A link between monoamine oxidase-A and apoptosis in serum deprived human SH-SY5Y neuroblastoma cells.

Increased monoamine oxidase (MAO) activity was recently shown to accompany apoptotic cell death of various neuronal cells following growth factor deprivation. Here we show that in serum deprived SH-SY5Y cells, MAO-A mRNA levels and catalytic activities are increased, linked with activation of the apoptotic executioner caspase-3. Importantly, specific inhibition of MAO-A activity resulted in loss of apoptotic cell morphology. Our study indicates that MAO catalytic activity is involved in apoptotic signalling in response to serum withdrawal in neuronal cells.

A comparative study of the expression of monoamine oxidase-A and -B mRNA and protein in non-CNS human tissues.

The distributions of monoamine oxidase (MAO)-A and -B proteins and mRNAs in human heart, lung, liver, duodenum, kidney and vasculature were compared using immunohistochemistry and cRNA in situ hybridisation. MAO-A and -B mRNA were detected in all tissues, to differing extents, but particularly in glomeruli, hepatocytes, and the crypts, muscularis mucosa and muscularis externa of duodenum. Renal proximal and distal tubules and loops of Henle had more intense labelling for mRNA of MAO-B than MAO-A; this was reflected in MAO protein expression. Little immunoreactivity was detected in glomeruli. Hepatocytes expressed MAO-A moderately, but MAO-B strongly. In lungs, similar moderately intense labelling for both MAO mRNAs and immunoreactivities was evident in pneumocytes, and epithelial and smooth muscle cells. Cardiomyocytes contained both MAO isoforms, but with more, albeit moderate, labelling for MAO-A. Both isoforms were expressed equally in duodenal villi, crypts, muscularis externa and mucosa; lower level expression occurred in mucosal and submucosal cells. MAO-A and -B mRNA were detected in endothelia, adventitia and media of a renal interlobular artery, but protein immunoreactivities were chiefly in the adventitia. The data reveal widespread tissue distribution of MAO mRNAs and proteins, but indicate that presence of MAO mRNAs does not invariably reflect quantitatively its protein expression.

Successful vaccination of BALB/c mice against human hookworm (Necator americanus): the immunological phenotype of the protective response.

In this murine (BALB/c) model of necatoriasis, high levels of protection against challenge infection by Necator americanus larvae (n=300) were afforded by successive vaccinations at 14-day intervals, either subcutaneously or percutaneously, with gamma-irradiated N. americanus larvae (n=300). Percutaneous vaccination was significantly more effective than the subcutaneous route, with pulmonary larval burdens at 3 days post-infection being reduced by 97.8 vs. 89.3%, respectively, after three immunisations (P<0.05). No worms were recovered from the intestines of thrice vaccinated mice. Two percutaneous vaccinations also reduced worm burdens, by 57% in the lungs and 98% in the intestines; P<0.05. In vaccinated animals, lung pathology (mainly haemorrhage) following infection was greatly reduced compared with non-vaccinated animals. In vaccinated mice (but not in non-vaccinated mice) mast cells accumulated in the skin and were degranulated. RT-PCR analyses of mRNAs in the skin of vaccinated animals indicated increased expression of interleukin (IL)-4 relative to gamma-interferon (gamma-IFN). Lymphocytes from the axillary (skin-draining) lymph nodes of vaccinated mice, stimulated in vitro with concanavalin A, exhibited enhanced secretion of IL-4 protein and a higher IL-4/gamma-IFN protein ratio than lymphocytes from non-vaccinated animals. In vaccinated mice, levels of IgG1 and IgG3 (directed against larval excretory/secretory products) were elevated for the most part compared with those in non-vaccinated animals. These data demonstrate the successful vaccination of BALB/c mice against human hookworm infection and suggest that a localised Th2 response may be important for conferring protection against necatoriasis.

Monoamine oxidase expression and activity in human placentae from pre-eclamptic and normotensive pregnancies.

A feature of pre-eclampsia is that circulating levels of maternal serotonin (5-hydroxytryptamine) are elevated and placental monoamine oxidase-A (MAO-A) activity, the major factor in the regulation of serotonin levels in pregnancy, is reduced. It is not known whether this is due to a reduced MAO-A protein content or a reduced catalytic turnover of the serotonin by MAO-A; this question has been addressed in the present work. Term placentae from normotensive and pre-eclamptic women were analysed for MAO-A specific mRNA expression (by semi-quantitative RT-PCR), MAO-A protein (by immunohistochemistry and quantitative ELISA, using a MAO-A specific monoclonal antibody), together with MAO activity (using [(3)H] labelled 5-hydroxytryptamine as substrate). Immunohistochemical analysis of placentae from both normotensive and pre-eclamptic women demonstrated that MAO-A protein is located in the cytoplasm of the placental syncytiotrophoblast layer, consistent with a mitochondrial location; no MAO-A protein was found in the nucleus. No MAO-B protein was detected in this placental layer, despite the presence of MAO-B mRNA. The results indicate that both total protein/g fresh weight and MAO-A protein/g fresh weight were approximately 40 per cent lower in pre-eclamptic than in normotensive placentae, but that there was no statistical difference in the expression of MAO-A mRNA in relation to GAPDH or actin mRNA or in MAO-A protein/mg total protein. However, MAO-A activity/g fresh weight was significantly reduced in pre-eclamptic placentae, in agreement with previous findings. This was found to be due to a 60 per cent reduction (P< 0.05) in the catalytic turnover (activity/molecule) of the enzyme. This study has therefore clearly shown that the expression of placental MAO-A specific mRNA and MAO-A protein are not specifically affected in pre-eclampsia, but that the catalytic efficiency of the expressed MAO-A enzyme in pre-eclamptic placentae is greatly reduced.

Cellular localization of monoamine oxidase A and B in human tissues outside of the central nervous system.

We studied the localization of monoamine oxidase (MAO) A and B in human heart, liver, duodenum, blood vessels and kidney by immunohistochemistry. The primary antibodies used were mouse monoclonal anti-human MAO-A (6G11/E1) and anti-human MAO-B (3F12/G10/2E3). Samples were obtained from six routine autopsy cases and fixed in 2% paraformaldehyde. All cardiomyocytes and hepatocytes showed MAO-A and MAO-B immunoreactivity. In the duodenum, both immunoreactivities were present in all cells of the villi, Lieberkühn crypts, muscularis mucosae and muscular layers, whereas Brunner glands were devoid of MAO-A and MAO-B staining. Endothelial cells of lymphatic vessels showed MAO-A but no MAO-B immunoreactivity, whereas arteries and veins presented MAO-A and MAO-B staining in muscular layers and fibroblasts but not in endothelial cells. In the kidney, renal tubuli showed MAO-A and MAO-B immunoreactivities, whereas collecting ducts and the Bowman's capsule showed only MAO-A staining. These data represent the first study of the cellular distribution of MAO-A and MAO-B in these human tissues. They show that both enzymes have a widespread distribution in the human body with a matching pattern in many, but not all tissues, and with strong differences from the pattern of distribution in rodents.

Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cells.

We have shown previously that subcytotoxic concentrations of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF-H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 microM) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP-induced cell death (cytotoxicity) and MPTP-induced NF-H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 microM) reduced the cytotoxic effect of MPTP, but blocked di-butyryl cyclic AMP (dbcAMP)-induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine oxidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP-induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF-H hyper-phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP-induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine on differentiating mouse N2a neuroblastoma cells.

The effect of the neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) was investigated in mouse N2a neuroblastoma cells, induced to differentiate by serum withdrawal and addition of dibutyryl cyclic AMP, over a 24-h period. Addition of MPTP (10 microM) during differentiation caused a change in cell morphology characterised by an inhibition of axon outgrowth, in the absence of cell death. Biochemical characterisation by western blotting revealed that MPTP had no significant effects on the levels of actin, alpha-tubulin, or total heavy-chain neurofilament (NF-H). However, NF-H phosphorylation appeared to increase following MPTP treatment when blots were probed with the phosphorylation state-specific antibodies RMd09 and Ta51. In addition, indirect immunofluorescence analysis revealed an accumulation of phosphorylated NF-H in the cell perikaryon, suggesting that altered NF-H distribution was associated with the observed effects of MPTP on cell morphology. These changes may represent a useful in vitro marker of MPTP neurotoxicity within a simple differentiating neuronal cell model system.

Localization of monoamine oxidase A and B in human pancreas, thyroid, and adrenal glands.

We studied monoamine oxidase (MAO) A and B localization in human pancreas, thyroid gland, and adrenal gland by immunohistochemistry. The primary antibodies used were mouse monoclonal anti-human MAO-A (6G11/E1) and anti-human MAO-B (3F12/G10/2E3). Samples were obtained from six routine autopsy cases and fixed in 2% paraformaldehyde. Exocrine pancreas showed a widespread distribution of MAO-A, whereas MAO-B was present only in centroacinar cells and epithelial cells of pancreatic ducts. In endocrine pancreas, MAO-A was observed in around 50% of islet cells, whereas MAO-B was less abundant and was restricted to the periphery of islets. Thyroid gland showed strong MAO-A immunoreactivity in all cell types and was MAO-B-negative. In adrenal gland, the capsule displayed MAO-A but not MAO-B immunoreactivity, whereas the cortex showed widespread MAO-A staining but was MAO-B-negative in interstitial cells. Finally, in the medulla only a few scattered cells showed either MAO-A or MAO-B immunoreactivity. To our knowledge, these data represent the first study of the cellular distribution of MAO-A and MAO-B in the three human tissues included.

Necator americanus (human hookworm) aspartyl proteinases and digestion of skin macromolecules during skin penetration.

The infective larvae of Necator americanus were shown to secrete all mechanistic classes of proteolytic enzymes with two overall pH optima of 6.5 and 8.5 using fluorescein isothiocyanate-labeled casein as the substrate. Since infective larvae are obligate skin penetrators, the effect of each of these enzyme classes against macromolecules derived from human skin was examined. Larval secretions were shown to degrade collagen types I, III, IV, and V, fibronectin, laminin, and elastin. All the skin macromolecules tested were hydrolyzed by aspartyl proteinase activity, which was inhibitable by pepstatin A. Collagen and elastin was also hydrolyzed by metalloproteinase activity, while the serine proteinase activity hydrolyzed only elastin. As a consequence of these experiments, the effect of proteinase inhibitors on the penetration of live larvae through hamster skin was tested. Larval penetration was significantly inhibited only by pepstatin A, confirming the importance of the aspartyl proteinase activity during the skin penetration process.

Localization of monoamine oxidase mRNA in human placenta.

Monoamine oxidase (MAO) oxidatively deaminates vasoactive and biogenic amines and exists in two distinct forms (A and B), coded for by separate genes, which exhibit distinct substrate specificities and inhibitor sensitivities. Using specific primers for MAO-A and MAO-B mRNA in a reverse transcription-polymerase chain reaction (RT-PCR) on RNA from human liver, the predicted products for both enzymes were detected. Furthermore, RT-PCR on RNA from human placenta, believed to contain predominantly (or only) MAO-A protein, also indicated the presence of both A and B gene transcripts. The cellular distribution of MAO mRNA in placental tissue was analyzed by in situ hybridization of MAO-A and MAO-B mRNA-specific cRNA probes on paraffin sections. MAO-A mRNA was mainly evident in the syncytiotrophoblastic layer. None was detected in the vascular endothelium/smooth muscles. Significantly, MAO-B mRNA signal was also evident in the placental villi, notably in the syncytiotrophoblasts, intermediate trophoblasts, cytotrophoblasts, and the vascular endothelium. To our knowledge, this is the first demonstration of the cellular distribution of MAO mRNA in human placenta via in situ hybridization. The expression of MAO-B in placental tissue rather than in blood elements within placenta is also unequivocally demonstrated. These highly specific cRNA probes can now be used to study the distribution of MAO-A and MAO-B expression in other tissues.

Human anti-mitochondria autoantibodies appearing in iproniazid-induced immunoallergic hepatitis recognize human liver monoamine oxidase B.

Anti-mitochondria (anti-M6) autoantibodies have been found in the serum of patients with immunoallergic iproniazid (Marsilid)-induced hepatitis, but to date the identity of the protein antigen has not been determined. Here we show, using immunoprecipitation of pargyline-labelled proteins, that among the mitochondrial proteins, liver MAO-B is specifically recognized by the sera containing anti-M6 antibodies. Moreover the enzymatic activity of MAO-B towards phenylethylamine and tyramine is also suppressed after this immunoprecipitation, contrary to the MAO-A activity towards 5-hydroxy-tryptamine. As MAO is irreversibly inhibited by iproniazid, these results suggest that the mechanism of iproniazid-induced appearance of anti-M6 antibodies could be another example of the reactive metabolite/enzyme haptenization mechanism already proposed in the case of tienilic acid for the appearance of anti-organelle antibodies in a drug-induced hepatitis.

An initial characterization of the proteolytic enzymes secreted by the adult stage of the human hookworm Necator americanus.

The proteolytic activities present in adult Necator americanus excretory-secretory products have been assessed using biologically relevant, naturally occurring substrates (haemoglobin and fibrinogen) and a number of synthetic fluorogenic and chromogenic substrates. One broad peak of activity was observed against haemoglobin in the pH range 5 to 7, with maximum activity at pH 6.6, while fibrinogenolytic activity was shown to be greater at pH 3.5. Inhibition studies against haemoglobin, fibrinogen and synthetic substrates using a battery of appropriate protease inhibitors indicated the presence of a mixture of aspartyl, cysteinyl and serine proteases. Metal ion (Ca2+, Zn2+ and Fe2+) stimulation was demonstrated, with stimulation by Zn2+ being the most marked. These results are discussed in the context of recent developments in the field of parasite proteolytic enzymes, where they have been suggested as targets for immuno- and chemotherapy.

An enzyme immunoassay for the measurement of human monoamine oxidase B protein.

A heterologous, competitive, solid phase ELISA has been developed which can measure monoamine oxidase (MAO) B concentration (both inactive and active) in human platelets and other tissue extracts. The assay is based on competition between a soluble form of MAO and MAO bound to a solid phase for binding to a limiting amount of a MAO B-specific monoclonal antibody, 3F12/G10. It utilises a crude and easily prepared sample of human liver mitochondrial membranes as the source of solid phase MAO. Optimal assay conditions allow detection of MAO B down to at least 0.5 ng (4.18 fmol; 6.25 ng/ml) of protein. As the assay is sensitive and simple to operate, it will allow a systematic assessment of the role of platelet MAO concentrations in the aetiology of psychiatric and neurologic conditions.

Purification of ornithine aminotransferase by immunoadsorption.

Ornithine aminotransferase was purified by conventional biochemical methods from rat kidney, rat liver, and human liver. Affinity-purified antibodies raised to the rat kidney enzyme were used to produce an immunoadsorbent enabling a one-step purification of ornithine aminotransferase to be made from crude human liver extracts. The harsh chemical conditions often required to desorb immunoadsorbents were avoided by isolating antibodies with low functional affinity and employing an electrophoretic desorption method which allowed the enzyme activity to be retained. The close structural similarity between human and rat ornithine aminotransferase was demonstrated by immunodiffusion reactions. It was therefore possible to purify the enzyme from human liver using immobilized antibodies raised against rat kidney ornithine aminotransferase. Furthermore, desorption was more readily achieved due to the lower affinity for the human enzyme.

Evaluation of reconstituted Sendai virus envelopes as intra-articular drug vectors: effects on normal and experimentally arthritic rabbit knee joints.

Fusogenic vesicles reconstituted from the envelopes of Sendai virus particles were injected into rabbit knee joints (both normal and experimentally arthritic) to evaluate the in-vivo biocompatibility of these putative drug carriers. The reconstituted Sendai virus envelopes (RSVE) were greater than 80% retained within the arthritic knee joints after 24 h and studies with 125I- and fluorescein-labelled RSVE both showed association of the vesicles with the synovia of arthritic and healthy joints. However, RSVE were found to cause inflammation after intra-articular injection, as judged by joint swelling and histological assessment, and these effects were exacerbated by successive administrations. RSVE-entrapped methotrexate, whether free or conjugated to human serum albumin, was ineffective in preventing the irritancy of RSVE or in reducing the chronic inflammation in joints affected by an experimentally induced arthritis.

A comparison of ornithine aminotransferase from human and rat sources.

Ornithine aminotransferase was purified from human liver, rat liver and rat kidney. Sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a subunit molecular weight of 45,000 in all three cases. Estimations of the native molecular weights of ornithine aminotransferase were determined by Sephadex G-200 chromatography in the presence and absence of 0.1% (w/v) Triton X-100. Human and rat enzymes were tetrameric in the presence of detergent but the rat subunits aggregated further in its absence. Characterisation of ornithine aminotransferase from the two rat sources indicated that they were the same protein. The human and rat enzymes were similar but not identical.

Sendai-viral HN and F glycoproteins as probes of plasma-membrane protein catabolism in HTC cells. Studies with fusogenic reconstituted Sendai-viral envelopes.

Reconstituted Sendai-viral envelopes (RSVE) were produced by the method of Vainstein, Hershkovitz, Israel & Loyter [(1984) Biochim. Biophys. Acta 773, 181-188]. RSVE are fusogenic unilamellar vesicles containing two transmembrane glycoproteins: the HN (haemagglutinin-neuraminidase) protein and the F (fusion) factor. The fate of the viral proteins after fusion-mediated transplantation of RSVE into hepatoma (HTC) cell plasma membranes was studied to probe plasma-membrane protein degradation. Both protein species are degraded at similar, relatively slow, rates (t1/2 = 67 h) in HTC cells fused with RSVE in suspension. Even slower degradation rates for HN and F proteins (t1/2 = 93 h) were measured when RSVE were fused with HTC cells in monolayer. Lysosomal degradation of the transplanted viral proteins is strongly implicated by the finding that degradation of HN and F proteins is sensitive to inhibition by 10 mM-NH4Cl (81%) and by 50 micrograms of leupeptin/ml (70%).

A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with transplanted Sendai-viral envelope proteins in HTC cells.

Reconstituted Sendai-viral envelopes (RSVE) were fused with hepatoma tissue-culture (HTC) cells, thereby introducing viral membrane glycoproteins into the plasma membrane [Earl, Billett, Hunneyball & Mayer (1987) Biochem. J. 241, 801-807]. Fractionation of homogenized cells on Nycodenz gradients shows that much of the viral 125I-labelled HN and F proteins were rapidly sequestered into a dense fraction distinct from fractions containing plasma membrane, lysosomes and mitochondria. Electron microscopy (results not shown) indicates that the dense fraction contains nuclear residues, multivesicular structures, dense bodies and fibrous structures. Both the dense fraction and a hexosaminidase-enriched fraction contain trichloroacetic acid-insoluble radioactivity, including intact 125I-labelled viral proteins. The viral proteins are progressively transferred from the dense fraction to the hexosaminidase-enriched fraction; the transfer is retarded by 50 micrograms of leupeptin/ml. Trichloroacetic acid-soluble radiolabel is progressively released into the culture medium as the proteins are degraded. Within 5 h after transplantation of viral HN and F proteins into recipient cells, a proportion (approx. 45%) of the 125I-labelled glycoproteins cannot be extracted by sequentially treating cells with digitonin (1 mg/ml), Triton X-100 (1%, w/v) and 0.3 M-KI. HN and F proteins in the non-extractable residue are tightly associated with nuclear-intermediate-filament (vimentin) material, as shown by Western blots and electron microscopy. The viral proteins are progressively transferred out of the nuclear-intermediate-filament residue; the transfer is slowed when cells are cultured with leupeptin. The data are consistent with the notion that transplanted viral HN and F proteins are sequestered to a perinuclear site in tight association with intermediate filaments before transfer into the autophagolysosomal system for degradation.