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PubMed2003

Spectral characterization of catechol estrogen quinone (CEQ)-derived DNA adducts and their identification in human breast tissue extract.

Estrogens, including the natural hormones estrone (E(1)) and estradiol (E(2)), are thought to be involved in tumor induction. Catechol estrogen quinones (CEQ) derived from 4-hydroxyestrone (4-OHE(1)) and 4-hydroxyestradiol (4-OHE(2)) react with DNA and form depurinating N7Gua and N3Ade adducts that might be responsible for tumor initiation (Cavalieri, E. L., et al. (2000) J. Natl. Cancer Inst. Monogr. 27, 75). Current detection limits for the CEQ-derived DNA adducts by high-performance liquid chromatography with multichannel electrochemical detection are in the picomole range. To improve the limit of detection (LOD) for CEQ-derived DNA adducts, spectrophotometric monitoring was investigated. Spectroscopic studies of 4-OHE(1)-1-N3Ade, 4-OHE(1)-1-N7Gua, 4-OHE(2)-1-N3Ade, and 4-OHE(2)-1-N7Gua adduct standards were performed at 77 and 300 K. Upon laser excitation at 257 nm, the 4-OHE(1)- and 4-OHE(2)-derived N7Gua and N3Ade adducts are strongly phosphorescent at T = 77 K. No phosphorescence was observed at 300 K. Both N3Ade and N7Gua adduct types have weak phosphorescence origin bands near 383 and 385 nm, respectively. The corresponding phosphorescence lifetimes are 1.11 +/- 0.05 and 0.37 +/- 0.05 s. The LOD, based on phosphorescence measurements, is in the low femtomole range. The concentration LOD is approximately 10(-9) M, i.e., similar to that recently obtained for CEQ-derived N-acetylcysteine conjugates (Jankowiak, R., et al. (2003) Chem. Res. Toxicol. 16, 304). The LOD in capillary electrophoresis (CE) with field-amplified sample stacking and absorbance detection is about 3 x 10(-8) M. To verify whether CEQ-derived DNA adducts are formed in humans or not, tissue extracts from two breast cancer patients were analyzed by CE interfaced with room temperature absorption and low temperature (laser-excited) phosphorescence spectroscopies. For the first time, formation of CEQ-derived DNA adducts is shown in humans. For example, the level of 4-OHE(1)-1-N3Ade in the breast tissue extract from a patient with breast carcinoma (8.40 +/- 0.05 pmol/g of tissue) is larger by a factor of about 30 than that in the breast tissue sample from a woman without breast cancer (0.25 +/- 0.05 pmol/g of tissue). In contrast, similar amounts of 4-OHE(2)-1-N3Ade were observed in both types of tissue. Although more breast tissue samples from women with and without breast cancer need to be studied, these results suggest that the N3Ade adducts could serve as biomarkers to predict the risk of breast cancer.

Breast Neoplasms↗
PubMed1966

A gas-liquid-chromatographic procedure for separating a wide range of metabolites occuring in urine or tissue extracts.

1. A gas-liquid-chromatographic procedure is described which permits separation and identification on the same chromatogram of a wide range of substances occurring in urine or tissue extracts. The method uses hydrogen flame ionization, which detects organic compounds whether free or conjugated with no requirement for specific reactive groups. 2. For chromatography, carboxyl groups are quantitatively converted into methyl esters or trimethylsilyl esters. Phenolic, alcoholic and potential enolic groups are converted into trimethylsilyl ethers. Separations are carried out on a 6ft. column of either 10% F-60 (a polysiloxane) or 1% F-60, temperature programming at 2 degrees /min. being used over such part of the temperature range 30 degrees -260 degrees as is required. Propionyl derivatives of hydroxy compounds can also be used, but only on a non-quantitative basis. Derivatives and columns have been selected for optimum range of usefulness when large numbers of samples are examined by using automated gas chromatography. 3. The method is applicable to: fatty acids above butyric acid; di- and tri-carboxylic acids; hydroxy acids and keto acids; polyhydroxy and alicyclic compounds such as glycerol, inositol, quinic acid, shikimic acid, ascorbic acid and sugar alcohols; aromatic hydroxy and acidic compounds, both benzenoid and indolic; sesquiterpenes; steroids; glycine conjugates; mercapturic acids; glucuronides. It is not satisfactory for sulphate conjugates, iminazoles or polypeptides. 4. Methylene units provide an accurate and reproducible parameter for characterizing peak position. Methylene unit values are reported for a large variety of substances occurring in, or related to those occurring in, urine and tissue extracts. 5. The nature of derivatives was confirmed by combining gas chromatography with mass spectrometry. Combined gas chromatography-mass spectrometry gives a diagnostic tool of great power in the evaluation of metabolic patterns, and various uses are discussed.

Journal Article↗
PubMed1984

Demonstration of placental and placental-like alkaline phosphatase in non-malignant human tissue extracts using monoclonal antibodies in an enzyme immunoassay.

The occurrence and nature of heat-stable placental-type alkaline phosphatase (Pl-ALP) in extracts from a variety of non-malignant human tissues has been investigated using monoclonal antibodies in a sensitive solid-phase enzyme immunoassay. The presence of Pl-ALP was confirmed in testicular, cervical and lung tissue extracts, and trace amounts were also detected in extracts from mammary and ovarian tissue. Evidence is presented that normal testis contains at least two forms of Pl-ALP, the major component being an L-leucine-inhibitable placental-like enzyme which is not the D-variant of Pl-ALP. These results have a bearing on the occurrence of Pl-ALP and placental-like ALP activity in malignancy.

Alkaline Phosphatase↗
PubMed1991

Ubiquitination of endogenous calmodulin in rabbit tissue extracts.

Previously we were able to show that purified calmodulins from vertebrates, plants (spinach) and the mold Neurospora crassa can be covalently conjugated to ubiquitin in a Ca(2+)-dependent manner. It was therefore pertinent to answer the question if a tissue extract contains all the components necessary for the endogenous synthesis of ubiquityl calmodulin (uCaM). Therefore [125I]ubiquitin, ATP/Mg2+ and Ca2+ were added to tissue extracts enriched by a single ion exchange step. In such extracts of red blood cells, skeletal muscle and testis a novel ubiquitin conjugate of 27-29 kDa is formed. This novel band could be identified as ubiquityl-calmodulin by the following methods: (i) identical Rf-value of novel conjugate and standard uCaM in SDS-PAGE; (ii) Ca(2+)-dependent conjugate formation; (iii) Ca(2+)-dependent adsorption to fluphenazine-Sepharose; (iv) Ca(2+)-dependent mobility change of the novel conjugate during SDS-PAGE; and (v) inhibition of conjugate band formation by phosphorylase kinase. These experiments clearly demonstrate that ubiquityl calmodulin can be endogenously generated in enriched cellular extracts and strongly indicate that this reaction is of importance in vivo.

Adenosine Triphosphate↗
PubMed1989

Suppression of lymphocyte proliferation in vitro by macromolecules in the vesicle fluid and tissue extracts of hydatidiform mole.

This study examines the effects of vesicle fluid and tissue extracts from hydatidiform mole trophoblast on lymphocyte proliferation in vitro. Samples were obtained by direct aspiration of vesicles (hydatidiform mole vesicle fluid (HMF] or homogenization of molar tissues (hydatidiform mole extract (HME] following therapeutic uterine evacuation of hydatidiform mole. Dialyzed and lyophylized HMF pooled from two patients exhibited a 30% suppression (P less than 0.05) of mitogen-induced lymphocyte proliferation at a concentration of 12.5 micrograms protein/ml. Similarly, lymphocyte transformation was significantly suppressed (P less than 0.05) by HME at concentrations of 500 and 250 micrograms/ml. Molecular weight chromatography of HME resolved 4 protein fractions. Fraction 3 (35--50 kDa) and fraction 4 (less than 35 kDa) significantly suppressed mitogen-induced lymphocyte transformation while fractions 1 and 2 demonstrated no immunosuppression. Heat treatment (56 degrees C, 30 min) abolished the immunosuppressive activity of HME as well as fractions 3 and 4. These results suggest that hydatidiform mole trophoblast contains heat-labile macromolecules which suppress mitogen-mediated lymphocyte transformation. Such trophoblast-derived factors may interfere with maternal rejection of the allograft.

Female↗
PubMed1977

A neuromuscular transmission block produced by a cancer tissue extract derived from a patient with the myasthenic syndrome.

The myasthenic syndrome occasionally is associated with bronchogenic carcinoma. The neuromuscular transmission defect in this syndrome is characterized by a reduction of acetylcholine release from motor nerve endings. This paper reports that an acetone extract of cancer tissue from a patient with the syndrome reduces the acetylcholine release from motor nerve endings and produces a neuromuscular transmission defect in the frog nerve-muscle preparation. This suggests that the pathogenic substance(s) contained in the extract may be produced by certain types of bronchogenic carcinoma and may cause the myasthenic syndrome.

Acetylcholine↗
PubMed2025

Systematic comparison of quantity and quality of RNA recovered with commercial FFPE tissue extraction kits.

BACKGROUND: FFPE tissue samples are commonly used in biomedical research and are a valuable source for next-generation sequencing in oncology, however, extracting RNA from these samples can be difficult the quantity and quality achieved can impact the downstream analysis. This study compared the effectiveness of seven different commercially available RNA extraction kits specifically designed for use with FFPE samples in terms of the quantity and quality of RNA recovered. METHODS: This study used 9 samples of FFPE tissue from three different types of tissue (Tonsil, Appendix and lymph node of B-cell lymphoma) to evaluate RNA extraction methods. Three sections of 20 µm of each sample were combined per sample. The slices were distributed in a systematic manner to prevent any biases. Each of the 7 commercially available RNA extraction kits were used according to manufacturer's instructions, with each sample being tested in triplicate resulting in a total of 189 extractions. The concentration, RNA quality score (RQS) and DV200 of each extraction was analysed using a nucleic acid analyser to determine the quantity and quality of the recovered RNA. RESULTS: This study found that despite processing the FFPE samples in the same standardized way, there were disparities in the quantity and quality of RNA recovered across the different tissue types. Additionally, the study found notable differences in the quantity of RNA recovered when using different extraction kits. In terms of quality, three of the kits performed better than the other four in terms of RQS and DV200 values. CONCLUSION: Though many laboratories have developed their own protocols for specific tissue types, using commercially available kits is still a popular option. Although these kits use similar processes and extraction procedures, the amount and quality of RNA obtained can vary greatly between kits. In this study, among the kits tested, while the Roche kit, provided a nearly systematic better-quality recovery than other kits, the ReliaPrep FFPE Total RNA miniprep from Promega yielded the best ratio of both quantity and quality on the tested tissue samples.

Paraffin Embedding↗
PubMedDate not supplied

A two-site immunoradiometric assay of proatrial natriuretic factor. Application to tissue extracts.

A "two-site" immunoradiometric assay (IRMA) was developed to specifically measure ANF (1-126), the precursor of ANF. This assay is based on the simultaneous use of antibodies against two different antigenic determinants: murine monoclonal antibody (2H2), which recognizes positions 101 through 103 of ANF, is linked to Immunobeads and employed to extract any ANF C-terminal; a second antibody, which is directed against positions 11 through 37, is radioiodinated and allows binding to any C-terminal-2H2-Immunobead material which bears the N-terminal antigenic site. A curvilinear relationship was obtained between radioactivity and the amount of proANF (1.5 to 400 fmol) added. Optimisation of IRMA was determined by the amount of 2H2-Immunobeads and labeled antibody used, incubation time as well as possible interference by both ANF (99-126) and ANF (1-98). Tissue extracts were used to validate the assay. proANF was detected in decreasing amounts in heart atria, heart ventricles, lungs, kidneys and adrenal glands. Its presence was further confirmed by reverse-phase HPLC followed by radioimmunoassay. IRMA is a simple and rapid method for the direct measurement of proANF in tissue extracts and chromatographic fractions. The presence of proANF in tissues strongly suggests local synthesis.

Adrenal Glands↗
PubMed2000

Effects of tissue extracts on oviduct contraction in the migratory grasshopper, Melanoplus sanguinipes.

The isolated oviduct of the migratory grasshopper, Melanoplus sanguinipes, shows a myogenic, rhythmic pattern of contraction. The pattern of contraction can be modified by treatment with hemolymph or extracts from a variety of tissues from animals of differing age, sex, and mated status. Extracts of oviducts and ventral nerve cord (VNC), as well as hemolymph, from both virgin and mated females were almost always stimulating, though their effect on frequency, amplitude and/or tonus varied. In contrast, whereas extracts of spermatheca and brain from mated females were stimulating, extracts of these tissues from virgin females inhibited contraction. All male material tested (long hyaline tubules (LHT), the male accessory gland complex less the LHT, testes, brain, VNC and hemolymph) stimulated oviduct contraction in a dose-dependent manner, usually enhancing at least two of frequency, amplitude and tonus. However, oviposition-stimulating protein, a purified product of the LHT, provoked only an increase in frequency when applied to the oviduct. LHT extract modulated the effects of virgin female tissue extracts, always in a stimulatory manner.

Journal Article↗
PubMed1987

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts.

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.

Adenine Phosphoribosyltransferase↗
PubMed2000

Induction of matrix metalloproteinases in keratinocytes by cholesteatoma debris and granulation tissue extracts.

Although it is generally accepted that destruction and remodeling of temporal bone associated with middle ear cholesteatoma is mainly caused by the action of osteoclasts, it has been shown that neutral collagenases also play a role in predigesting the osteoid layer and exposing the mineralized bone to osteoclastic activity. Here we show that gelatinase B (matrix metalloproteinase-9) is over-expressed in cholesteatoma compared to external ear canal skin (EACS). Expression of MMP-9 in cholesteatoma mainly occurs in suprabasal layers, and more rarely in basal layers of cholesteatoma epithelium, as well as in inflammatory cells of the perimatrix. We further analyzed the influence of cholesteatoma debris, cholesteatoma granulation tissue, and cholesteatoma components such as keratin, cholesterol and bacterial endotoxin on the expression of MMPs in EACS keratinocytes. We show that cholesteatoma debris and granulation tissue extract both induced the secretion of MMP-9 by EACS keratinocytes, while keratin. bacterial lipopolysaccharide (LPS) or cholesterol did not show any effect. We further performed co-incubation and immunoprecipitation experiments using neutralizing interleukin-1alpha, EGF, TGF-beta, TGF-alpha, interleukin-6 and TNF-alpha antibodies. Inhibition of MMP-9 up-regulation by debris or granulation tissue extract could be revealed with diverse cytokine antibodies. The results are discussed with regard to previously published studies.

Antibodies↗
PubMed1978

The nature of the immunoreactive lipotropins in human plasma and tissue extracts.

This study was designed to establish definitively the nature of immunoreactive lipotropin (IR-LPH) in human plasma and tissue extracts. Using gel filtration, gel filtration under denaturing conditions, cationic exchange chromatography, immunoprecipitation, and radioimmunoassay, we have studied normal and tumorous human pituitaries, ectopic ACTH- and LPH-secreting tumors, plasma from normal subjects before and after dexamethasone administration, and plasma from patients with primary adrenal insufficiency and pituitary and nonpituitary ACTH- and LPH-secreting tumors. Except in the plasma and tumors of occasional patients with ectopic ACTH syndrome, the smallest IR-LPH appears to be lambda-lipotropin (lambdaLPH), which is often the predominant and occasionally the only IR-LPH present. The other major peptide appears to be betaLPH, a 91-amino acid molecule that contains lambdaLPH as its 1-58 sequence. Larger immunoreactive materials were observed in some specimens, but the "big" LPH in one plasma was shown to be lambdaLPH bound to IgG.The weak melanocyte-stimulating activity of LPH suggests that ACTH may be the principal pigmentary hormone in man. The fact that lambdaLPH, rather than betaLPH, is the predominant form in plasma suggests that the enkephalin-endorphin opiate peptides, which are contained in the "missing" 59-91 sequence from the betaLPH precursor of lambdaLPH, may be secreted in parallel with ACTH under both physiological and pathological conditions in man.

Chromatography, Gel↗