Biomedical subjects
G I Murray
Publications and source records attributed to G I Murray.
Dehydrogenase enzyme histochemistry on freeze-dried or fixed resin-embedded tissue.
A method has been developed for the histochemical demonstration of a variety of dehydrogenases in freeze-dried or fixed resin-embedded tissue. Seven dehydrogenases were studied. Lactate dehydrogenase, NADH dehydrogenase and NADPH tetrazolium reductase were all demonstrable in sections of paraformaldehyde-fixed resin-embedded tissue. Freeze-dried specimens were embedded, without fixation, in glycol methacrylate resin or LR Gold resin at either 4 degrees C or -20 degrees C. All the dehydrogenases except succinate dehydrogenase retained their activity in freeze-dried, resin-embedded tissue. Enzyme activity was maximally preserved by embedding the freeze-dried tissue specimens in glycol methacrylate resin at -20 degrees C. The dehydrogenases were accurately localized without any diffusion when the tissue sections were incubated in aqueous media. Addition of a colloid stabilizer to the incubating medium was not required. Freeze-drying combined with low-temperature resin embedding permits accurate enzyme localization without diffusion, maintenance of enzyme activity and excellent tissue morphology.
The immunocytochemical localisation and distribution of cytochrome P-450 in normal human hepatic and extrahepatic tissues with a monoclonal antibody to human cytochrome P-450.
1. The localisation and distribution of cytochrome P-450 in human tissues has been studied by immunocytochemistry using a monoclonal antibody to a major form of human hepatic cytochrome P-450, P-450hA7, which is closely related to cytochromes P-450 HLp and P-450NF. 2. Strong immunoreactivity was identified in hepatocytes, columnar absorptive epithelial cells of the small intestine, polymorphonuclear leucocytes and their precursors in the bone marrow, and in mast cells. 3. Weak immunoreactivity was present in the proximal tubules of the kidney, pancreatic acini, gall bladder epithelium, squamous epithelium and sebaceous glands of the skin, interstitial cells of the testis and luteal cells of the ovary. 4. Immunoreactivity could not be demonstrated in the adrenal gland, placenta, colonic epithelium and alveolar type II cells and Clara cells of the lung.
Enzyme histochemical demonstration of NADH dehydrogenase on resin-embedded tissue.
We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.
gamma-Glutamyl transpeptidase demonstrated in tissue sections embedded in glycol methacrylate resin.
A method is described for the histochemical demonstration of gamma-glutamyl transpeptidase in tissue sections embedded in glycol methacrylate at low temperature. Enzyme activity was preserved by a short (3 h) fixation of tissue in 4% paraformaldehyde at 4 degrees C prior to embedding at 4 degrees C. Tissue embedded in glycol methacrylate combined good morphology with accurate enzyme localization. Blocks of the embedded tissue could be stored at room temperature for at least 3 months without loss of enzyme activity. The resin is non-fluorescent, allowing the use of the fluorescent coupling agent 5-nitrosalicylaldehyde to visualize the reaction product.
Glutathione localisation in benign and malignant human breast lesions.
Reduced glutathione (GSH) has been demonstrated in benign and malignant human breast lesions using a newly developed histofluorescence technique. GSH was present in every lesion and in each case was localised to the epithelium. A semi-quantitative assessment revealed a moderate amount of GSH in normal epithelium and fibroadenoma and a high level in apocrine metaplasia, epitheliosis and intraduct carcinoma. Invasive ductal carcinoma contained a variable amount of GSH. Correlation between fluorescence intensity and histological grade of ductal carcinomas was almost statistically significant but a relationship to oestrogen receptor status was not detected. The rapid assessment of GSH in breast cancer may aid in the selection of optimum chemotherapeutic regimens.
Cytochrome P-450 immunolocalization in human liver.
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Glutathione localization by a novel o-phthalaldehyde histofluorescence method.
Glutathione in tissues forms an intense fluorophore with a solution of o-phthalaldehyde at room temperature. We have studied the loss of glutathione from tissue sections and find that it is not measurable from thick sections. The fluorescence spectra of the induced fluorophore between glutathione and o-phthalaldehyde are identical in model and tissue sections, while depletion of hepatic glutathione by diethyl maleate produces a comparable fall in fluorescence measured biochemically or histochemically. This simple method is specific as interfering substances, such as spermine and spermidine, produce very weak fluorescence under the conditions employed.
Fluorescence-microscopic measurement of intracellular cytochrome P-450 enzyme activity (ethoxyresorufin O-de-ethylation) in unfixed liver section.
A fluorescence-microscopic method has been developed for measurement of the intracellular kinetics of the cytochrome P-450 reaction, ethoxyresorufin O-de-ethylation, in individual hepatocytes in unfixed non-frozen liver sections obtained from control or 3-methylcholanthrene-pretreated mice. De-ethylation was enhanced in the presence of salicylamide, but inhibited in the presence of alpha-naphthoflavone, A total of 21 reaction rate curves (fluorescence of the metabolite, resorufin, versus time) were constructed for different individual hepatocytes and groups of two or three hepatocytes, in a total of 12 sections from four control and eight 3-methylcholanthrene-induced mice. No two reaction curves were identical, but the curves were classified, by similarity of curve shape and fluorescence intensity, into three control categories and four 3-methylcholanthrene-induced categories. It was considered that the data represented a total of seven types of hepatocytes, which differed in their ethoxyresorufin de-ethylase reaction characteristics. Hepatocytes in the sections from 3-methylcholanthrene-pretreated mice showed higher de-ethylase activities than the hepatocytes from control mice, but the increase in activity covered a wide range of values, from 3-fold to 17-fold higher than the mean control value, suggesting that 3-methylcholanthrene did not induce ethoxyresorufin de-ethylation to the same extent in all hepatocytes.
Cytochrome P-450 localization in normal human adult and foetal liver by immunocytochemistry using a monoclonal antibody against human cytochrome P-450.
Immunocytochemical studies with a monoclonal antibody (MAb-HL3), which recognises a major isozyme of human hepatic cytochrome P-450, have demonstrated this cytochrome in both cryostat and formalin-fixed paraffin-embedded sections of normal human adult liver. Prior trypsin digestion of the formalin-fixed sections prevented staining. There was a zonal distribution of immunoreactive cytochrome P-450, with localization predominantly in the hepatocytes of zone 3 of the hepatic acinus (the centrilobular region). Cytochrome P-450 was also demonstrated in foetal liver, but all foetal hepatocytes contained immunoreactive cytochrome P-450 and there was no zonal distribution of the protein. The biliary epithelium of adult liver contained a small amount of immunoreactive cytochrome P-450 whereas there was no immunoreactivity in the epithelium of foetal bile ducts.
Expression of matrix metalloproteinases 1, 2, 9 and their tissue inhibitors in stage II non-small cell lung cancer: implications for MMP inhibition therapy.
Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, with a very poor survival rate. Therefore there is intense scrutiny to provide a better understanding of the molecular and cellular processes involved in this aggressive disease. The matrix metalloproteinases (MMPs) are a large family of extracellular matrix degrading enzymes believed to play a crucial role in tumor invasion and metastasis. MMP inhibitors are now under development as an adjuvant approach to surgical control of NSCLC. However, there is little data available on MMPs or their tissue inhibitors (TIMPs) in NSCLC. Expression of MMP1, MMP2, MMP9, TIMP1 and TIMP2 was assessed in 44 stage II NSCLC. All proteins were found to be expressed at high levels and significant co-expression was observed. These results suggest that a broad spectrum MMP inhibitor is worthy of evaluation as a therapeutic method of reducing tumor invasion and metastasis in stage II NSCLC.