Auto-antigenicity of connective tissue extracts.
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Antibodies↗
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[Principal problems in tissue-extract therapy].
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Cell- and Tissue-Based Therapy↗
Direct and simultaneous determination of reduced and oxidized glutathione and homoglutathione by liquid chromatography-electrospray/mass spectrometry in plant tissue extracts.
A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/mass spectrometry (time of flight) method has been developed for the direct and simultaneous determination of glutathione and related compounds such as homoglutathione in different plant tissues. These compounds are low-molecular mass antioxidants involved in cellular redox homeostasis in plants, and efforts are being made to develop methods to determine the concentrations of oxidized and reduced forms of these compounds and their ratio. Many of the methodologies developed so far, however, are time-consuming and complex; therefore, analytes can decompose and their redox status can change during the analysis process. The method we have developed allows the simultaneous determination of reduced forms (glutathione [GSH] and homoglutathione [hGSH]) and oxidized forms (glutathione disulfide [GSSG]) of these compounds and is also suitable for the determination of ascorbic acid (ASA) and S-nitrosoglutathione (GSNO). Quantification was done using isotopically labeled GSH and ASA as internal standards. All compounds were base peak resolved in less than 6 min, and limits of detection were 60 pmol for GSH, 30 pmol for hGSH, 20 pmol for GSSG, 100 pmol for ASA, and 30 pmol for GSNO. The intraday repeatability values were approximately 0.4 and 7% for retention time and peak area, respectively, whereas the interday repeatability values were approximately 0.6 and 9% for retention time and peak area, respectively. Analyte recoveries found were between 92 and 105%. The method was used to determine the concentrations of GSH, GSSG, hGSH, and ASA in extracts from several plant tissues.
Ascorbic Acid↗
IMP dehydrogenase. I. Studies on regulatory properties of crude tissue extracts based on an improved assay method.
Inhibition of conversion from IMP to uric acid, which interferes with both spectrophotometric and radioisotopic assays of IMP dehydrogenase, by addition of allopurinol (0.1 mM), an inhibitor of xanthine oxidase, to the incubation system made it possible to determine the enzyme activity in crude liver extracts. With this improved assay method, the regulatory properties of the enzyme in crude extracts of liver and Yoshida sarcoma ascites cells were examined. In both tissues IMP dehydrogenase was found in the postmicrosomal supernatant. However, further centrifugation resulted in precipitation of the enzyme, the enzyme from Yoshida sarcoma ascites cells being precipitated more easily than that from rat liver. It was also found that IMP dehydrogenase activity increased during liver regeneration and that this increase was associated with the precipitate from the postmicrosomal fraction. These findings suggest that such a large sedimentable complex including IMP dehydrogenase might be formed in relation to cell growth. Most of the enzyme activity in rat liver and Yoshida sarcoma ascites cells was extracted in the supernatant obtained by centrifugation at 105,000 X g for 4 h after treatment of tissue homogenates with 1 M KCl, 0.75 M (NH4)2SO4, 2 M dimethylsulfoxide, 2 M KSCN, 25% glycerol, or 0.8 M guanidine-HCl. Treatment with 2% deoxycholate, 2% Triton X-100 or 2 M urea gave limited extraction. The enzyme was retained on a phenyl-Sepharose CL-6B or octyl-Sepharose CL-6B column and eluted with 0.8 M guanidine-HCl. These results suggested that the enzyme molecule has not only ionic but also hydrophobic domains, through which it interacts with other molecules of the enzyme itself and/or postmicrosomal cellular components.(ABSTRACT TRUNCATED AT 250 WORDS)
Allopurinol↗
Demonstration of specific cell-mediated anti-tumor immunity in lung cancer to autologous tissue extracts.
Cell-mediated immunity (CMI) of lung cancer patients to autologous tumor antigens was assessed by mixed lymphocyte tumor interactions (MLTI) as measured in a microculture (200 microliter) lymphocyte proliferation (LP) assay. Positive lymphoproleferative responses were observed with cryopreserved intact mitomycin-C-treated autologous tumor cells (8/12 or 67% patients reactive) and with hypotonic membrane extracts (HMP) of tumor cells (28/40 or 70%). Good correlation was found between reactivity to tumor cells and extracts in parallel testing. In contrast, HMP of autologous normal lung tissue elicited very little LP reactivity, with only one patient giving a weak response by the SI criterion and low level of n cpm. Upon repeat testing, many patients gave reproducibly positive LP responses to tumor HMP. Patients at all clinical stages of disease and with different histologic tumor types had a similar proportion of HMP reactivity. Most reactive patients responded to a broad range of protein concentrations of tumor HMP, and LP responses were frequently elicited with 1 microgram or less of HMP. Thus, HMP appear to afford a convenient source of reactive tumor antigen for assessing anti-tumor immunity.
Adenocarcinoma↗
Radioimmunological determination of cellular retinol-binding protein in human tissue extracts.
A sensitive and specific radioimmunoassay was developed for human cellular retinol-binding protein (CRBP) using antibodies raised in rabbits against purified human liver CRBP. The assay could determine concentrations of CRBP in extracts down to 8 micrograms/liter. CRBP could be demonstrated in all tissues investigated except in serum, and the same immunoreactive CRBP seemed to be present in all tissues. The highest concentrations were found in the gonads. In the gastrointestinal tract, the small intestine had a higher concentration than did stomach or colon. Muscle and skin had low concentrations of CRBP.
Antibodies↗
Properties of hepatic tissue extracts: 2. An approach to the artificial liver.
In the previous paper [3], the lethal effect of normal rabbit liver extract intravenously infused into rabbits was described. By comparing to the course of destruction of the liver tissue in fulminant hepatitis, the toxicity of the liver extract was recently studied. Attempts were also made to adsorbing the liver extract by activated clay and detoxifying the liver extract with a formaldehyde solution, and the results suggested that the findings would be applicable as an approach to the artificial liver.
Adsorption↗
Effect of irrigating fluids and prostate tissue extracts on isolated cardiomyocytes.
OBJECTIVES: A higher mortality probably due to myocardial infarction has been demonstrated after transurethral resection of the prostate (TURP) compared with open prostatectomy. We used an experimental model system to study the possibility that this difference might be due to cardiotoxic effects of two events that frequently occur during TURP, namely, dissemination of electrolyte-free irrigating fluid and release of prostate tissue substances by electrocutting. METHODS: Cardiomyocytes were isolated from male Sprague-Dawley rats. Cell morphology and viability were examined repeatedly during 3 hours of incubation. Control experiments were compared with 34 preparations mixed with one of five different irrigating fluids and with 28 preparations mixed with a prostate extract, either from the same rat or from patients undergoing TURP. RESULTS: Most irrigating fluids reduced the viability of the myocytes. The fraction of viable cells in the incubation mixture averaged 83% (glycine 1.5%), 88% (glycine 1.5% plus ethanol 1%), 92% (glycine 2.2%), 92% (mannitol 3% plus ethanol 1%), and 99% (sorbitol 2% plus mannitol 1%) of that found in the respective control incubations. In contrast, the prostate extracts did not decrease the viability of the cardiomyocytes. Extract from the rats even seemed to have a trophic effect. CONCLUSIONS: Our results show that electrolyte-free irrigating fluids but not prostate extracts have mild cardiotoxic properties. This opens up the possibility that fluid absorption during TURP has a devitalizing effect on the heart.
Animals↗
Single-run high-performance liquid chromatography of nucleotides, nucleosides, and major purine bases and its application to different tissue extracts.
A high-performance liquid chromatography (HPLC) method is described for the separation and quantitation of nucleotides, nucleosides, purine bases, and related compounds in one single run. The separation of a standard mixture of at least 24 components is achieved within 35 min on glass columns (30 cm, 3-mm i.d.) with C-18 reversed-phase particles of 5 micron, and ammonium dihydrogen phosphate (0.15 M, pH 6.00) and a slow linear gradient of methanol/acetonitrile (to 15%) as eluting solvent. The method has been applied to microsamples of different cells and tissues. Samples (2.5 mg dry wt) were cooled in liquid nitrogen, lyophilized, and extracted with 0.6 N perchloric acid. After neutralization with potassium bicarbonate, the extract (20 microliter) was directly injected into the column. To illustrate the wide applicability of the method, representative chromatograms are shown of extracts of biopsies from heart tissue, skeletal muscle, and brain and liver and from hepatocytes, erythrocytes, and yeast cells, under different conditions, known to induce changes in purine metabolism.
Animals↗
Tissue extraction procedures for investigation of urokinase plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in human breast carcinomas.
Urokinase type plasminogen activator (uPA) and its inhibitors, plasminogen activator inhibitor type I (PAI-1) and type II (PAI-2), are supposed to be involved in the expression of the invasive and metastatic phenotype of cancer cells. However, clinical investigations on the prognostic significance of their levels in tumor tissue are difficult to realize because of the absence of a convenient method of measurement of these parameters. The aim of the present investigation was to set up a method allowing the measurement of these enzymes and of sex steroid receptor status in appropriate subcellular fraction(s) in conditions easily reproducible in routine. We found that a tissue homogenate prepared according to the method recommended [5] for current measurement of sex steroid receptors is appropriate for further distinct preparations. One aliquot is used for cytosol preparation; another can be treated by 2% Triton X-100 (vol/vol) and provide an extract containing the totality of uPA and PAI-1. The advantage of this procedure is that appropriate subcellular fractions can be derived from a unique homogenization step. Total uPA and PAI-1 are measured in a Triton extract with good performance as compared to previous investigations [4]. PAI-2 is measured in the same cytosol fraction used for sex steroid receptors and other parameters. Because of its simplicity and its high reliability, this method could be a useful tool in the investigation of uPA family proteases and analysis of their prognostic significance in early breast tumors.
Breast↗
Type II phospholipase A2 in human gestational tissues: extractable immuno- and enzymatic activity in fetal membranes.
In this study, we have established the presence of immunoreactive (ir) Type II PLA2 in human amnion and choriodecidua obtained from women at term prior to the onset of labour. The content of irType II PLA2 present in 1 M NaCl extracts of choriodecidua and amnion averaged 3.5 +/- 3.1 and 10.6 +/- 5.2 ng/mg tissue protein (n = 3), respectively. PLA2 enzymatic activity present in the same tissues averaged 1.3 +/- 0.2 and 1.9 +/- 0.7 nmol phosphatidylethanolamine (PE) hydrolysed/mg tissue protein per h (n = 3), respectively. To allow intra-patient comparison of the relative distribution in gestational tissues, irType II PLA2 and PLA2 enzymatic activity was also determined in placenta obtained from the same group of women, and averaged 26.0 +/- 7.0 ng/mg tissue protein and 3.5 +/- 1.0 nmol PE hydrolysed/mg protein per h (n = 3), respectively. As has been previously reported for human placenta, the recovery of Type II PLA2 and PLA2 enzymatic activity from amnion and choriodecidua was increased between 16- and 25-fold when tissues were homogenized in high-ionic strength media (i.e., 10% (w/v) ammonium sulphate or 1 M NaCl) compared with that recovered when tissues were homogenized in low-ionic strength media (i.e., 0.32 M sucrose-20 mM Hepes). The data obtained represent the first quantitative estimates of immunoreactive Type II PLA2 in human amnion and choriodecidua, and support the conclusion that previous analyses of the PLA2 enzymatic activity present in gestational tissues have essentially excluded the contribution made by this PLA2 isozyme to net enzymatic activity. We suggest that this isozyme represents a major component of the PLA2 enzymatic activity present in human gestational tissues at term and that it contributes significantly to the phospholipid metabolism and arachidonic acid release which occurs during late pregnancy and at the time of labour.
Extraembryonic Membranes↗
Insulin and adipose tissue extracts.
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Adipose Tissue↗
High performance liquid chromatographic separation of anabolic oestrogens and ultraviolet detection of 17 beta-oestradiol, zeranol, diethylstilboestrol or zearalenone in avian muscle tissue extracts.
A multi-residue analysis was developed to discern oestrogens (17 beta-oestradiol or zeranol) used as anabolic drugs and diethylstilboestrol (DES) from the mycotoxins zearalenone and zearalenol. A mixture of 17 beta-oestradiol, oestrone, DES, zeranol, zearalenol, zearalenone and zearalanone was analysed using a 5 micron silica column with gradient elution from hexane to hexane:methanol:2-propanol, 40:45:15 (v/v) and ultraviolet detection at 280 nm. Only six peaks were obtained since zearalenone and zearalanone had identical elution times. Minimum detectabilities were 5-10 ng for the standards. Chicken muscle tissues (2.5 g) were extracted with acetone:water (95:5, v/v). The extracts were spiked with oestradiol and zeranol at 50, 100 or 200 ppb; and DES or zearalenone at 50 ppb; cleaned-up through dual columns of basic alumina and phosphate exchanged AGMP-1 resin and analysed by high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Minimum detectability was 10 ng for oestradiol, zeranol, DES or zearalenone when tissues were spiked at 50 ppb. Recoveries were greater than or equal to 86%, greater than or equal to 83%, 60% and 100% for oestradiol, zeranol, DES and zearalenone, respectively.
Animals↗
An affinity labeling of ras p21 protein and its use in the identification of ras p21 in cellular and tissue extracts.
We have carried out photoaffinity labeling of the ras p21 protein, a ras oncogene product, with [alpha-32P]GTP. Based on our studies, a sensitive, rapid, and specific assay for the detection of multiple forms of ras p21 has been developed. The specificity of this protocol is shown by (a) sensitivity of affinity labeling of ras p21 to known inhibitors of GTP binding and (b) immunoprecipitation of affinity labeled protein with anti-ras p21 serum. Detection and semiquantitation of ras p21 by this method is accomplished in less than 24 h and requires as little as 100,000 cells or about 5 mg of tissue sample from skin tumor, liver, and mammary tumor tissues. Furthermore, using this approach, we were able to detect the selective loss of one species of ras p21 in transplanted Morris hepatoma cells.
Affinity Labels↗
Antibodies to saline extractable tissue antigen in sera of patients with renal diseases.
Multiple serum samples originating from 110 renal allograft recipients were examined against saline extract of normal human kidney by means of double diffusion gel precipitation. Eleven recipients were found to be positive; 99 of 106 sera from these patients were positive. Pretransplantation sera were available from 7 of these recipients and 6 patients were found "positive." The precipitation reaction was composed of one line. Identity reactions were formed between the lines produced by sera from all patients except 1. Sera of patients from end-stage renal disease produced similar reaction; however, only 3 of 234 sera from patients with nonrenal diseases precipitated the kidney extract. None of 154 normal sera were positive. Several positive sera also were positive in complement fixation tests with human kidney extract. Evidence was presented that the antibodies under study combined with a nonorgan-specific but species-restricted tissue antigen. The hypothesis was advanced that these antibodies are autoantibodies formed in response to a sequestered antigen released as a result of tissue damage. Apparently, the antigen is released frequently in immunogenic form from injury to kidney but infrequently from injury to other organs.
Autoantibodies↗
Single-stage clean-up of animal tissue extracts for organochlorine residue analysis.
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Adipose Tissue↗
Antigenicity of connective tissue extracts.
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Antigens↗
Immunofluorometric assay of prostaglandin D synthase in human tissue extracts and fluids.
A two-site sandwich-type assay for human prostaglandin D (PGD) synthase (beta-trace) was developed with two monoclonal antibodies and using time-resolved fluorometry as the detection technique. The assay is precise (CVs < 10%), accurate, and highly specific for PGD synthase and has a detection limit of 0.05 microgram/L. Using this assay, we measured PGD synthase concentrations in serum, urine, amniotic fluid, cerebrospinal fluid (CSF), seminal plasma, breast cyst fluid, breast discharge fluid, breast milk, and breast tumor extracts. The highest concentrations were found in CSF. We identified proteolytic degradation of PGD synthase in amniotic fluid. Fetal tissues contained various amounts of the enzyme, with the highest values being found in brain and heart. In placental extracts, PGD synthase content was greatest at 11-28 weeks of gestation-in accordance with the concentrations measured in amniotic fluids for this gestational period. We conclude that PGD synthase is ubiquitous and is present in many fluids and tissues of adults and fetuses. This first quantitative and sensitive assay of PGD synthase should facilitate expansion of knowledge on this enzyme and possibly will have applications for diagnosis and monitoring of human diseases.
Adult↗