Immunochemical detection of different isoenzymes of cytochrome P-450 induced in chick hepatocyte cultures.

This study investigated whether the same cytochrome P-450 (P-450) isoenzymes were inducible in cultures of chick-embryo hepatocytes as in the liver of chicken embryos. We purified two isoenzymes of cytochrome P-450 from the livers of 17-day-old-chick embryos: one of molecular mass approx. 50 kDa induced in vivo by the phenobarbital-like inducer glutethimide, and the second of approx. 57 kDa induced by 3-methylcholanthrene. Rabbit antiserum against the 50 kDa protein inhibited benzphetamine demethylase activity in hepatic microsomes (microsomal fractions) from glutethimide-treated chick embryo. Antiserum to the 57 kDa protein inhibited ethoxyresorufin de-ethylase activity in hepatic microsomes from methylcholanthrene-treated chick embryo. Cultured chick hepatocytes were treated with chemicals known to induce isoenzymes of P-450 in rodent liver. The induced P-450s were quantified spectrophotometrically and characterized by immunoblotting and enzyme assays. From these studies, chemical inducers were classified into three groups: (i) chemicals that induced a P-450 isoenzyme of 50 kDa and increased benzphetamine demethylase activity: glutethimide, phenobarbital, metyrapone, mephenytoin, ethanol, isopentanol, isobutanol, lindane, lysodren; (ii) chemicals that induced a P-450 isoenzyme of 57 kDa and increased ethoxyresorufin de-ethylase activity: 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl; and (iii) the mono-alpha-substituted 2,3',4,4',5-pentabromobiphenyl, which induced both proteins and both activities. The immunochemical data showed that chick-embryo hepatocytes in culture retain the inducibility of glutethimide- and methylcholanthrene-induced isoenzymes of P-450 that are inducible in the liver of the chicken embryo.     

Ha-ras-1 alleles in Norwegian lung cancer patients

Summary We have examined DNA restriction fragment length polymorphisms (RFLP) of the Ha-ras-1 gene in DNA from 118 lung cancer patients and 123 unaffected controls. When DNA samples were digested with MspI/ HpaII restriction endonucleases. Southern blot analysis demonstrated 4 common, 4 intermediate and 7 different rare alleles in the combined population after hybridization to the pGDa1 probe. Six of the rare alleles were unique for the lung cancer group and 1 rare allele for the control group. The frequency of rare alleles in lung cancer patients (10/236) was significantly different (P<0.01) from the control group (1/246). The lung cancer group also had a significantly lower frequency of the common 2.57 kb fragment than the controls (P<0.02). The results thus indicate that Ha-ras genotyping may be of value in lung cancer risk assessment.     

Neuropeptides in free-living and parasitic flatworms (Platyhelminthes). An immunocytochemical study

The nervous systems of the turbellarians Microstomum lineare and Polycelis nigra and of the cestodes Diphyllobothrium dendriticum and Schistocephalus solidus were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the neuropeptides FMRF-amide, vasotocin, leu-enkephalin, met-enkephalin, neurotensin, somatostatin, and VIP, and to the bioamine serotonin. Anti-FMRF-amide positive perikarya and fibers occurred in all species, while the occurrence of the vertebrate brain-gut peptides and serotonin varied between the species. Anti-somatostatin and anti-VIP gave a negative result. Anti-FMRF-amide and anti-vasotocin positive immunoreactivity was found in the brain and gut of M. lineare, and in the CNS and the peripheral nerve net of the cestodes. We suggest that the brain-gut peptides of free-living flatworms act on the subtegumental region in the cestodes, which lack a gut but absorb their nutrients directly through the tegument.     

Differences between basophils and mast cells: failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil granule major basic protein in human mast cells

Human eosinophils contain several distinctive proteins including eosinophil granule MBP and the membrane-associated CLC protein (lysophospholipase). Human basophils also contain these proteins, indicating biochemical similarities between eosinophils and basophils. To determine whether MBP or CLC protein is present in connective tissue mast cells, we studied human lung and cutaneous mast cells by immunofluorescence by utilizing specific antibodies to CLC and MBP. Cytocentrifuge slides of enriched lung mast cells and mast cells in sections of formalin-fixed, paraffin-embedded cutaneous tissue from urticaria pigmentosa lesions were stained for CLC and MBP. Neither pulmonary nor cutaneous mast cells stained for CLC protein or MBP. In contrast, lung and cutaneous eosinophils in the same preparations showed bright staining for both proteins. The failure to find CLC protein and MBP in mast cells provides additional evidence of dissimilarity between mast cells and basophils, and an immunochemical means to distinguish between them.     

Immunocytochemical demonstration of neuropeptides and serotonin in the tapeworm Diphyllobothrium dendriticum

Summary The present immunocytochemical study concerns the distribution of four neuropeptides, FMRF-amide, vasotocin, leu-enkephalin and neurotensin, and of the bioamine serotonin in the plerocercoid larva of Diphyllobothrium dendriticum. Anti-FMRF-amide and vasotocin-reactivity occurs in perikarya and nerve fibres in the CNS and PNS of this worm. The peptide-containing fibres surround and seem to innervate the musculature and to terminate beneath the basal lamina of the tegument at the inner surface of the bothridia, suggesting a neurotransmitter function. Antileu-enkephalin reaction occurs in perikarya and fibres in the main nerve cords and in the PNS. Anti-neurotensin reactive fibres were observed in the neuropile of the nerve cords. Serotonin immunoreactivity was found in neurons in the ganglionic commissure of the brain and along the main nerve cords. This study is the first immunocytochemical identification of neuropeptides and serotonin in a parasitic flatworm and the information gained may be of importance for the development of new antihelminthics.     

Nasal Tumor in a Fallow Deer (Dama Dama L)

A nasal, so called ethmoidal, tumor from a fallow deer is described. It appears to be the first reported case of that species. The etiology is discussed.     

Altered prostanoid formation in human umbilical vasculature in response to variations in oxygen tension

Prostanoid formation in human umbilical vessels perfused was assessed at different oxygen tensions. At an atmosphere of 5% oxygen the production rate of prostacyclin (measured as 6-keto-PGF_1α) was higher, while those of thromboxane A₂ (measured as TXB₂), PGE₂ and PGF_2α were lower than with 20%, 50% and 95% oxygen. The stimulatory effect of angiotensin II on prostanoid production was found to be independent on the prevailing oxygen tension. Vascular formation of prostanoids thus seems to be at least partially affected by the ambient oxygen tension. Though altered oxygen tension does not seen to affect angiotensin induced prostanoid formation, the action of other vasoactive agents influencing vascular formation of prostanoids may respond differently to hypoxia or hyperoxia.     

Mixtures of polycyclic aromatic compounds inhibit mutagenesis in the Salmonella/microsome assay by inhibition of metabolic activation

We observed that complex mixtures of aromatic compounds isolated from a coal-derived oil suppressed the mutagenic activity of the indirect mutagens benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 2-aminofluorene, and 2-acetylaminofluorene as measured in the Salmonella/microsome mutagenicity assay, using strain TA98 and metabolic activation with Aroclor-induced rat-liver S9 or microsomes. The mixture also inhibited S9-dependent benzo[a]pyrene metabolism and covalent binding to DNA in a cell-free system. The mixture did not suppress the activity of either the direct acting mutagens 2-nitrofluorene and benzo[a]pyrene diol-epoxide, or of the indirect mutagen N-hydroxy-2-acetylaminofluorene which requires a microsomal deacetylase for metabolic activation. Spectrophotometric measurements showed that components of the mixture bound to microsomal cytochrome P-450. The mixture did not inhibit microsomal NADPH-cytochrome c (P-450) reductase. These observations show that the mixtures inhibited metabolic activation by the microsomal monooxygenase system, probably by binding of unidentified components to cytochrome P-450. The resulting inhibition of mutagenesis may have implications for risk estimates for the mixtures we examined as well as for other types of complex mixtures for which similar inhibitory effects have been observed.     

The nervous system of Tricladida. III. Neuroanatomy ofDendrocoelum lacteum andPolycelis tenuis (Plathelminthes,Paludicola): an immunocytochemical study

The organization of the nervous system ofDendrocoelum lacteum (Tricladida, Paludicola, Dendrocelidae) andPolycelis tenuis (Tricladida, Paludicola, Planariidae) was studied by immunocytochemical double staining, using neuropeptide RFamide and serotonin (5-HT) antisera on cryosections. The study confirmed the status of the main nerve cords (MCs) as the most important and stable of the longitudinal cords and supported the hypothesis of a common phylogenetic origin of the MCs in flatworms. The ganglion-like structures along the MCs at the beginning of transverse commissures and laterla branches showed a close contact with ventral fibres of the submuscular nerve plexus indicating an origin from crossing points of insunken ring commissures. The distributional pattern and morphology of the RFamide-IR cell bodies inD. lacteum corresponded to that of neurosecretory cells. Most RFamide-IR cells were unipolar and rounded while 5-HT-IR cells were uni- bi- and multipolar. The neutropile consisted of a dense RFamide-IR and a loose 5-HT-IR network. RFamide dominated in all parts of the genital plexus.     

Phospholipase C activation in rat pituitary adenoma (GH) cells

The presence of the pertussis toxin (PTX) insensitive GTP-binding proteins (G-proteins) G_q and/or G₁₁ has been demonstrated in three different prolactin (PRL) and growth hormone (GH) producing pituitary adenoma cell lines. Immunoblocking of their coupling to hormone receptors indicates that G_q and/or G₁₁ confer throliberin (TRH) responsive phospholipase C (PL-C) activity in these cells. The contention was substantiated by immunoprecipitation analyses snowing that anti G_q/11-sera coprecipitated PL-C activity. In essence, only G_q/11 (but neither G_i2, G_i3 nor G_o) seems to mediate the TRH-sensitive PL-C activity, while G_o may be coupled to a basal or constitutive PL-C activity. Immunoblocking studies imply that the B-complex also, to some extent, may stimulate GH₃ pituitary cell line PL-C activity. Finally, the steady state levels of G_q/11 mRNA and protein were downregulated upon long term exposure of the GH3 cells to TRH (but not to vasoactive intestinal peptide = VIP).