The immediate activator of the NADPH oxidase is arachidonate not phosphorylation

Superoxide generation is rapidly triggered following the addition of a stimulus to neutrophils. The signal-transduction pathway culminates in the activation of protein kinase C, whose phosphorylation of a protein component is considered to activate the oxidase. Arachidonate stimulated the oxidase in a concentration-dependent manner but, unlike phorbol-12-myristate-13-acetate (PMA), was not inhibited by staurosporine, a protein kinase inhibitor. Increase protein phosphorylation, apparent with PMA, was not observed when superoxide generation was triggered by arachidonate. Inhibitors of phospholipase A2 inhibit the PMA activation of the oxidase. Therefore, we propose that arachidonate and not phosphorylation is the immediate stimulus for superoxide generation.     

A gene encoding the plant-like alternative oxidase is present in Phytomonas but absent in Leishmania spp

OBJECTIVE: To develop a unique strain of Pasteurella haemolytica, selectable from nasopharyngeal respiratory tract secretions, that retains the ability to efficiently colonize the respiratory tract of calves. ANIMALS: 26 calves that each weighed approximately 200 kg. PROCEDURE: Rifampicin-resistant mutants of P haemolytica were developed and tested for in vitro growth rate and leukotoxin production. After instillation into the tonsils of calves, an isolate that was efficient at colonizing was selected and transformed, using electroporation, with a 4.2-kilobase (kb) plasmid encoding for streptomycin resistance. This isolate was instilled into the tonsils of 4 of 14 commingled calves to examine transmission of organisms. Nasal secretion and tonsil wash specimens were collected, cultured, and examined for P haemolytica. Serum antibody concentration was measured by means of indirect hemagglutination. RESULTS: Selected P haemolytica organisms colonized the tonsils and nasal passages for more than 2 weeks. Exposed calves and contact calves shed the organism, which was recovered from specimens of nasal secretions and tonsil washes. The 4.2-kb plasmid was lost during in vivo colonization. CONCLUSIONS AND CLINICAL RELEVANCE: The selected rifampicin-resistant P haemolytica organism colonized tonsils and nasal passages in a manner similar to the wild-type organisms. Selective media suppressed other bacterial flora to the extent that a single colony-forming unit was detectable from 200 microl of specimen, a 100-fold improvement in detection sensitivity. The selectable strain spread rapidly among commingled calves. A 4.2-kb plasmid marker was unstable when P haemolytica replicated in vivo.     

Independently derived IgG anti-DNA autoantibodies from two lupus-prone mouse strains express a VH gene that is not present in most murine strains

The origin and structure of two clonally unrelated IgG anti-DNA autoantibodies from lupus-prone MRL/Ipr mice were examined. One of these antibodies, H241, binds dsDNA and glomeruli and deposits in the kidneys of normal mice, whereas the other, H102, binds only ssDNA and does not deposit in kidneys. The VH genes of these two antibodies were almost identical to each other and were frequently expressed in anti-DNA antibodies derived from lupus-prone mice. Six other clonally unrelated anti-DNA antibodies from the literature or from data banks expressed nearly identical VH genes (< or = 4 nucleotide differences) and eight others had nearly identical protein sequences (< or = 3 amino acid differences). Analysis of the germ line with oligonucleotide probes from the CDR regions suggests that all 10 autoantibodies are derived from a single member of the J558 gene family, which is present only in mice with the j haplotype for the J558 gene family. The amount of somatic mutation in these VH genes appears to be low, suggesting that some V, N, and D gene combinations can generate high affinity IgG anti-DNA auto-antibodies with little or no somatic mutation. Unusual reading frames, D-D fusions, and inversions were common in the IgG antibodies and may have been co-selected. Although the N and D regions of one IgM and all five IgG autoantibodies contained Arg residues, the presence of Arg residues was not correlated with binding to dsDNA or with pathogenicity. These results suggest that differences in the Ag-binding properties and the pathogenicity of these antibodies are determined by the CDR3 region and the L chain.     

Quinolinate immunoreactivity in experimental rat brain tumors is present in macrophages but not in astrocytes

Experimental tumors of the central nervous system were investigated with antibodies to quinolinate to assess the cellular distribution of this endogenous neurotoxin. In advanced F98 and RG-2 glioblastomas and E367 neuroblastomas in the striatum of rats, variable numbers of quinolinate immunoreactive cells were observed in and around the tumors, with the majority being present within tumors, rather than brain parenchyma. The stained cells were morphologically variable, including round, complex, rod-shaped, and sparsely dendritic cells. Neuroblastoma and glioma cells were unstained, as were neurons, astrocytes, oligodendrocytes, ependymal cells, endothelial cells, and cells of the choroid plexus and leptomeninges. Glial fibrillary acidic protein immunoreactivity was strongly elevated in astrocytes surrounding the tumors. Dual labeling immunohistochemistry with antibodies to quinolinate and glial fibrillary acidic protein demonstrated that astrocytes and the cells containing quinolinate immunoreactivity were morphologically disparate and preferentially distributed external and internal to the tumors, respectively, and no dual labeled cells were observed. Lectin histochemistry with Griffonia simplicifolia B4 isolectin and Lycopersicon esculentum lectin demonstrated numerous phagocytic macrophages and reactive microglia in and around the tumors whose distribution was similar to that of quinolinate immunoreactive cells, albeit much more numerous. Dual labeling studies with antibodies to quinolinate and the lectins demonstrated partial codistribution of these markers, with most double-labeled cells having the morphology of phagocytes. The present findings suggest the possibility that quinolinate may serve a functional role in a select population of inflammatory cell infiltrates during the immune response to brain neoplasms.     

A functional soluble form of the murine mannose receptor is produced by macrophages in vitro and is present in mouse serum

A soluble form of the mannose receptor (sMR) has been found in conditioned medium of primary macrophages in vitro and in mouse serum. sMR was released as a single species, had a smaller size than the cell-associated form, and accumulated in macrophage-conditioned medium, in a cytokine-regulated manner, to levels comparable with those found for cell-associated mannose receptor. Pulse-chase experiments showed that sMR production in culture occurred by constitutive cleavage of pre-existing full-length protein. A binding assay was developed to determine the sugar specificity of sMR and its ability to interact with pathogens and particulate antigens (i.e. Candida albicans and zymosan). Protease inhibitor studies suggested that sMR was produced by cleavage of an intact mannose receptor by a matrix metalloprotease or ADAM metalloprotease. A role for sMR in the immune response is proposed based on its binding properties, regulation by cytokines, and the previous discovery of putative ligands for the cysteine-rich domain of the mannose receptor in lymph nodes and spleen.     

Immunoreactivity of glycoprotein IIb is present in metastasized but not in non-metastasized primary malignant melanoma

Glycoprotein IIb-IIIa (integrin alpha IIb beta3) is an adhesive receptor involved in platelet aggregation and adhesion to the extracellular matrix. Previous studies showed the presence of IIb-IIIa-like glycoproteins on cells of melanoma cell lines and on cells of lymph node metastases. This study evaluates the presence of glycoprotein IIb-IIIa subunits on cells of primary cutaneous malignant melanomas with (n = 4) and without (n = 9) metastases over a period of 6 years and on naevus cells (n = 4). Monoclonal antibodies directed against the subunits of the glycoprotein IIb-IIIa receptor were used on paraffin-embedded sections and evaluated by means of immunohistochemistry. The glycoprotein IIb subunit was exclusively present on cells of metastatic melanomas. It was not found on non-metastatic melanomas or benign melanocytes. These data favour the role of the integrin receptor glycoprotein IIb-IIIa in the metastatic behaviour of malignant melanomas.     

Platelet monoamine oxidase, plasma benzylamine oxidase and liver mixed function oxidase in cirrhotic patients

Because it has been suggested that an accumulation of false neurochemical transmitters plays a part in the cardiovascular and neurological complications of cirrhosis it appeared worthwhile to study the level of monoamine oxidase in the liver and platelets of cirrhotic patients. In fact a decrease of such activities might explain the increase of false neurotransmitters observed in cirrhotic patients. Other enzyme were also tested: the plasma benzylamine oxidase activity and the liver mixed function oxidases. 13 cases of severe cirrhosis (B, C according to Child classification) and 10 cases of less severe cirrhosis (A according to Child classification) were studied in comparison to patients affected by cholelithiasis. A defect in the level of monoamine oxidase activity of platelets and liver was observed in some cirrhotic patients together with a decrease of the level of the liver mixed function oxidases.     

Cytochrome c oxidase from eucaryotes but not from procaryotes is allosterically inhibited by ATP

The activity of reconstituted cytochrome c oxidase from bovine heart but not from Rhodobacter sphaeroides is allosterically inhibited by intraliposomal ATP, which binds to subunit IV. The activity of cytochrome c oxidase of wild-type yeast and of a subunit VIa-deleted yeast mutant, measured with Tween 20-solubilized mitochondria in the presence of an ATP-regenerating system, was also allosterically inhibited by ATP, indicating the general validity of this mechanism of "respiratory control" in eucaryotic cytochrome c oxidases (Arnold and Kadenbach, Eur. J. Biochem. (1997) 249, 350-354). Deletion of subunit VIa changes the biphysic into monophysic kinetics of the yeast enzyme in the presence of ADP. A tenfold higher amount of horse heart cytochrome c, as compared to yeast cytochrome c, was required to relieve the ATP inhibition of the yeast enzyme.     

NADPH oxidase is not essential for low density lipoprotein oxidation by human monocyte-derived macrophages

NADPH oxidase has been reported to be involved in low density lipoprotein (LDL) oxidation by monocytes. We have investigated the ability of monocyte-derived macrophages from four chronic granulomatous disease patients, which lack NADPH oxidase, to oxidise LDL. All the cells oxidised LDL to significantly increase its uptake by mouse macrophages. We conclude therefore that NADPH oxidase is not essential for LDL oxidation by macrophages.     

Hepatocellular carcinoma in an orthotopic mouse model metastasizes intrahepatically in cirrhotic but not in normal liver

Prognosis of hepatocellular carcinoma (HCC) still remains poor mainly because of intrahepatic metastasis. In the majority of cases, HCC is found in conjunction with liver cirrhosis. It is, therefore, of great importance to investigate the invasive and metastatic behavior of HCC in cirrhotic liver. To examine this, a liver cirrhosis model was produced by injecting thioacetamide i.p. into mice. Murine HCC cells were labeled with the fluorescent carbocyanine dye, DiI, and implanted directly under the capsule of cirrhotic and normal livers of syngeneic mice. DiI-labeled HCC cells in the liver were observed under fluorescent and confocal microscopy. Histological analysis of cirrhotic and normal livers revealed that implanted HCC cells migrated to and invaded the adjacent periportal regions, but not the adjacent centrolobular areas. This characteristic behavior of HCC was more evident in cirrhotic liver than in normal liver. Furthermore, intrahepatic metastasis to unimplanted hepatic lobes was observed in cirrhotic liver as early as 7 days after implantation, while it was not detected in normal liver even 4 weeks later. Thus, an orthotopic animal model for HCC with cirrhosis described here may be suitable for investigating the invasive and metastatic behavior of HCC. Importantly, labeling tumor cells with a fluorescent dye before orthotopic implantation may be a convenient and useful method to investigate the invasive and metastatic behavior of various types of cancer.     

Aspartate-407 in Rhodobacter sphaeroides cytochrome c oxidase is not required for proton pumping or manganese binding

Several pathways for proton transport in cytochrome c oxidase have been proposed on the basis of mutational analysis and X-ray structure: at least one for moving "pumped" protons from the interior to exterior of the membrane and a separate route for transporting "substrate" protons from the interior to the binuclear metal center to combine with oxygen to make H2O. According to the crystal structures of cytochrome c oxidase, Asp407 (Rhodobacter sphaeroides numbering) is at the interface of subunit I and subunit II of the oxidase, in a negative patch proposed to be the proton exit site in a pumping pathway, as well as a possible ligand to Mg [Iwata et al. (1995) Nature 376, 660-669]. Three mutants at the Asp407 position of R. sphaeroides cytochrome oxidase, Asp407Ala, Asp407Asn, and Asp407Cys, have been purified and characterized. All showed electron transfer activity, and pH dependence of activity, similar to that of the wild type enzyme and no major structural changes, as evidenced by visible, EPR, and resonance Raman spectroscopy. When reconstituted into artificial vesicles, the purified mutants pumped protons with normal efficiency and responded to the membrane pH and electrical gradients in a manner similar to that of wild type. Furthermore, the EPR spectra and Mn quantitation analysis of mutants grown in high Mn indicated no significant alteration in the Mn/Mg site. These results suggest that Asp407 does not play a critical role in proton translocation or in Mn/Mg binding.     

Prohibitin expression is decreased in the regenerating liver but not in chemically induced hepatic tumors in rats

Expression of prohibition, a growing-regulatory protein, was immunohistochemically investigated in normal rat tissues, regenerating livers, and chemically induced preneoplastic and neoplastic hepatic lesions. Specific cell types including hepatocytes, striated and smooth muscle cells, salivary gland duct epithelial cells, chondrocytes, immature spermatocytes and oocytes were found to be positive. In regenerating livers, prohibitin protein disappeared as early as 3 h after two-thirds hepatectomy and returned to near the original level by 24 h, while its mRNA level did not markedly vary. The timing of the disappearance was coincident with the expression of c-myc, suggesting a relation to quiescent hepatocytes entering the cell cycle. However, no pronounced decrease was evident in the most hyperplastic hepatic nodules and hepatocellular carcinomas investigated. Examination of 9 rat hepatocellular carcinoma cell lines, 6 hyperplastic hepatic nodules and 5 hepatocellular carcinomas revealed a single case of a base substitution in prohibition cDNA, identified as a synonymous sense change. The observed abundant expression of prohibitin in quiescent hepatocytes and its rapid loss under conditions of regeneration indicate a growth-regulatory function, but our results do not suggest any critical role in rat hepatocarcinogenesis.     

Dopamine-degrading activity of monoamine oxidase is not detected by histochemistry in neurons of the substantia nigra pars compacta of the rat

Monoamine oxidase (MAO) activity was examined in neurons of the substantia nigra pars compacta (SNC) of the rat using a histochemical method, and compared to MAO activity in neurons of the locus coeruleus (LC) and dorsal raphe nucleus (DR). Using dopamine as a substrate, dopamine-degrading MAO activity was not detected in any SNC neurons, although LC and DR neurons were intensely stained for this activity. We further examined MAO activity in these neurons using other substrates, including serotonin (an MAO type A preferential substrate), beta-phenylethylamine (an MAO type B preferential substrate), and tyramine (a substrate common to both MAO types A and B). As for dopamine, no SNC neurons were stained for MAO activity using any of these other substrates. In contrast, LC neurons were intensely stained when either serotonin or tyramine was used, and DR neurons were darkly stained when either beta-phenylethylamine or tyramine was used. The lack of evidence of MAO activity in the SNC is surprising given that there are densely packed tyrosine hydroxylase (TH)-immunoreactive neurons in the SNC (i.e., dopaminergic neurons). By comparison, in the LC and DR the distribution patterns of the MAO-stained neurons were similar to those of TH-immunolabeled neurons (i.e., noradrenergic neurons) and serotonin-immunoreactive neurons, respectively. Our results suggest that dopamine-degrading MAO activity and MAO types A and B activities in SNC dopamine neurons are very low compared to MAO activity in LC noradrenaline neurons and in DR serotonin neurons.