathode configurations.

Shift in pH of biological fluids during storage and processing: effect on bioanalysis

The pH of ex vivo plasma, bile and urine was monitored at different times and temperatures of storage, and following different sample processing methods such as ultrafiltration, centrifugation, precipitation and evaporation. The results showed that the pH of ex vivo plasma, bile and urine increased upon storage, and following sample processing and could lead to significant degradation of pH-labile compounds. Several compounds were used to illustrate the impact of pH shifts on drug stability and interpretation of results obtained from in vivo studies. For example, after 1 h of incubation (37 °C) in rat plasma (pH 8.3), about 60% of I was lost. However, in phosphate buffer, losses were about 12% at pH 7.4 and 40% at pH 8.0. Plasma pH also increased during ultrafiltration, centrifugation and extraction. After methanol precipitation of plasma proteins, and evaporation of the supernatant and redissolution of the residue, the resulting solution had a pH of 9.5. Under these conditions, II was degraded by 60% but was stable when phosphate buffer was used to maintain the pH at 7.4. The shift in plasma pH can yield misleading results from in vivo studies if the pH is not controlled. For example, the major circulating metabolite of II was also formed in plasma ex-vivo. This ex vivo degradation was prevented when blood samples were collected into tubes containing 0.1 volume of phosphate buffer (0.3 M, pH 5). The pH of ex vivo plasma can best be stabilized at physiological conditions using 10% CO 2 atmosphere in a CO 2 incubator. Changes in pH of ex vivo urine and bile samples can have similar adverse effect on pH-labile samples. Thus, processing of plasma samples under a 10% CO 2 atmosphere is a method of choice for stability or protein binding studies in plasma, whereas citrate or phosphate buffers are suitable for stabilizing pH in bile and urine and for plasma samples requiring extensive preparations or long term storage.

Determination of apomorphine in human plasma by alumina extraction and high-performance liquid chromatography with electrochemical detection

Apomorphine is a powerful agonist of dopaminergic receptors which several years ago was introduced into the therapy of Parkinson's Disease. The pharmacological activity of apomorphine already appears significant at low doses. Unfortunately, the difficulty in determining the drug in plasma at low concentrations hampers the completion of accurate pharmacokinetic studies in humans. Considering the analogy of apomorphine with the molecular structure of catecholamines, the extraction of the drug from plasma was optimized by using adsorption on alumina, a technique widely used for noradrenaline and adrenaline analysis in clinical chemistry laboratories. This method proved particularly efficient and selective in apomorphine extraction from plasma prior to high-performance liquid chromatographic analysis. After pretreatment of 200 μl of plasma sample with 40 mg of alumina and 10 μl of tris buffer (pH 8.6), the drug was eluted with 200 μl of an acidic-organic solution. One volume of the supernatant was mixed with two volumes of phosphate buffer (pH 3.6), and 100 μl of the obtained mixture were injected into the HPLC system. The chromatograph was equipped with a C18 reversed-phase column and with an electrochemical coulometric detector fitted with a high-sensitivity cell (first electrode 0.00 volts, second electrode +0.35 volts). Sensitivity (20 pg of injected drug), precision (CV within assay and between assays of 3.7% and 5.6%, respectively) and accuracy were comparable to more complex analytical procedures. The miniaturisation of the entire sample pretreatment proved very advantageous for pharmacokinetics studies and, in principle, for therapeutic drug monitoring and toxicological investigations.

Adsorption and physical state of medium- and long-chain fatty acids in neutral aqueous buffer solution

Varying amounts of radiolabelled fatty acids, lauric, myristic, palmitic, stearic, and oleic acids, were agitated with 5-ml volumes of phosphate buffer (pH 7.4) at 37°C in glass tubes, producing visibly clear solutions. A portion of the fatty acid was recovered from the aqueous phase, while the rest was adsorbed to the glass wall and the water surface. Stearate and oleate could hardly be demonstrated in the aqueous phase. The adsorbed amount, a, of the other fatty acids was related to the concentration, c, in the aqueous phase by a = βc α , where α was 0.87 for laurate, 0.73 for myristate and 1.17 for palmitate. The relation is identical to Freundlich's adsorption isotherm except that the numerical value of α, when dealing with adsorption equilibria, is less than unity. The observations can be explained if laurate and myristate are present in genuine aqueous solution in equilibrium with the adsorbed substances; palmitate is aggregated in the buffer phase as vesicles, liposomes, in equilibrium with monomer palmitate in lower concentration, the latter equilibrium as well as the adsorption obeying Freundlich's equation with α < 1. Even at a concentration as low as 10 −11 M, palmitate appears to be present mainly in an aggregated state.

Glutathione S-transferase-mediated metabolism of glyceryl trinitrate in subcellular fractions of bovine coronary arteries.

The possible role of glutathione S-transferases (GTSs) in vascular glyceryl trinitrate (GTN) metabolism was investigated. GTN degradation to form its dinitrate metabolites (GDNs) in the 9000g (9k) supernatant fraction of bovine coronary arteries (BCA) was examined. BCAs were homogenized with a 3x volume of phosphate buffer, and the 9k fraction was obtained by centrifugation. GTN (40 ng/ml; 1.76 x 10(-7) M) was incubated for 2 hr in the 9k fraction of BCA in the presence of reduced glutathione (2 x 10(-3) M). Samples were taken at 10, 20, 40, 60, and 120 min. GTN was observed to degrade readily, exhibiting a half-life of 26 min in the incubate. While both 1,2- and 1,3-GDNs were generated from GTN, formation of 1,3-GDN was predominant (GDN ratio, as 1,2/1,3-GDN, = 0.7-0.8). Coincubation with 2 x 10(-5) M concentrations of two GST inhibitors, sulfobromophthalein (SBP) and ethacrynic acid (ECA), decreased the rate of GTN loss. The GTN half-lives in SBP- and ECA-treated incubations were 66 and 84 min, respectively. In addition, the pattern of GDN formation was also altered. The resultant GDN ratios exceeded unity in the presence of these inhibitors, indicating that 1,3-GDN formation was attenuated to a greater extent than that of 1,2-GDN. These data suggest that vascular GTN metabolism in BCA is carried out by cytosolic GST isozymes which possess a preference for C-2 denitration of GTN.

Quantitation of Indomethacin in Serum by HPLC Using Fluorescence Detection

Abstract An improved, sensitive and accurate HPLC procedure using fluorescence detection for quantitation of indomethacin in serum has been developed. After addition of an equal volume of phosphate buffer, pH 6.6 to serum along with the internal standard, the samples were extracted with methylene chloride. Prior to chromatography, the extracted indomethacin was deacylated to its fluorescent product (DBI) in 0.01 N-NaOH. The mobile phase consisted of methanol and pH 4 acetate buffer (3:7 V/V) and the separation was achieved on a C18 reversed phase column. The retention times of DBI and the internal standard were 7.5 and 16.0 min. respectively. The fluorometric excitation and emission wavelengths were 278 and 358 nm, respectively. The sensitivity of the assay was 1 ng/ml of serum and the CV at this concentration was 2.46%. The standard plot was linear (r > 0.999) for indomethacin concentrations between 1 and 500 ng/ml. The inter-and intra-day studies showed high reproducibility (CV = 2.8%, F = 0.89, p > 0.0...

Corrections

Abstract p 1131: Under Alkaline picrate reagents, "...four volumes of phosphate buffer ... " should read "... two volumes of phosphate buffer..."

Corrections

Abstract p 1131: Under Alkaline picrate reagents, "...four volumes of phosphate buffer ... " should read "... two volumes of phosphate buffer..."

Effect of starvation upon the constitution of bacteria.

WHEN bacteria, which have grown in a mineral salts medium, are transferred to fresh medium of the same composition a lag period may develop before growth is resumed1. Lag is reduced by addition of sterile culture filtrates or by certain compounds shown to be present in such filtrates2. We have recently observed changes within the cells themselves when they are transferred to phosphate buffer. Esch. coli was harvested from a growth medium containing 0.5 per cent peptone, 0.2 per cent glucose and mineral salts and the crop was divided into two equal parts. One half was disrupted immediately in the Hughes bacterial press3 without abrasive and the crushed cells extracted with phosphate buffer. The other half of the crop was suspended in the same volume of phosphate buffer as that of the medium in which the cells had grown. After incubation at 30° C. for 2 hr. the bacteria were harvested, crushed under the same conditions as those used for the first half of the original crop and the two extracts were adjusted to the same protein content by addition of buffer. The ultracentrifuge pattern for the extract from the cells crushed immediately after harvesting from their growth medium is shown in Fig. 1a. It agrees closely with patterns reported by previous authors4–6. When the cells are incubated in the absence of compounds necessary for growth, however, a dramatic modification occurred in the distribution of macromolecules in the extract. The leading boundary, which has an uncorrected sedimentation coefficient of 40S, was largely abolished while that sedimenting at 20S was somewhat augmented (Fig. 1b).

Lactic Acid Formation by Muscles of Scorbutic Guinea Pigs

SummaryThe excised muscle of scorbutic guinea pigs suspended in an equal volume of phosphate buffer plus glucose, produces only half as much lactic acid as the muscle of normal animals. Addition of 100 mg ascorbic acid to 100 g of muscle had no effect upon the rate or the final amount of lactic acid produced, neither with normal nor with scorbutic muscle.