Olfactory cytochrome P-450. Studies with suicide substrates of the haemoprotein.

1. The olfactory epithelium of male hamsters has been found to be extremely active in the cumene hydroperoxide-supported oxidation of tetramethylphenylenediamine, and this peroxidase activity has been shown to be cytochrome P-450-dependent. 2. The interaction of a series of suicide substrates of cytochrome P-450 with the hepatic and olfactory mono-oxygenase systems has been assessed by determination of peroxidase, 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) activities after treatment in vivo with these compounds. Chloramphenicol, OOS-trimethylphosphorothiolate and two dihydropyridines [DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) and 4-ethyl DDC (3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine)] all caused similar percentage inhibitions of hepatic and olfactory activities, but the absolute amounts of enzymic activity lost were considerably greater in the latter tissue. In contrast, halothane had little effect upon hepatic cytochrome P-450-dependent reactions, whereas it severely inhibited those of the olfactory epithelium. 3. The time course of loss and recovery of hepatic and olfactory peroxidase, ECOD and EROD activities after a single dose of 4-ethyl DDC was studied. The rates of loss of activity observed were very similar, irrespective of tissue or reaction examined. In the olfactory epithelium, all three activities recovered concurrently and at a rate similar to that of the hepatic peroxidase activity. In contrast, the hepatic de-ethylation of 7-ethoxycoumarin and 7-ethoxy-resorufin recovered significantly more rapidly. 4. It is suggested that this behaviour is due to 4-ethyl DDC acting not only as a suicidal inhibitor but also as an inducer of certain forms of cytochrome P-450 in the liver; in the olfactory epithelium, however, inactivation, but not induction, occurs. Classical inducing agents were reported to have no effect upon olfactory cytochrome P-450, and in the present study neither phenobarbitone nor beta-naphthoflavone treatment had any effect upon olfactory cytochrome P-450-dependent reactions, although it induced those of the liver.     

The use of automated image analysis for rapid measurement of the in vitro attachment of Candida albicans to transparent acrylic

A rapid method for measuring the in vitro attachment of Candida albicans to the surface of transparent acrylic is described. The method involves use of the 'Magiscan' automated image analysis system which measures attachment in terms of percentage area coverage. These measurements correlate highly significantly ( P < 0·001) with the number of adherent yeast cells.     

Morphometrical analysis of the gap-junctional area in parenchymal cells of the rat liver after administration of dibutyryl cAMP and aminophylline

Summary In view of the presumed involvement of gap junctions in the coordination of metabolic activities, the influence of cAMP as a regulatory signal of cell metabolism on gap junctions of hepatocytes has been examined. Male rats received two intraperitoneal doses of 10 mg dibutyryl cAMP/100 g body weight with a time interval of 2.5 h and were decapitated 2.5 h later. After this 5-h interval, analysis of freeze-fracture replicas of fixed liver tissue revealed an increase in the mean (± SEM) gap-junctional membrane portion on the lateral hepatocyte membranes from 0.049 + 0.003 (n = 66) in controls to 0.061 ± 0.003 (n = 70) in treated rats, while the configuration of the connexons appeared unaltered. This effect could not be reinforced by prior administration of aminophylline: the relative gapjunctional area is similarly extended from 0.054 ± 0.003 (n = 126) in the control group to 0.065 ± 0.004 (n = 105) in the experimental animals. Probing for the time course of the junctional response, a group of rats was sacrificed 3 h after the onset of treatment. Already within this time, the gapjunctional area is augmented from 0.042 ± 0.004 (n = 63) in the concurrent controls to 0.069 ± 0.006 (n = 42) in the treated rats. These statistically significant increases in area may suggest a stimulating effect of cAMP on gap junctions of hepatocytes in vivo.This investigation was supported by grant No. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium)     

Cystic fibrosis typing with DNA probes: experience of a screening laboratory

Summary A sample of 125 individuals from 37 British cystic fibrosis (CF) families with at least one living affected child were typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene. These probes were MetD, MetH, pJ3.11 and 7C22. Using this combination of probes, 30 out of the 37 families were sufficiently informative to enable prenatal diagnosis of the disease. Linkage analysis has also proved to be useful in excluding CF in two cases where diagnosis of the disease was equivocal in the sibling of an affected child.     

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.     

Allometric shape change and heterochrony in the freeliving coral Trachyphyllia bilobata (Duncan)

Regression analysis has been used to study the relationship between age, size, shape, and surface area in two ancestral-descendant populations of the Neogene Caribbean coral Trachyphyllia bilobata. Analyses of the relationship between size and age show that the relationship is isometric and that little difference occurs between populations in mean corallite length or height and in their rates of growth. Onset of columella growth is significantly earlier, however, in the descendant population. Studies of the relationship between size and shape show that growth is allometric, with shape change occurring in both corallum elongation and pinching of the corallite wall during ontogeny. In the descendant population, pinching and elongation initiate earlier in the ontogeny of the coral. These results suggest that the evolutionary development of the meandroid form in freeliving corals has been accomplished by heterochrony, involving a complex set of disassociated peramorphic changes in ontogeny accompanied by paedomorphic changes in astogeny. Further analyses show that the observed heterochronic changes serve to decrease corallum surface area which may in turn enhance sediment removal and nutrition in unstable habitats.     

Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Pneumocystis carinii shows DNA homology with the ustomycetous red yeast fungi

Pneumocystis carinii causes life-threatening pneumonia in T-lymphocyte-immunodeficient subjects in transplant and oncology units or with acquired immune deficiency syndrome (AIDS). Recent DNA homology studies show P. carinii to be a fungus. To investigate the biology and epidemiology of this parasite further, we elected to determine for it a more precise taxonomic assignment within the fungal kingdom. We screened a wide range of organisms representing the major orders of fungi using DNA amplification and subsequently sequenced a portion of the mitochondrial gene encoding the large subunit ribosomal RNA. Our data show that the opportunistic pulmonary pathogen P. carinii is closely related to the ustomycetous red yeast fungi, a group which includes organisms that are extensively distributed throughout the environment and which release many widely dispersed airborne spores.     

Retention of oxidized glutathione by isolated rat liver mitochondria during hydroperoxide treatment

The addition of tert-butyl hydroperoxide (t-BuOOH) to isolated mitochondria resulted in oxidation of approximately 80% of the mitochondrial reduced glutathione (GSH) independently of the dose of t-BuOOH (1-5 mM). Concomitant with the oxidation of GSH inside the mitochondria was the formation of GSH-protein mixed disulfides (protein-SSG), with approximately 1% of the mitochondrial protein thiols involved. A dose-dependent rate of GSH recovery was observed, via the reduction of oxidized GSH (GSSG) and a slower reduction of protein-SSG. Although t-BuOOH administration affected the respiratory control ratio, the mitochondria remained coupled and loss of the matrix enzyme, citrate synthase, was not increased over the control and was less than 3% over 60 min. A slow loss of GSH out of the coupled non-treated mitochondria was not increased by t-BuOOH treatment, in fact, a dose-dependent drop of GSH levels occurred in the medium. However, no GSSG was found outside the mitochondria, indicating the necessary involvement of enzymes in the t-BuOOH-induced conversion of GSH to GSSG. The absence of GSSG in the medium also suggests that, unlike the plasma membrane, the mitochondrial membranes do not have the ability to export GSSG as a response to oxidative stress. Our results demonstrate the inability of mitochondria to export GSSG during oxidative stress and may explain the protective role of mitochondrial GSH in cytotoxicity.