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V Riveros-Moreno

Publications and source records attributed to V Riveros-Moreno.

35 records · Page 2Linked to original sources
PubMed1994

Nitric oxide synthase activity in human gynecological cancer.

Nitric oxide is generated by the NO synthases, a family of isoenzymes expressed in a wide range of mammalian cells. In the vascular and nervous systems distinct isoforms generate NO to act as a signal transduction mechanism. The isoform induced by cytokines, on the other hand, provides a sustained release of NO which mediates some cytotoxic and cytostatic effects of the immune system. Solid tumors are a heterogeneous population of cell types, including tumor, vascular, and infiltrating immune cells. Studies in vitro show that NO synthase can be present in many of these cells. However, its presence in situ in solid human tumors has not been reported. In this study, we have investigated NO synthase activity and its cellular localization in malignant and nonmalignant human gynecological tissue. Nitric oxide synthase activity was observed in malignant tissue, was highest (> or = 250 pmol/min/g tissue) in poorly differentiated tumors, and was below detectable levels in normal gynecological tissue. Furthermore, investigations with a polyclonal NO synthase antibody revealed immunoreactivity only in malignant tissue. This was associated with NO synthase activity and localized to tumor cells. Thus NO synthase is present in human gynecological tumors, and its presence seems to correlate inversely with the differentiation of the tumor.

Amino Acid Oxidoreductases↗
PubMed1994

Inducible nitric oxide synthase is increased in murine lung epithelial cells by cytokine stimulation.

Nitric oxide (NO) is detectable in exhaled air. To elucidate whether airway epithelial cells could be a source of NO, we investigated the expression of inducible nitric oxide synthase (iNOS) by the murine lung epithelial cell line, LA-4, in response to cytokine stimulation and the ability of corticosteroids to modulate this effect. Stimulation with cytomix, a combination of interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma, elevated nitrite levels by 873% in the culture supernatants and enhanced the conversion of arginine to citrulline by 273% at 24 h. An increased number of cells stained for iNOS and an increase in iNOS mRNA was also observed. Dexamethasone decreased the cytokine-induced increase in nitrite levels, NOS activity, iNOS immunoreactivity, and mRNA but did not change the half life of iNOS mRNA. These results show that lung epithelial cells can release NO, a process which can be inhibited by dexamethasone.

Amino Acid Oxidoreductases↗
PubMed1994

Induction of nitric oxide synthase in rat immune complex glomerulonephritis.

Nitric oxide (NO) is a biological mediator which is synthesized from L-arginine by a family of nitric oxide synthases (NOS). Previously we have shown that NO is synthesized ex vivo by glomeruli obtained from animals with acute immune complex glomerulonephritis. We have now sought evidence for the in vivo induction of NOS in glomeruli by immunohistochemistry using specific antisera raised against a peptide sequence of inducible mouse macrophage NOS and by in situ hybridization. The expression of the enzyme was studied in kidneys of rats with acute unilateral immune complex glomerulonephritis, induced by cationized IgG, by immunohistochemistry. Inducible NOS (iNOS) was present in glomeruli in nephritic (left) kidneys at the time of maximum macrophage infiltration, both within intraglomerular mononuclear cells and cells emigrating into Bowman's space. iNOS expressing cells were also present in interstitial infiltrates. There was no expression in normal rat kidneys or in glomeruli in the non-nephritic (right) kidneys of experimental rats. In situ hybridization confirmed the immunohistochemical localization. These results provide the first direct evidence for the presence and localization of inducible NOS in glomeruli and support a significant role for NO in the pathogenesis of immune complex glomerulonephritis.

Amino Acid Oxidoreductases↗
PubMed1993

Cloning and expression of a rat neuronal nitric oxide synthase coding sequence in a baculovirus/insect cell system.

A DNA sequence encoding rat neuronal NO synthase (nNOS) was isolated and cloned into the baculovirus expression vector pVL1393 to generate pVLRBNOS. Transfection of Spodoptera frugiperda Sf-21 cells with the construct pVLRBNOS resulted in the synthesis of high levels of neuronal NO synthase. Analysis of the expression pattern revealed soluble, enzymatically active NO synthase in the cytoplasm of cell extracts. Active enzyme could also be purified from culture supernatants using 2'-5' ADP sepharose affinity chromatography. This enzyme was recognised by antibodies to the native nNOS and showed a similar degree of inhibition by arginine analogs as the native nNOS. The majority of the NOS synthesised had accumulated as insoluble "inclusion-body" material. The purification of recombinant nNOS from insect cells should facilitate characterisation of neuronal NO synthase.

Amino Acid Oxidoreductases↗
PubMed1993

Localisation of nitric oxide synthase within non-adrenergic, non-cholinergic nerves in the mouse anococcygeus.

Immunocytochemical staining of whole mount preparations of the mouse anococcygeus muscle, using antibodies to rat brain nitric oxide synthase (NOS), revealed a dense network of NOS-immunoreactive nerve fibres running through the tissue. These fibres were resistant to the sympathetic neurotoxin 6-hydroxydopamine and are therefore likely to be the non-adrenergic nerves which mediate relaxation of this smooth muscle. Further, NOS-immunoreactive fibres were absent following denervation by cold-storage (4 degrees C; 72 h), which has been shown to abolish non-adrenergic, non-cholinergic (NANC) relaxations. The results provide strong support for the hypothesis that the L-arginine:NO pathway is responsible for the generation of the NANC transmitter in the anococcygeus.

Amino Acid Oxidoreductases↗
PubMed1993

Nitric oxide synthase. Structural studies using anti-peptide antibodies.

The amino acid sequence for the constitutive rat brain nitric oxide (NO) synthase was analysed by a set of computer programs that estimate and display physicochemical properties such as hydrophilicity, flexibility, accessibility, hydrophilic periodicity and conformation [Comerford, S. A., McCance, D. J., Dougan, G. & Tite, J. P. (1991) J. Virol. 65, 4681-4690]. Overall, they allow prediction of whether each peptide region will be an alpha-helix, a beta-strand or a less regular coil and also whether the region will be buried in the protein core or exposed to water at the surface of the protein molecule. Ten peptide regions were chosen; the majority were predicted to be exposed areas of the molecule and therefore likely to be immunogenic. The peptides were chemically synthesised, coupled to keyhole limpet haemocyanin carrier protein and injected into rabbits to raise antibodies. These antibodies have been used by us and others to locate the NO synthase in different tissues and species. Here we present the characterisation of the antibodies in relation to the possible conformation of the enzyme and an immunological comparison between two isoforms of NO synthase: constitutive (rat brain) and inducible (macrophage). Peptide regions predicted to be exposed, flexible or substantially in core, have produced antibodies that were able to recognise the native protein. Peptides of mixed characteristics possibly involved in the binding site tended to produce antibodies with low recognition for the tertiary structure of the native, purified NO synthase, although these peptides were all highly immunogenic. We postulate that either the peptides when conjugated to the carrier protein attain a different conformation to that in the native NO synthase, or alternatively the accessibility of the antibodies to substrate binding sites is highly restricted by steric hindrance. This latter seems to be more likely since a mixture of antibodies against this area of the protein molecule was able to achieve a similar neutralisation of the enzyme activity as the antibodies against the whole enzyme (approximately 50%). Most of the selected anti-peptide antibodies were not able to cross-react with the inducible macrophage enzyme; only two that have 60% sequence identity showed a weak reaction in Western blot. The polyclonal antibody against the complete brain enzyme showed cross-reaction in a Western blot with inducible enzyme. The macrophage enzyme was able to compete weakly with the binding of the brain enzyme to its own antibody, but 10 times more inducible protein was required.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Oxidoreductases↗
PubMed1993

Induction of nitric oxide synthase in the neo-vasculature of experimental tumours in mice.

Maintenance of blood flow is an important factor in sustaining tumour growth. Functional studies have previously demonstrated a reduction in tumour blood flow with selective inhibitors of nitric oxide (NO) synthesis, L-NAME (NG-nitro-L-arginine-methylester) and L-NMMA (NG-monomethyl-L-arginine), when administered locally to tumours derived from murine colon 26 adenocarcinoma and B16 melanoma cells. The type of NO synthase which might be responsible for this locally-derived NO and the site of synthesis was not described. Here we have investigated the distribution of immunoreactivity and the biochemical characteristics of the enzymes synthesizing NO in the same murine model. Adenocarcinoma (colon 26) or melanoma (B16) cells were introduced into a sponge matrix implanted subcutaneously in mice. After 7, 12, and 14 days, the implants were removed and frozen sections were immunostained with rabbit antisera to constitutive and inducible isoforms of NO synthase. Immunoreactivity with antisera to inducible NO synthase was detected in the vasculature of neoplastic implants, with and without the sponge, at 12 and 14 days. The enzyme was not evident in 7-day-old tumours, in non-neoplastic implants, in areas of tissue outside the tumour, or in adenocarcinoma or melanoma cells. Enzyme activity was measurable in homogenates of neoplastic implants removed at day 7 and was found to be Ca2+/calmodulin-independent. Immunoreactivity with antisera to inducible NO synthase was seen principally in the endothelium of newly-formed capillaries, identified by immunostaining for von Willebrand factor in serial sections. Immunoreactivity with antiserum to constitutive NO synthase was not evident in either neoplastic or non-neoplastic implants.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1993

Immunohistochemistry of nitric oxide synthase demonstrates immunoreactive neurons in spinal cord and dorsal root ganglia of man and rat.

The presence of nitric oxide synthase in spinal cord and dorsal root ganglia was investigated by immunohistochemistry using antibodies against the constitutive neuronal form of nitric oxide synthase (NOS). NOS immunoreactivity was present in both man and rat with similar distribution, being present in primary sensory neurons of dorsal root ganglia and their afferent terminals in dorsal horn of spinal cord. NOS immunoreactive interneurons were found in the superficial layer of the dorsal horn, around the central canal and in the intermediolateral cell column. NOS immunoreactivity was also present in numerous motoneurons in the ventral horn. The widespread distribution of NOS in both sensory and motor nervous system is indicative of the involvement of nitric oxide in different neural functions.

Adult↗
PubMed1993

Patients with achalasia lack nitric oxide synthase in the gastro-oesophageal junction.

The abnormal function of the lower oesophageal sphincter in achalasia is likely to be due to impaired nonadrenergic, noncholinergic (NANC) inhibitory input. Since recent studies in animals suggest that nitric oxide (NO) is implicated physiologically in the inhibitory responses of the lower oesophageal sphincter, we have investigated whether the synthesis of NO is altered in the gastro-oesophageal junction of patients with achalasia. NO synthase activity was investigated in samples of tissue from the gastro-oesophageal junction obtained during surgery in eight patients with typical achalasia and six non-achalasic controls who underwent oesophagectomy for reasons other than sphincter dysfunction. The NO synthase activity was determined by the transformation of 14C-L-arginine into 14C-L-citrulline in tissue homogenates. In addition, immunohistochemical staining of the tissues was performed using a polyclonal antibody raised against a peptide sequence of rat brain NO synthase. Furthermore, the relaxant response to an exogenous NO donor (sodium nitroprusside, SNP) was measured in vitro in muscle strips obtained from two patients with achalasia and in two non-achalasic controls. NO synthase activity was detected in each of the samples obtained from six control patients (0.59 +/- 0.21 pmol mg-1 min-1; mean +/- SE). By contrast, none of the samples obtained from the eight patients with achalasia had any detectable NO synthase activity. Immunohistochemical studies confirmed the presence of NO synthase in the myenteric plexus of the gastro-oesophageal junction of control patients and its absence in achalasia. SNP relaxed muscle strips precontracted with bethanechol in both control samples and those from patients with achalasia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms.

Nitric oxide (NO) is generated from L-arginine by NO synthases. Localization of the brain enzyme has been carried out in the rat; however, despite data suggesting that NO is a major regulator of vascular and neural functions in man, there is no information about the localization of NO synthase in human tissues. Rabbit antisera to NO synthase purified from rat brain (antisera A and B) were raised, tested by Western blotting, affinity purification and enzyme immunoprecipitation assay, and used to investigate the distribution of the enzyme in a variety of human tissues by immunohistochemistry. Antisera to two synthetic peptides from cloned neural NO synthase were used to aid specificity testing. Anti-sera A and B reacted with a approximately 160-kDa protein in Western blots of human brain extracts, gave immunostaining of nerves, and precipitated enzyme activity from rat brain homogenates. Antiserum B to NO synthase also reacted with proteins of M(r) between 125 and 140 kDa in extracts of well-vascularised tissues, and immunostained vascular endothelium; the neural and vascular immunoreactivity persisted after affinity purification of antiserum B with the approximately 160 kDa protein. Endothelial staining with antiserum B was seen in respiratory tract, liver, skin and umbilicus; syncytial trophoblasts stained in the placenta. Neural staining with antiserum A and B was seen in the myenteric and submucous plexus, and in nerve fibres in smooth muscle of the gut and in many areas of the central nervous system, particularly cortex, hippocampus, hypothalamus, cerebellum, brain stem and spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1986

Structural diversity of the major surface antigen of Plasmodium falciparum merozoites.

The structures of the major merozoite surface antigen of Plasmodium falciparum and the gene encoding it were indistinguishable for the Wellcome strain and the Thai clone T9/94 but different for clones T9/96, T9/98, and T9/101. The central portion of the gene is subject to the greatest variation in structure. The protein from all five lines was found to be posttranslationally modified by covalent addition of both carbohydrate and fatty acid.

Animals↗
PubMed1979

Chemical properties of the principle in C. parvum that produces splenomegaly in mice.

Suspensions of Wellcome C. parvum strain 6134 produce splenomegaly in mice when injected i.p. in amounts as low as 20 microgram. This lymphoreticular stimulatory activity is extremely sensitive to cell breakage and is abolished by heating for 4 h at 100 degrees. Periodate oxidation of the bacteria destroys their capacity to produce splenomegaly and abrogates the agglutination of intact C. parvum by Con A. Mild HCl hydrolysis also abolished the splenomegaly but phenol:chloroform:ether and chloroform:methanol extractions did not. These results suggest that the relevant stimulatory principle in C. parvum is of carbohydrate nature, and most probably present on the surface of the bacterium.

Animals↗
PubMed1978

Antitumor activity of purified cell walls from Corynebacterium parvum.

Cell walls (CW), containing peptidoglycan and carbohydrate, were prepared from Corynebacterium parvum and tested for lymphoreticular stimulation and antitumor effects in CBA-T6T6 mice. CW did not induce splenomegaly. Peritoneal macrophages became cytostatic to Rl leukemia cells in vitro after ip injection of CW or of peptidoglycan but not of carbohydrate; however, on a dry-weight basis the activity was low (less than 10%) compared with that of C. parvum. Tumor outgrowth was significantly suppressed after sc injection of mixture of M4 fibrosarcoma cells and CW, but again the activity of CW was less than 10% of the of C. parvum. In contrast to injection of C. parvum, intratumor injection of CW failed to retard tumor growth in normal mice, although a suppressive effect was found in mice presensitized to C. parvum. Again, unlike C. parvum, CW did not act as an adjuvant for tumor-specific transplantation antigen, as judged by a lack of enhanced resistance to tumor challenge after injection of mixtures of CW and irradiated M4 cells. The distribution and persistence of 125l-labeled C. parvum and CW after sc or ip injection were similar. CW activity was not restored by attachment to oil droplets or emulsification in Freund's incomplete adjuvant.

Animals↗
PubMedDate not supplied

Induction of nitric oxide synthase in asthma.

Nitric oxide (NO) is a mediator of vasodilatation and bronchodilatation synthesised from L-arginine by the enzyme NO synthase, which is either constitutive or induced by lipopolysaccharides and/or cytokines. The presence and function of NO synthase in normal or diseased lung is not yet clear. Asthma is characterised by bronchial hyperresponsiveness, epithelial damage, inflammation, and increased cytokine production. To investigate the presence of NO synthase in asthma, we immunostained bronchial biopsies from non-steroid-treated people with asthma and non-asthmatic controls with specific polyvalent antisera to purified inducible NO synthase and to a selected peptide sequence of the same enzyme. Immunoreactivity was seen in the epithelium and some inflammatory cells in 22 of 23 biopsies from people with asthma, but in only 2 of 20 controls. To assess the relation of cytokines to NO synthase induction, bronchial epithelial cells in culture were stimulated with tumour necrosis factor (TNF alpha). Inducible enzyme immunoreactivity was found only in the treated cells. The existence of inducible NO synthase in human lungs suggests that increased production of NO, probably induced by cytokines, may be relevant to the pathology of asthma.

Adult↗
PubMedDate not supplied

Primary structure of the precursor to the three major surface antigens of Plasmodium falciparum merozoites.

Recently, a class of protein antigens of high relative molecular mass (Mt) which can induce protective immunity against blood-stage malaria has been identified. In Plasmodium falciparum the protein has a Mr of approximately 195,000 (P195). It is the precursor of three proteins of Mr 83,000 (83K), 42K and 19K which are the major surface antigens of merozoites; thus it may also be useful for immunization against P. falciparum. Three studies describing the isolation of single short complementary DNA clones for part of the P195 gene sequence have been reported. Here we describe the complete structure of the P195 gene determined from further DNA clones, its organization within genomic DNA and the location of the specific processing fragments within the primary amino-acid sequence.

Amino Acid Sequence↗