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Transforming growth factor beta 1 prevents cytokine-mediated inhibitory effects and induction of nitric oxide synthase in the RINm5F insulin-containing beta-cell line.

The aim of this study was to examine if the growth factor, transforming growth factor beta 1 (TGF beta 1), could prevent induction of nitric oxide synthase and cytokine-mediated inhibitory effects in the insulin-containing, clonal beta cell line RINm5F. Treatment of RINm5F cells for 24 h with interleukin-1 beta (IL-1 beta) (100 pM) induced expression of nitric oxide synthase and inhibited glyceraldehyde-stimulated insulin secretion. Combinations of IL-1 beta (100 pM), tumour necrosis factor-alpha (100 pM) and interferon-gamma (100 pM) reduced RINm5F cell viability (determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium (MTT) reduction assay) and de novo protein synthesis, as measured by incorporation of radiolabelled amino acids into perchloric acid-precipitable protein. Pretreatment of RINm5F cells with TGF beta 1 (10 pM) for 18 or 24 h, prior to the addition of either IL-1 beta or combined cytokines, prevented cytokine-induced inhibition of insulin secretion, protein synthesis and the loss of cell viability. TGF beta 1 pretreatment inhibited cytokine-induced expression and activity of nitric oxide synthase in RINm5F cells as determined by Western blotting and by cytosolic conversion of radiolabelled arginine into labelled citrulline and nitric oxide. Chemically generated superoxide also induced expression of nitric oxide synthase possibly due to direct activation of the nuclear transcription factor NF kappa B, an effect prevented by both an antioxidant and TGF beta 1 pretreatment. In conclusion, the mechanism of action of TGF beta 1 in blocking cytokine inhibitory effects was by preventing induction of nitric oxide synthase.

Animals↗

Experimental gingivitis in ODU plaque-susceptible rats. V. The presence of bradykinin in the gingival tissue and the bradykinin inactivating factor in rat dental plaque.

Plaque formation and gingival inflammation were found in ODU plaque-susceptible rats. A study was performed to determine the relationship between the presence of bradykinin in gingival tissue and the inflammatory capacity of rat plaque. Bradykinin content in gingival tissue and the bradykinin inactivating activity of dental plaque of 12-month-old ODU rats were measured. The presence of bradykinin was observed in the gingival tissue of the ODU rat and a bradykinin inactivating factor was found in the rat plaque. The factor was destroyed at 60 degrees C for 30 minutes, at pH 1.5 or by treatment with perchloric acid. The optimum pH of the factor was approximately 8.0. Chelate reagents, such as O-phenanthroline and EDTA . 4Na, strongly inhibited the bradykinin inactivating factor. The protease inhibitor, Trasylol, also inhibited this factor. The SH reagent, L-cysteine, inhibited weakly and Zn++ inhibited less. The investigation suggested that the rat plaque posseses the ability to inactivate bradykinin and plays an important role in modifying the inflammatory response caused by bradykinin in gingival tissue.

Acids↗

Fluorometric determination of khellin in human urine and serum by high-performance liquid chromatography using postcolumn photoirradiation.

For the determination of khellin in urine and serum, fluorometry using HPLC-postcolumn photoirradiation has been developed. Khellin and visnagin of similar structure were separated on a column of Capcell Pak C8. The mobile phase consisted of 40%(v/v) ethanol containing 75 mmol l(-1) H2O2. The postcolumn reagent, 70 mmol l(-1) KH2PO4-NaOH buffer (pH 12.7) containing 50%(v/v) ethanol, were mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The fluorescence was monitored with excitation at 378 nm and emission at 480 nm. The calibration graph for khellin was linear over the range of 65 - 2620 ng ml(-1) using an injection volume of 20 microl. The pretreatment of the urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively.

Calibration↗

A novel separation and preconcentration method for traces of manganese, cobalt, zinc and cadmium using coagulation of colloidal silica.

A separation and preconcentration method has been developed for traces of heavy metals using coagulation of colloidal silica. An appropriate amount of colloidal silica was added to a sample solution, the pH was adjusted to 11 with tetramethylammonium hydroxide solution and calcium chloride solution was then added to coagulate the silica. The coagulated silica and solution were separated by centrifugation, and the silica was then treated with hydrofluoric and perchloric acids. The residue was taken up in dilute nitric acid and subjected to ICP-AES to determine manganese, cobalt, zinc and cadmium. The proposed method was successfully applied to analysis of river-water.

Journal Article↗

Automation of liquid-liquid extraction-spectrophotometry using prolonged pseudo-liquid drops and handheld CCD for speciation of Cr(VI) and Cr(III) in water samples.

A simple spectrophotometric system, based on a prolonged pseudo-liquid drop device as an optical cell and a handheld charge coupled device (CCD) as a detector, was constructed for automatic liquid-liquid extraction and spectrophotometric speciation of trace Cr(VI) and Cr(III) in water samples. A tungsten halogen lamp was used as the light source, and a laboratory-constructed T-tube with two open ends was used to form the prolonged pseudo-liquid drop inside the tube. In the medium of perchloric acid solution, Cr(VI) reacted with 1,5-diphenylcarbazide (DPC); the formed complex was automatically extracted into n-pentanol, with a preconcentration ratio of about 5. The organic phase with extracted chromium complex was then pumped through the optical cell for absorbance measurement at 548 nm. Under optimal conditions, the calibration curve was linear in the range of 7.5 - 350 microg L(-1), with a correlation coefficient of 0.9993. The limit of detection (3sigma) was 7.5 microg L(-1). That Cr(III) species cannot react with DPC, but can be oxidized to Cr(VI) prior to determination, is the basis of the speciation analysis. The proposed speciation analysis was sensitive, yet simple, labor-effective, and cost-effective. It has been preliminarily applied for the speciation of Cr(VI) and Cr(III) in spiked river and tap water samples. It can also be used for other automatic liquid-liquid extraction-spectrophotometric determinations.

Automation↗

Determination of sulfur compounds in hydrotreated transformer base oil by potentiometric titration.

A method was developed to analyze the distribution of sulfur compounds in model sulfur compounds by potentiometric titration, and applied to analyze hydrotreated transformer base oil. Model thioethers were oxidized to corresponding sulfoxides by tetrabutylammonium periodate and sodium metaperiodate, respectively, and the sulfoxides were titrated by perchloric acid titrant in acetic anhydride. The contents of aliphatic thioethers and total thioethers were then determined from that of sulfoxides in solution. The method was applied to determine the organic sulfur compounds in hydrotreated transformer base oil.

Journal Article↗

Electrocatalytic reduction of nitrate in water with a palladium-modified copper electrode.

A highly active electrocatalytic electrode for nitrate reduction was prepared by the electro-deposition of palladium onto a copper electrode. The capacity of nitrate reduction by a palladium-modified copper electrode has been studied using cyclic voltammetry (CV). The existence of a reduction peak at -0.605 V versus saturated calomel electrode in 0.1-M sodium nitrate + 0.1-M perchloric acid solution (pH = 0.86) can be found in the CV measurement. The influence of solution properties, such as pH, nitrate concentration, and other anions in solution, on nitrate reduction was determined in detail. Results showed that nitrate reduction was suppressed in alkaline solution, while it was beneficial to nitrate reduction in acid or neutral solution. At low nitrate concentrations (0.01 to 0.5 M), nitrate reduction current increased with increasing nitrate concentration, but was hindered by sulfate. At high nitrate concentrations (1 to 5 M), no significant difference on nitrate reduction was observed. Compared with other different electrodes prepared in our work (copper, titanium, and palladium-modified titanium electrodes), the palladium-modified copper electrode showed the highest electrocatalytic capacity and stability in the nitrate-reduction process.

Catalysis↗

In vitro fluoride uptake by enamel and dentin. A comparative study of two varnishes.

The uptake of fluoride in enamel and dentin after application of an experimental fluoride varnish has been studied and compared with the uptake from a commercially available varnish (Duraphat). Each varnish was applied to 10 extracted human cuspid teeth. Successive etchings with perchloric acid were carried out separately in enamel and root dentin to obtain samples at three different depths. The concentrations of fluoride and calcium in the samples were determined, and the fluoride uptake was calculated. The experimental varnish gave a significantly greater fluoride uptake at all three sample depths in both enamel and dentin.

Cuspid↗

Rapid micromethod for flourometric determination of neutral lipids on thin-layer chromatograms.

A rapid fluorometric procedure for in situ evaluation of neutral lipids on thin-layer chromatograms is described. The lipids are rendered fluorescent by treatment with perchloric acid, and the intensity of the emitted light is recorded by flying spot scanning. Detection limits (2.8 x SD) are 0.10, 0.11, and 0.17 nmol for triglycerides, cholesterol, and cholesterol esters, respectively. Range of linearity is 0--3.4 nmol for triglycerides, 0--2.0 nmol for cholesterol, and 0--4.0 nmol for cholesterol esters. When standard in triplicate and 2 samples of each unknown are applied to the chromatogram, standard error of the mean is 2.7% for triglycerides, 1.7% for cholesterol, and 1.0% for cholesterol esters. The sensitivity of the method allows simultaneous determination of the three lipid classes in 5 microliter lipid extract representing 0.238 microliter human plasma.

Animals↗

Preparation and partial characterization of a pancreatic cancer-associated glycoprotein expressing CA50.

In our previous study, two antigens associated with pancreatic cancer were prepared from ascites of the patients by using a newly developed affinity chromatography technique; one expressed CA19-9, CA50, Span-1, sialyl SSEA-1, and Dupan-2. This report describes the other part of the antigens, which was scarcely found in normal sera. This antigen was a glycoprotein with a high molecular weight of 1,000,000 in its native state, estimated by size exclusion chromatography on Sephacryl S400. After sodium dodecyl sulfate polyacrylamide gel electrophoresis and blotting analysis, the antigenic activity was observed on the 200-Kd band, and monoclonal antibody of CA50 showed intense reactivity to it. By means of an enzyme immunoassay, CA19-9 determinant was also recognized on it to a lesser extent, although sialyl SSEA-1 and Dupan-2 determinants were scarcely observed. From these observations, the present antigen was considered to be a serum glycoprotein carrying type-1 polylactosamine determinant and differs from previously reported glycoprotein carrying CA19-9 in its insolubility in perchloric acid solution because of its lesser degree of glycosilation. Serum levels of the antigen measured by enzyme-linked immunosorbent assay demonstrated that highly positive rates were observed in 78% of pancreatic cancers, 82% of biliary tract cancers, and 96% of hepatocellular carcinomas.

Antigens, Neoplasm↗

Concentric membranous bodies in hepatocytes from a patient with systemic lupus erythematosus.

Concentric membranous bodies (CMB) are described in the cytoplasm of hepatocytes in a liver biopsy from a patient with systemic lupus erythematosus (SLE). Histologically, the biopsy showed binucleated hepatocytes and rosette formation with lymphocytic infiltrates in portal fields. Hepatocyte cytoplasm showed focal fatty metamorphosis, focal staining for RNA with azure B at pH 4.0 (which was perchloric acid labile), diffuse alloxan-Schiff staining for protein, and PAS-positive, diastase-sensitive glycogen. By electron microscopy one to three CMB were present in 10-15% of hepatocytes. CMB appeared as concentric, "fingerprint" or parallel arrays of particle-studded, membranous profiles. Other membranous configurations included mazelike forms. The space of Disse was dilated. Bile ductular changes included basement membrane thickening and redoubling and luminal bulging of ductular epithelium devoid of microvilli. CMB have been rarely reported in human hepatoma and have not heretofore been observed in non-neoplastic human liver. Their appearance in hepatocytes in a patient with SLE may reflect an increase in protein synthesis during regeneration.

Adult↗

Technical note: effect of removal of microbial cells by centrifugation on peptide and alpha-amino nitrogen concentrations in ruminal fluid.

We evaluated the effect of centrifuging rumen fluid prior to analysis on concentrations of alpha-amino N (AAN) and peptides. Rumen fluid was collected from steers fed grain-based diets at either various times after feeding or after dosing the rumens with solubilized casein. Fluid was either directly processed for peptide analysis by acidifying 10 ml of rumen fluid with 0.5 ml of 70% (wt/wt) perchloric acid, or first centrifuged at 500 x g for 20 min to remove protozoa and then at 30,000 x g for 15 min to remove bacterial cells prior to further processing. By removing microbial cells, intracellular AAN and peptides were not included in subsequent analyses. Concentrations of AAN were determinedusing an automated trinitrobenzene sulfonic acid assay, and peptides were determined as the increase in AAN following acid hydrolysis of the samples. When casein was not dosed, removal of microbial cells prior to analysis decreased concentrations of both AAN andpeptides, and the decrease was greater for AAN (2.2 mM)than for peptides (1.2 mM). Dosing with casein led to much higher concentrations of ruminal peptides and AAN. After casein dosing, decreases in AAN and peptidecon-centrations due to prior centrifugation (2.1 mM and 1.0 mM for AAN a nd pept ides, respectively) were similar to the decreases observed before the casein dosing. Results suggest that the contribution of intracellular AAN and peptides to the concentrations in ruminal fluid are relatively constant across broad ranges of dietary protein supply for cattle fed corn-based diets.

Amino Acids↗

Magnetic resonance spectroscopy of brain hemangiopericytomas: high myoinositol concentrations and discrimination from meningiomas.

OBJECT: Hemangiopericytomas are a rare type of brain tumor that are very similar to meningiomas in appearance and symptoms but require different treatment. It is not normally possible to distinguish between them by using magnetic resonance (MR) imaging and computerized tomography studies. However, discrimination may be possible by using in vivo MR spectroscopy (MRS) because the biochemical composition of these two lesions is different. The goal of this study was to describe the use of MRS in discriminating between these similar tumor types. METHODS: In vivo MRS spectra were acquired in 27 patients (three with hemangiopericytomas and 24 with meningiomas) by using a single-voxel proton brain examination system at 1.5 teslas with short- (20-msec) and long- (135-msec) echo times. In addition, brain biopsy specimens obtained by open craniotomy were frozen within 5 minutes of resection and stored in liquid nitrogen until they were used. The specimens were powdered, extracted with perchloric acid, redissolved in 2H2O2 and high-resolution in vitro MRS was used at 9.4 teslas to record their spectra. CONCLUSIONS: In this study the authors show that hemangiopericytomas could be clearly distinguished from meningiomas because they have a larger peak at 3.56 ppm. Measurements of extracts of the tumors and comparison of spectra acquired with MRS at long- (135-msec) and short- (20-msec) echo times established that this was due to the much higher levels of myoinositol in the hemangiopericytomas.

Brain Neoplasms↗

Formation of guanine ribonucleotidyl-(3'-5')-adenosine in a flavinogenic strain of Eremothecium ashbyii.

The addition of caffeine caused the accumulation of a new nucleotide compound simultaneously with the rigid inhibition of ribofalvin production in non-growing cells of Eremothecium ashbyii. In the present study we tried to identify the structure of the nucleotide compound using non-growing cells of the mold. 1) It became possible to obtain a large amount of mycelia by masscultivation in a reagent tank. 2) A new nucleotide compound, referred to as compound A in the paper, was extracted with perchloric acid solution and purified by the following subsequent procedures: 1) Dowex 1 x 2 (HCOO-) column, 2) charcoal treatment, 3) DEAE-Sephadex A25 (CI-) column, 4) Dowex 1 x 2 (C1-) column, and 5) DEAE-Sephadex A25 (HCO3-) column. 3) The structure of the new nucleotide compound was proved to be guanine ribonucleotidyl-(3'-5')-adenosine (GpA) from the results of the following analyses: 1) alkaline degradation, 2) UV-spectra, IR-spectra and NMR-spectra, and 3) enzymatic treatments with RNase T2 and phosphodiesterase. 4) The roles of caffeine and guanine ribonucleotidyl-(3'-5')-adenosine in connection with flavinogenesis of this mold were discussed.

Adenosine↗

Measurement of plasma adenosine concentration: methodological and physiological considerations.

This study tested the hypothesis that measurements of plasma adenosine concentration made on samples of blood obtained in dipyridamole and EHNA (i.e., "stopping solution") may be falsely elevated as a result of ongoing in vitro production and accumulation of adenosine during sample processing. Studies were performed with samples of anticoagulated blood obtained from anesthesized domestic swine. Adenosine concentration of ultra filtrated plasma was determined by high-pressure liquid chromatography (HPLC). The following parameters were evaluated: (i) rate of clearance of [3H]adenosine added to plasma, (ii) endogenous adenosine concentration of matched blood samples obtained in "stopping solution" alone, "stopping solution" plus EDTA, and perchloric acid (PCA), (iii) plasma and erythrocyte endogenous adenosine concentration in nonhemolyzed samples, and (iv) plasma adenosine concentration of samples hemolyzed in the presence of "stopping solution" alone or "stopping solution" plus EDTA. We observed that (i) greater than or equal to 95% of [3H]adenosine added to plasma is removed from it by formed elements of the blood in less than 20 s, (ii) plasma adenosine concentration of samples obtained in "stopping solution" alone is generally 10-fold greater than that of matched samples obtained in "stopping solution" plus EDTA, (iii) deliberate mechanical hemolysis of blood samples obtained in "stopping solution" alone resulted in substantial augmentation of plasma adenosine levels in comparison with matched nonhemolyzed specimens--addition of EDTA to "stopping solution" prevented this, and (iv) adenosine content of blood samples obtained in PCA agreed closely with the sum of plasma and erythrocyte adenosine content of samples obtained in "stopping solution" plus EDTA. The data obtained demonstrate that (i) plasma adenosine concentrations are falsely elevated in samples of blood obtained in "stopping solution" alone, and (ii) addition of EDTA to "stopping solution" blocks in vitro production and accumulation of adenosine. Finally, rapid removal of adenosine from plasma by formed elements of blood may make it difficult to employ measurements of plasma adenosine concentration to assess physiological processes even in the absence of in vitro production of the nucleoside.

Adenine↗

Catabolism of 5-fluoro-2'-deoxyuridine by isolated rat intestinal epithelial cells.

The kinetics of conversion of 5-fluoro-2'-deoxyuridine (FdUrd) to 5-fluorouracil (FUra) by isolated rat intestinal epithelial cells was investigated. Also, the effects of potential inhibitors of this reaction, which is catalyzed by uridine phosphorylase and thymidine phosphorylase, were determined. A 2.5% suspension of isolated cells was incubated with FdUrd or FUra, and at specific times cells were lysed with perchloric acid and fluoropyrimidines were determined by high-performance liquid chromatography. During a 25-min incubation with either FdUrd or FUra, the amount of drug in the incubation system (total volume 0.8 ml) fell by less than 5%. However, in the presence of FdUrd, the amount of FUra increased linearly over 25 min. The apparent Vmax and Km for FUra formation were 17-27 nmole/mg DNA/min and 1.6-2.5 mM, respectively. With each nucleoside phosphorylase inhibitor, the apparent Km increased but Vmax was unaffected. The apparent Ki values were as follows (in mM): 5-nitrouracil (an inhibitor of both uridine phosphorylase and thymidine phosphorylase), 0.12; 4-thiothymine (a uridine phosphorylase-selective inhibitor), 1.52; and 6-benzyl-2-thiouracil (a thymidine phosphorylase-selective inhibitor), 0.73. It was concluded that intestinal epithelial cells are capable of degrading FdUrd to FUra and that the cells possess both uridine phosphorylase and thymidine phosphorylase activity.

Animals↗

[An enzymatic determination of serum citrate with citrate lyase].

A method for the enzymatic determination of the serum citrate using citrate lyase is described. The optimum pH was equal to or higher than 8.6. Prior to the determination of the serum citrate, the samples were deproteinized with perchloric acid. After the precipitation of the proteins, potassium hydroxide was added under an ice-cooling condition in order to neutralize the solution. The recovery rates of the citrate added to serum ranged from 92% to 108%, and the coefficient of variation of triplicate assay was 4.2%. The mean serum value of 17 healthy subjects was 1.57 mg/dl, with a range of 1.03-2.03 mg/dl. In seven healthy subjects, 3 g of sodium-potassium citrate was administered orally and the serum citrate was measured 0, 15, 30, 45, 60, and 120 min later. The serum citrate was significantly increased in the periods between 15 and 60 min.

Adult↗

[Study of the "continuous cell antigen" and the human gastric mucosa].

The antigen common for continuous epithelial cell lines and gastric mucosa of humans described earlier was studied. This antigen was revealed in one more cell line, namely in that prepared from human mammary carcinoma MDA-MB-231, noncontaminated with HeLa cells. The antigen described can be detected in the exophytely growing adenocarcinomas of the stomach and in the mucosa of the carcinoma affected stomach at a distance of 10--12 cm from the site of affection; no such antigen was revealed in the endophytely growing carcinoma of the stomach and in mucosa areas surrounding gastric ulcer. The antigen is not a glycoprotein since glycoprotein fractions obtained by means of 1.2 M perchloric acid from the normal stomach mucosa homogenate and the E 16b extract were inactive in immunodiffusion with a sensitive serum. The electrophoretic mobility of the antigen was similar to that of globulin alpha1-beta2. This antigen is of interest since its detection or absence would possibly aid in determination of the initial type of cells from which development of carcinoma occurred, and in more precise recognition of the histological form of carcinoma of the stomach.

Antigens↗

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