The Cultivation of Symbiote-free Marine Ciliates in Axenic Medium

SYNOPSIS. Media have been developed for axenic cultivation of 10 strains belonging to 7 species of small marine ciliates. A medium containing cerophyl extract, proteose peptone, trypticase, yeast nucleic acid, biotin, calcium pantothenate folic acid, nicotinamide, pyridoxal HCl, riboflavin, thiamine HCl, and DL-thioctic acid in sea water supports the growth of Uronema nigricans strains Pc and 34/2, Parauronema virginiatum strains 2/1 and 19/1, Miamiensis avidus strains Ma and 19/3, Miamiensis sp. strain 1/1, a strain of "G" ciliate, and strains 33/8 and 34/7 of 2 unidentified species. By substituting a mixture of asolecithin, cephalin, and Tween 80 for cerophyl in the medium, luxurious growth of all except the strains of the 2 unidentified species can be obtained. A defined medium consisting of 18 amino acids, 5 purine derivatives, 8 vitamins, asolecithin, cephalin, and Tween 80 in synthetic sea water also has been developed for 6 of the strains: M. avidus Ma and 19/3, Miamiensis sp. 1/1, P. virginiatum 2/1 and 19/1, and U. nigricans Pc. In general the ciliates grow best at pH 7.2 in the dark at 27 C in media containing sea water of density = 1.015. Under these conditions maximum populations are reached in 4–5 days and range from several hundred thousand to 3 or 4 × 10⁶ depending upon the strain. Electronmicroscopic observations for the presence of endosymbiotes gave negative results.     

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The levels of individual free and conjugated ecdysteroids and ecdysteroid acids, labeled from [¹⁴C]cholesterol, in five different age groups of male Manduca sexta during pupal-adult development were determined by HPLC. Eight free ecdysteroids, eight ecdysteroid phosphates, and two ecdysteroid acids were identified. Newly ecdysed pupae contained predominantly 3-epiecdysteroids in each of the free, conjugated, and acidic ecdysteroid fractions. The titer of each ecdysteroid fraction rose sharply by day 4, and this was particularly noteworthy with respect to free ecdysone and 3-epi-20-hydroxyecdysonoic acid. This stage demonstrated high degrees of ecdysone biosynthesis, oxidative catabolism, and phosphorylation. As development proceeded to day 16, total ecdysteroid titer remained constant; a decreasing free ecdysteroid titer was accompanieid by increasing titers of both conjugates and acids resulting from the metabolic processes of hydroxylation, oxidation, epimerization, and phosphorylation. The predominant metabolites throughout development were 3-epi-20-hydroxyecdysonoic acid and the phosphate conjugates of 3-epi-20-hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone. The ultimate inactivation of the ecdysteroids of M. sexta during pupal-adult development is possibly mediated by two pairs of metabolically-linked processes, one leading to a 3-epiecdysteroid acid, and the other to 3-epiecdysteroid phosphates.     

Synergistic inhibition of metabolic cooperation by oleic acid or 12-0-tetradecanoylphorbol-13-acetate and dichlorodiphenyltrichlorethane (DDT) in Chinese hamster V79 cells: Implication of a role for protein kinase C in the regulation of gap junctional intercellular communication

The effects of TPA and/or DDT and oleic acid and/or DDT on gap junction-mediated intercellular communication (i.e. metabolic cooperation) between Chinese hamster V79 cells was examined. Addition of TPA, DDT or oleic acid alone to cocultures of 6t-hioguanine-resistant (6-TG ^R ) and 6-thioguanine-sensitive (6-TG ^S ) V79 cells significantly increased the recovery of 6-TG ^R cells indicating inhibition of metabolic cooperation. In the presence of TPA and DDT or oleic acid and DDT the observed recovery of 6-TG ^R cells was significantly greater than the expected (calculated) additive 6-TG ^R cell recovery. No synergistic increases in 6-TG ^R cell recovery were observed when co-cultures of V79 cells were exposed to dieldrin and DDT. These results indicate that TPA and DDT or oleic acid and DDT can act synergistically to inhibit metabolic cooperation. These data suggest a role for protein kinase C in the regulation of gap junction-mediated intercellular communication.Abbreviations DDT dichlorodiphenyltrichlorethane - MC metabolic cooperation defective - 6-TG 6thioguanine - TPA 12-0-tetradecanoylphorbol-13-acetate     

The mutagenic potential of high flash aromatic naphtha

Catalytic reforming is a refining process that converts naphthenes to aromatics by dehydrogenation to make higher octane gasoline blending components. A portion of this wide boiling range hydrocarbon stream can be separated by distillation and used for other purposes. One such application is a mixture of predominantly 9-carbon aromatic molecules (C9 aromatics, primarily isomers of ethyltoluene and trimethylbenzene), which is removed and used as a solvent — high-flash aromatic naphtha. A program was initiated to assess the toxicological properties of high-flash aromatic naphtha since there may be human exposure through inhalation or external body contact. The current study was conducted partly to assess the potential for mutagenic activity and also to assist in an assessment of carcinogenic potential. The specific tests utilized included the Salmonella/mammalian microsome mutagenicity assay, the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) forward mutation assay in CHO cells, in vitro chromosome aberration and sister chromatid exchange (SCE) assays in CHO cells, and an in vivo chromosome aberration assay in rat bone marrow.There was no evidence that high-flash aromatic naphtha was either a gene or chromosomal mutagen. Thus it is unlikely to be a genotoxic carcinogen.Abbreviations Brdu 5-Bromo-2-deoxyuridine - C9 Aromatic species with 9 carbons (i.e., ethyl toluene and trimethyl benzene isomers) - CE Cloning efficiency - CHO Chinese hamster embryo - CP Cyclophosphamide - DMSO Dimethyl sulfoxide - HGPRT Hypoxanthine-guanine phosphoribosyl transferase - HVAC Heating, Ventilation, Air Conditioning - 3MC 3 Methylcholanthrene - MMC Mitomycin C - MMS Methyl methanesulfonate - S9 S9 Mammalian microsomal enzyme activation mixture - SCE Sister chromatid exchange     

Scanning Electron Microscopy of Xiphinema,Longidorus, and Californidorus Stylet Morphology

Stylet ultrastructure of five Xiphinema, four Longidorus, and three Californidorus species was compared by scanning electron microscopy. Morphological differences were seen in the odontophores and odontostyle bases between the genera and some of the species. All Xiphinema studied had well-developed odontophore flanges; the Longidorus species lacked flanges, except for weakly developed ones in L. diadecturus; and none of the Californidorus had flanges. Three sinuses were present in the odontophores of all species. The sinuses varied in length depending upon species. In Xiphinema and Californidorus the odontostyle bases had distinct overlapping collars, but in Longidorus the collars were absent except for L. diadecturus. The odontostyle-odontophore junction from a lateral view appeared as a slanted transverse line in all the species, but in a dorsal view of Xiphinema and Californidorus it was V-shaped. Dorsal longitudinal seams of the odontostyle and odontophore were observed in all the species. The dorsally located odontostyle aperture was ca. 1 μm from the anterior end in all species, except in one Longidorus sp. it was ca. 4 μm from the end.     

Decreased Protein Kinase C Activity During Cerebral Ischemia and After Reperfusion in the Adult Rat

The possible activation of protein kinase C (PKC) during total cerebral ischemia was investigated in the rat. Translocation of PKC activity from the soluble to the particulate fraction was used as an index of PKC activation. There was a drop in the proportion of particulate PKC activity from 30% for controls to 20% by 30 min of ischemia (p less than 0.01). By 20 min of cardiac arrest, there was a 40% decline of the total cellular PKC activity (p less than 0.01). This was not accompanied by an increase in activator-independent activity, a finding indicating PKC was not being converted to protein kinase M. These data suggest that PKC was not activated during ischemia, but rather that ischemia causes a reduction in cellular PKC activity. Translocation of PKC activity to the particulate fraction was not observed in the cerebral cortex or hippocampus of reperfused brain for up to 6 h of recovery following 11-13 min of total cerebral ischemia. The level of total, soluble, and particulate PKC activity in the cerebral cortex was reduced (p less than 0.05), corresponding to the decrease observed by 15 min of ischemia without reflow. A similar decline in activity was also observed in the hippocampus. No increase in activator-independent activity was observed. These data suggest that PKC was inhibited during cerebral ischemia and that this reduced level of PKC activity was maintained throughout 6 h of recovery. We conclude that pathological activation of PKC was not responsible for the evolution of ischemic brain damage.     

Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

α1-Adrenergic Receptor Mediates Arachidonic Acid Release in Spinal Cord Neurons Independent of Inositol Phospholipid Turnover

The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.