[L-amino acid oxidases: properties and molecular mechanisms of action].

During previous decade L-amino acid oxidases (LAAO) attracted the steady interest of researchers due to their poly functional effects on different biological systems. The review summarizes information concerning the sources, structure, phisico-chemical and catalytical properties of LAAO which exhibit antibacterial, antifungal, antiprotozoal, antiviral effects as well as the ambiguous action on platelet aggregation. Special attention is devoted to the elucidation of molecular mechanisms of LAAO action. It is proposed that the unique properties of LAAO) are based on their catalytic reaction, which causes the decrease of L-amino acid levels, including the essential amino acids and formation of hydrogen peroxide. The action of liberated H2O2 on cells involves the synthesis of oxygen reactive species and the development of necrotic and apoptotic pathways of cell death. The presence of carbohydrate moieties in LAAO molecules promotes their attachment to cell's surface and creation of high H2O2 local concentrations. The wide range of LAAO biological effects is undoubtedly connected with their important functional roles in the organism. In particular, it was shown that in the mice brain the LAAO-catalyzed reaction is the single pathway of L-lysine degradation, while in the mice milk LAAO carry out the antibacterial effect and in human leucocytes LAAO take part in fulfilling their defending role. Protector action may be also attributed to the oxidases from the other numerous sources: microscopic fungi, snake venoms and sea inhabitants.

Atomic force microscopy-based antibody recognition imaging of proteins in the pathological deposits in pseudoexfoliation syndrome.

The phenomenon of protein aggregation is of considerable interest to various disciplines, including the field of medicine. A range of disease pathologies are associated with this phenomenon. One of the ocular diseases hallmarked by protein aggregation is the Pseudoexfoliation (PEX) Syndrome. This condition is characterized by the deposition of insoluble proteinaceous material on the anterior human lens capsule. Genomic and proteomic analyses have revealed an association of specific genetic markers and various proteins, respectively, with PEX syndrome. However, the ultrastructure of the protein aggregates is poorly characterized. This study seeks to build capacity to determine the molecular nature of PEX aggregates on human lens capsules in their native state by AFM-based antibody recognition imaging. Lysyl oxidase-Like 1 (LOXL1), a protein identified as a component of PEX aggregates, is detected by an antibody-modified AFM probe. Topographical AFM images and antibody recognition images are obtained using three AFM-based techniques: TREC, phase and force-volume imaging. LOXL1 is found to be present on the lens capsule surface, and is localized around fibrous protein aggregates. Our evaluation shows that TREC imaging is best suited for human tissue imaging and holds significant potential for imaging of human disease tissues in their native state.

IL4I1: an inhibitor of the CD8⁺ antitumor T-cell response in vivo.

The L-phenylalanine oxidase IL4I1 inhibits T-cell proliferation in vitro through H(2) O(2) production, and is highly expressed in tumor-associated macrophages. IL4I1 is also detected by immunohistochemistry in neoplastic cells from several B-cell lymphomas and some non-lymphoid tumors. To evaluate IL4I1's effect on tumor growth, we developed a mouse melanoma model constitutively coexpressing IL4I1 and the GP33 epitope. After GP33 vaccination, tumors developed more frequently in mice injected with IL4I1-expressing cells in comparison with mice receiving control cells. Tumor escape was preceded by a rapid diminution of IFN-γ-producing cytotoxic antitumor CD8(+) T cells. Moreover, tumor incidence was already increased when only 20% of the injected cells expressed IL4I1. The minimal IL4I1 activities leading to tumor escape were close to those detected in human melanoma and mesothelioma. Thus, we demonstrate the immunosuppressive functions of IL4I1 in vivo and suggest that IL4I1 facilitates human tumor growth by inhibiting the CD8(+) antitumor T-cell response.

Immunological responses induced by asd and wzy/asd mutant strains of Salmonella enterica serovar Typhimurium in BALB/c mice.

Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate beta-semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer's patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 10(6) higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1x10(10)CFU) or i.p. (1x10(7) CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD(50) (5x10(6) CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.

Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor.

In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.

Human IL4I1 is a secreted L-phenylalanine oxidase expressed by mature dendritic cells that inhibits T-lymphocyte proliferation.

Interleukin-4-induced gene 1 (IL4I1) was first described as a B-cell IL4-inducible gene and is highly expressed in primary mediastinal B-cell lymphomas. We established stable HEK293 clones expressing human and mouse IL4I1 to examine their biochemical properties and function. Both proteins were secreted into the culture medium, and we observed the secretion of endogenous human IL4I1 (hIL4I1) protein in a mediastinal lymphoma B-cell line, MedB-1. We showed that IL4I1 has l-amino acid oxidase activity, optimal at physiological pH and primarily directed toward phenylalanine. Immunohistochemical analysis of secondary lymphoid organs showed staining of germinal center macrophages and inflammatory myeloid cells. In vitro, functional enzyme was highest in mature dendritic cells (DCs), suggesting a role in antigen-presenting cell/T-lymphocyte cross-talk. Indeed, hIL4I1 inhibited the proliferation of CD3-stimulated T lymphocytes with a similar effect on CD4(+) and CD8(+) T cells. In contrast, memory T cells were more strongly affected by hIL4I1 and its catabolite H(2)O(2) than naive T cells. hIL4I1 inhibitory effect was dependent on enzymatic activity and H(2)O(2) production and associated with a transient down-regulation of TCRzeta expression. Altogether these data suggest IL4I1 as a new immunomodulatory enzyme produced by DCs.

Antibodies to a fragment of the Bothrops moojenil-amino acid oxidase cross-react with snake venom components unrelated to the parent protein.

It is widely accepted that immunological cross-reactivity of snake venoms is mediated by antibodies that recognize venom components bearing either amino acid sequence homology or similar biological functions. However, here we demonstrate that polyspecific Bothrops antivenom is a source of cross-reactive antibodies that interact with venom proteins of distinctive primary structures and biological functions. The homoserine lactone derivative of the undecapeptide IQRWSLDKYAM (Ile1-Hse11), excised from the l-amino acid oxidase (LAAO) of the Bothrops moojeni venom, was the ligand of an affinity resin used to isolate specific anti-Ile1-Hse11 antibodies which were instrumental in revealing immunological cross-reactivity among unrelated venom proteins. We examined the extent of the cross-reactivity of these antibodies by probing electroblots of venoms from representative snakes of genera Bothrops, Lachesis, Crotalus and Micrurus, and by unambiguous structural characterization of the affinity-purified proteins of B. moojeni venom recovered from an agarose-anti-Ile1-Hse11 column. Our results indicate that all venoms tested had at least three reactive components toward anti-Ile1-Hse11 antibodies, among which we identified two serine proteases, one phospholipase A2 homologue, and LAAO. We hypothesize that the cross-reactivity of the anti-Ile1-Hse11 antibodies to unrelated venom proteins derives from their mechanism of antigen recognition, whereby complementarity is achieved through reciprocal conformational adaptation of the reacting molecules. Also, we believe these findings have implications both in the development of improved antivenoms and the preparation of immunochemical reagents for diagnostic and scientific investigation purposes in the field of snake venoms.

[Biological properties of L-lysine alpha-oxidase in native and conjugated form]. / Biologicheskie svoistva L-lizin-alpha-oksidazy i ee kon''iugatov.

The significant difference between biological properties of L-lysine-alpha-oxidase from Trichoderma harzianum Rifai (LO) and L-asparaginase from E. coli has been observed in vitro and in vivo. High antitumor activity was shown against 8 types of murine and rat transplanted tumors with a wide range of LO therapeutic doses: 35-350 U/mg. The LO conjugates with monoclonal antibodies CD5 specific to the surface of cell line Yurkat were obtained without significant loss of either enzymatic and cytotoxic activity or immunological specificity. The further perspective investigation for the clinical application of the native or conjugated enzymes is discussed.

HLA-A2-restricted CD8+-cytotoxic-T-cell responses to novel epitopes in Mycobacterium tuberculosis superoxide dismutase, alanine dehydrogenase, and glutamine synthetase.

Major histocompatibility complex class I-restricted CD8(+) cytotoxic T lymphocytes (CTL) are implicated in protective Th1 immunity to Mycobacterium tuberculosis infection. We report the identification of three novel HLA-A*0201-restricted CTL epitopes within mycobacterial superoxide dismutase (SodA), L-alanine dehydrogenase (AlaDH), and L-glutamine synthetase (GlnS) proteins.

Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.

Elastic fibers are components of the extracellular matrix and confer resilience. Once laid down, they are thought to remain stable, except in the uterine tract where cycles of active remodeling occur. Loss of elastic fibers underlies connective tissue aging and important diseases including emphysema. Failure to maintain elastic fibers is explained by a theory of antielastase-elastase imbalance, but little is known about the role of renewal. Here we show that mice lacking the protein lysyl oxidase-like 1 (LOXL1) do not deposit normal elastic fibers in the uterine tract post partum and develop pelvic organ prolapse, enlarged airspaces of the lung, loose skin and vascular abnormalities with concomitant tropoelastin accumulation. Distinct from the prototypic lysyl oxidase (LOX), LOXL1 localizes specifically to sites of elastogenesis and interacts with fibulin-5. Thus elastin polymer deposition is a crucial aspect of elastic fiber maintenance and is dependent on LOXL1, which serves both as a cross-linking enzyme and an element of the scaffold to ensure spatially defined deposition of elastin.

Autoepitope mapping and reactivity of autoantibodies to the dihydrolipoamide dehydrogenase-binding protein (E3BP) and the glycine cleavage proteins in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial antibodies (AMA) directed primarily against the E2 subunits of the pyruvate dehydrogenase complex, the branched chain 2-oxo-acid dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, as well as the dihydrolipoamide dehydrogenase-binding protein (E3BP) of pyruvate dehydrogenase complex. The autoantibody response to each E2 subunit is directed to the lipoic acid binding domain. However, hitherto, the epitope recognized by autoantibodies to E3BP has not been mapped. In this study, we have taken advantage of the recently available full-length human E3BP complementary DNA (cDNA) to map this epitope. In addition, another lipoic binding protein, the H-protein of the glycine cleavage complex, was also studied as a potential autoantigen recognized by AMA. Firstly, the sequence corresponding to the lipoic domain of E3BP (E3BP-LD) was amplified by polymerase chain reaction and recombinant protein and then purified. Immunoreactivity of 45 PBC sera (and 52 control sera) against the purified recombinant E3BP-LD was analyzed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Secondly, reactivity of PBC sera was similarly analyzed by immunoblotting against H-protein. It is interesting that preabsorption of patient sera with the lipoic acid binding domain of E3BP completely removed all reactivity with the entire protein by immunoblotting analysis, suggesting that autoantibodies to E3BP are directed solely to its lipoic acid binding domain. Fifty-three percent of PBC sera reacted with E3BP-LD, with the majority of the response being of the immunoglobulin G (IgG) isotype (95%). Surprisingly, there was little IgM response to the E3BP-LD suggesting that the immune response was secondary because of determinant spreading. In contrast, H-protein does not appear to possess (or expose) autoepitopes recognized by PBC sera. This observation is consistent with structural data on this moiety.

L-amino acid oxidase (LOX) modulation of melphalan activity against intracranial glioma.

These studies evaluated the efficacy of sequential pretreatment with L-amino acid oxidase (LOX) and LOX antiserum in the modulation of melphalan activity against intracranial glioma in athymic nude mice. LOX produced statistically significant (P < 0.01) depletion of the large neutral amino acids isoleucine, leucine, methionine, phenylalanine, tyrosine, and valine in murine plasma at doses of 100 and 200 micrograms administered intravenously. Polyclonal anti-LOX antibody was successfully produced in mice, rabbits, and goats subsequent to immunization with LOX. Staphylococcal protein A-purified rabbit anti-LOX serum inhibited approximately 50% of LOX activity in vitro relative to control samples. This antiserum was used in vivo to inactivate LOX after it had depleted the large neutral amino acids, thereby preventing LOX-mediated catabolism of melphalan. Inoculation of three mice with rabbit anti-LOX serum after the treatment with LOX (100 micrograms) reduced LOX activity by 100%, 89%, and 100% at 6 h compared with reductions of 80%, 59%, and 52% over the same period in animals receiving LOX alone. In three separate studies using groups of eight to ten mice bearing intracranial human glioma xenografts, pretreatment with LOX followed by anti-LOX serum increased the antitumor activity of melphalan as compared with treatments with melphalan plus LOX, melphalan plus anti-LOX serum, or melphalan alone.

Nitric oxide synthase-immunoreactive vagal afferent fibers in rat superior cervical ganglia.

Chronic (5-14 days) preganglionic denervation of the rat superior cervical ganglia by sectioning the cervical sympathetic trunk resulted in a time-related partial or complete loss of nitric oxide synthase (isoform I)-immunoreactive fibers and terminals surrounding many sympathetic ganglionic neurons. Unexpectedly, denervation unmasked many varicose nitric oxide synthase-immunoreactive fibers, some of which could be traced the entire length of the superior cervical ganglia. Injection of the retrograde tracer Fluorogold into the superior cervical ganglia labeled a population of nodose ganglion cells and of dorsal root ganglion cells from C8 to T3 segments. When the same sections were processed for nitric oxide synthase-immunoreactivity, 40% of the Fluorogold-containing nodose ganglion cells also expressed nitric oxide synthase-immunoreactivity, whereas colocalization was observed in only a few dorsal root ganglion cells. Similarly, injection of Fluorogold into denervated superior cervical ganglia labeled a population of nodose ganglion cells. Sectioning of all nerve trunks associated with the superior cervical ganglion prior to injection of Fluorogold, except the cervical sympathetic trunk, resulted in no detectable labeling of Fluorogold in the ipsilateral nodose ganglion cells. These results indicate that a population of rat nodose ganglion cells contain nitric oxide synthase and that some of these neurons project their axons through the superior cervical ganglion and terminate in the peripheral target tissues. The possibility that nitric oxide synthase-immunoreactive vagal afferent fibers may participate in nociception is considered.

Co-localization of neurotransmitter immunoreactivities in putative nitric oxide synthesizing neurones of the cat brain stem.

The distribution of nitric oxide producing neurones in the medulla oblongata of the cat was investigated using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, and nitric oxide synthase (NOS) immunohistochemistry. The pattern of staining obtained with both methods was found to be similar. Strongly diaphorase and NOS reactive neurones were present in the paramedian and lateral tegmental fields, including the regions occupied by the A1/C1 catecholamine cell groups, the nucleus ambiguus and lateral reticular nucleus, and in a number of sensory nuclei including the nucleus of the tractus solitarius and the dorsal column nuclei. The extent of co-localization of NADPH-diaphorase with a number of neuropeptides and neurotransmitters was investigated by combining NADPH-diaphorase histochemistry with immunocytochemistry for neuropeptide Y, somatostatin, glutamate, cholecystokinin and tyrosine hydroxylase. NADPH-diaphorase reaction product was observed in neurones immunoreactive for glutamate and somatostatin. These double-labelled cells were found in the paramedian region, lateral reticular field, the nucleus prepositus hypoglossi and in the rostral nucleus of the tractus solitarius. In the rostral ventrolateral medulla NADPH-diaphorase/somatostatin immunoreactive cells were found in the paragigantocellular nucleus. NADPH-diaphorase/glutamate immunoreactive cells overlapped the nucleus ambiguus, the lateral reticular nucleus and the A1/C1 catecholaminergic cell groups. In addition, a few NADPH-diaphorase/glutamate immunoreactive cells were found in the paraolivary area and gigantocellular tegmental field, in the external cuneate and infratrigeminal nuclei. The functional implications of the co-localization of nitric oxide with these neurotransmitters in areas of the medulla concerned with cardiovascular regulation is discussed.

Expression of ferredoxin-dependent glutamate synthase in dark-grown pine seedlings.

Pine seedlings are able to accumulate chlorophylls and develop green plastids in a light-independent manner. In this work, we have characterized ferredoxin-dependent glutamate synthase (EC 1.4.7.1; Fd-GOGAT), a key enzyme in nitrogen interconversion during this process. Fd-GOGAT has been purified about 170-fold from cotyledons of maritime pine (Pinus pinaster). As occurs in angiosperms, the native enzyme is a single polypeptide with an apparent molecular mass of 163-168 kDa that is confined to the chloroplast stroma. Polyclonal antibodies generated against the purified enzyme were used to immunoscreen a lambda gt11 expression library from Scots pine (Pinus sylvestris) seedlings and partial cDNA clones were isolated and characterized. The clone with the longest cDNA insert (pGOP44) contained the codification for the C-terminal (550 amino acids) of the pine Fd-GOGAT polypeptide. Immunological cross-reactivity and comparative amino sequence analysis revealed that Fd-GOGAT is a well conserved protein in higher plants. Western blot analyses showed that protein was expressed in chloroplast-containing pine tissues and this expression pattern was not affected by exogenously supplied nitrogen. Fd-GOGAT mRNA, polypeptide and enzyme activity accumulated in substantial amounts in dark-grown pine seedlings. The presence of a functional Fd-GOGAT may be important to provide the required glutamate for the biosynthesis of nitrogen compounds during chloroplast biogenesis in the dark.

Localisation of neuronal nitric oxide synthase-immunoreactivity in rat and rabbit retinas.

The distribution of neuronal nitric oxide synthase (NOS) immunoreactivity was examined in rat and rabbit retinas and was compared with the distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactivity and vasoactive intestinal peptide (VIP) immunoreactivity. An antibody raised against a C-terminal fragment of a cloned rat cerebellar NOS was used to localise NOS immunoreactivity. NOS immunoreactive cells were not detected in rat retinas at postnatal day 1 or 4, but were seen from postnatal day 7 onwards. NOS immunolabelling was seen in a small population of cells in the proximal inner nuclear layer. Most of the labelled cells had the position of amacrine cells and were seen to send processes into the inner plexiform layer. A few labelled cells were at times also seen in the ganglion cell layer, which are likely to correspond to displaced amacrine cells. The same NOS-labelling pattern was seen in rat and rabbit retinas. NADPH-diaphorase staining was observed in both species, in photoreceptor inner segments, in cells with the position of horizontal cells, in a subset of amacrine and displaced amacrine cells, in large cell bodies in the ganglion cell layer, in both plexiform layers, and in endothelium. Colocalisation of NOS immunoreactivity and NADPH-diaphorase staining was only observed among amacrine cells. However, not all NADPH-diaphorase-reactive amacrine cells were found to be NOS immunoreactive. VIP immunoreactivity was also localised in rat retinas in a subpopulation of amacrine cells, but no colocalisation of NOS and VIP immunoreactivity was observed. Our observations indicate that only amacrine cells contain the NOS form recognisable by the antibody used, and suggest that different isoforms of neuronal NOS may be present in retinal cells. Further, the onset of NOS expression in rat amacrine cells appears to occur independently of neuronal activity.

Immunohistochemical expression of inducible nitric oxide synthase (iNOS) in reversible endotoxic shock studied by a novel monoclonal antibody against rat iNOS.

An antirat monoclonal antibody (mAb) against inducible nitric oxide synthase (iNOS), ANOS11, was used for immunohistochemistry to examine the expression of iNOS in various organs and tissues of adult rats in experimental endotoxic shock induced by lipopolysaccharide (LPS) injection. The phenotype of iNOS-expressed cells was also examined immunohistochemically using various mAbs. In control rats, very few cells were positive for ANOS11 except in the thymus. After intravenous injection of LPS, the number of iNOS-positive cells increased rapidly in almost all organs, except the thymus and brain, peaked 6 h after the injection, and decreased slowly. Of the numerous inflammatory cells that infiltrated the lungs, liver, and spleen after LPS injection, many were positive for ANOS11. Besides inflammatory cells, hepatocytes and endothelial cells of the aorta were also positive for ANOS11 but only around 6 h after injection. The cellular composition of iNOS-positive infiltrated cells changed along with the progression of endotoxic shock. At 4 to 6 h after injection, most iNOS-positive cells were considered polymorphonuclear leukocytes judging by their positive reactivity to OX42 and their nuclear morphology. The population of iNOS-positive macrophages positive for ED1 or ED2 increased with time. After 24 h, many iNOS-positive macrophages were found around the focal necrosis in the liver and spleen. These results indicate that the expression of iNOS in neutrophils, endothelial cells, and hepatocytes precedes that of macrophages in experimental endotoxic shock. The expression of iNOS in various cells and organs is closely associated with the progress and pathological changes of endotoxic shock.

The extravascular contractile system in the human placenta. Morphological and immunocytochemical investigations.

In the human placenta, besides the fetal blood vessel system a second extravascular contractile system exists. It is localized in the chorionic plate and runs in a longitudinal direction and adjacent to fetal blood vessels into the stem villi, where it forms perivascular contractile sheaths. Characteristically, cells of the extravascular contractile system are extremely long and spindle-shaped and give rise to fine cell processes, by which they obviously contact each other or insert into the basement membrane of the trophoblast. They show immunoreactivity with desmin, vimentin, alpha-actin, myosin, nitric oxide synthase type I (brain form) and dipeptidyl peptidase IV. The ultrastructure suggests that cells of the extravascular contractile system are related to smooth muscle cells, including subpopulations with myofibroblastic features. In stem villi a few cells are nitric oxide synthase type I immunoreactive. These cells are thought to be specialized smooth-muscle-like cells of the extravascular contractile system or cells of the extravascular contractile system related to paraneurons that generate nitric oxide, which, in turn, may modulate the tone of perivascular contractile sheaths. The high dipeptidyl peptidase IV activity suggests that modulation of the extravascular contractile system may also occur by substance P.

Immunohistochemical demonstration of a paracrine role of nitric oxide in bronchial function.

We addressed the controversial role of nitric oxide (NO) in bronchial function by an immunohistochemical study of the localization of NO synthase (NOS) and its effector protein, soluble guanylate cyclase, in rat bronchus. For this study, a monoclonal antibody to the bovine constitutive neuronal NOS was developed and characterized. In Western blot analysis, this monoclonal antibody (anti-NOS antibody) reacted with bovine cerebellum NOS (150 kDa) as well as with structurally different NOSs from cultured bovine aortic endothelial cells (130 kDa) and cultured RAW 264.7 macrophages (130 kDa). The reactivity of anti-NOS antibody was confirmed by immunohistochemical staining of rat cerebellum, arterial endothelial cells, and cultured stimulated macrophages. When the distribution of NOS in rat airway was characterized, the anti-NOS antibody showed immunoreactivity within respiratory epithelium but not in the bronchial smooth muscle. The NADPH-diaphorase staining correlated with the immunostaining. In contrast, a monoclonal antibody to the rat lung-soluble guanylate cyclase immunostained respiratory smooth muscle but not epithelium. This study suggests a paracrine role for NO in bronchial function analogous to the function of the NOS-soluble guanylate cyclase pathway in blood vessels.

Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation.

In this study, we wished to clarify the distribution and co-localization of nitric oxide synthase and NA-DPH-diaphorase (NADPH-d) in nerve cells, nerve fibres and parenchymal cells in exocrine and endocrine pancreas, and to assess the influence of fixation on the staining pattern obtained. For this purpose, we applied nitric oxide synthase immunocytochemistry and NADPH-d histochemistry to rat and human pancreas under different fixation conditions. Antibodies to neuronal and endothelial nitric oxide synthase were similarly applied. We found complete co-localization of neuronal nitric oxide synthase and NADPH-d in ganglion cells, and in nerve fibres around acini, excretory ducts, blood vessels and in islets of Langerhans of rat and human pancreas. Immunoreactivity for endothelial nitric oxide synthase was co-localized with NADPH-d in endothelial cells. However, in NADPH-d reactive islet and ductal epithelial cells we could detect neither brain nor endothelial nitric oxide synthase immunoreactivity with any fixation protocol applied. There were marked differences in NADPH-d staining of both neurons and parenchymal cells under different fixation conditions. These results indicate the existence of different types of NADPH-d, which are associated or not associated with nitric oxide synthase(s), and which are differently influenced by various fixation procedures in rat and human pancreas.