Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4

o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of ¹⁸O- and ¹⁶O-labeled 3-methylcatechol from toluene in an atmosphere of ¹⁸O₂ and ¹⁶O₂ established that G4 catabolizes toluene by successive monooxygenations at the ortho and meta positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the ortho position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities.     

Influence of cyanobacterial diet on Asplanchna predation risk in Brachionus calyciflorus

Asplanchna sylvestrii does not discriminate between groups of Brachionus calyciflorus fed either the cyanobacterium Anabaena flos-aquae or a control diet of Euglena gracilis. We based our analysis on the observed probabilities of attack, capture and ingestion during encounters between predator and prey. While A. sylvestrii was very sensitive to brachionid size, we found no significant affects of prey diet on predatory behavior. Thus, cyanobacterial diet did not influence the short-term predation risk of B. calyciflorus exposed to an effective predator. On the other hand, matched cohorts of A. sylvestrii fed B. calyciflorus cultured on the cyanobacterium reproduced more slowly than those fed the same prey cultured on the control food. With prolonged sympatry, therefore, the long-term risk of Asplanchna predation may be reduced for Brachionus by the latter's consumption of cyanobacteria.     

Bioethics and academic freedom

The author describes the events surrounding his attempts to lecture on the subject of euthanasia in West Germany in June 1989. Singer, who defends the view that active euthanasia for some newborns with handicaps may be ethically permissible, had been invited to speak to professional and academic groups. Strong public protests against Singer and his topic led to the cancellation of some of his engagements, disruptions during others, and harrassment of the German academics who had invited him to speak. These incidents and the subject of euthanasia became matters of intense national debate in West Germany, but there was little public or academic support for Singer's right to be heard. Singer argues that bioethics and bioethicists must have the freedom to challenge conventional moral beliefs, and that the events in West Germany illustrate the grave danger to that freedom from religious and political intolerance.     

Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to -galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.Issued as NRCC No. 31634     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7     

Extracellular matrix influences hormone and protein production by human chorionic villi

Summary Increasing evidence confirms that the extracellular matrix greatly influences cell behaviour and function. Collagen and fibrin are in contact with trophoblast throughout pregnancy. To investigate whether these two matrices influence hormon production by the trophoblast, explants from first-trimester chorionic villi were cultured for up to 30 days either a) in medium with agitation, b) embedded in type-I collagen (three-dimensional gels), or c) embedded in fibrin (three-dimensional gels). The supernatant culture medium was changed every 48 h and tested by radioimmunoassay for hCG, progesterone and pregnancy-associated plasma protein A. In addition, after 3, 7, 15, and 30 days of culture villi were fixed and studied by light and electron microscopy. Embedding in the extracellular matrix showed higher and longer-lasting production rates of all measured products and superior structural preservation as compared to cultures with agitation. Collagen matrix proved to be superior to fibrin. As established by several tests, this difference was neither due to thrombin used to polymerize fibrinogen, nor to differences in the diffusion rates through the two different matrices used. We conclude that extracellular matrix, particularly collagen, influences the synthesis of trophoblastic products. Embedding of the villous explants in three-dimensional gels constitutes a new method for long-term cultures of chorionic villi.This study was presented at the workshop Placental-and decidual-specific protein synthesis and secretion: regulation, role and interaction, Zemun, Belgrade, Yugoslavia, 19–20 May, 1988 (Bischof and Castellucci 1988; see also J. Aplin 1989), and at the 11th Rochester Trophoblast Conference, Rochester, N.Y. USA, 9–12 October 1988 (Castellucci et al. 1988)     

Evaluation of strain competitiveness in Rhizobium leguminosarum bv phaseoli using a nod + fix− natural mutant

Competition from native soil rhizobia is likely to be an important factor limiting Phaseolus vulgaris L. inoculant response in Latin America. We used UMR 1116, a nod ⁺ fix^– natural mutant of Rhizobium leguminosarum bv phaseoli strain CC511, as a reference strain to study competition for nodulation sites in this species. When P. vulgaris cv Carioca was planted in soils containing different proportions of UMR 1116 and the effective and competitive strain UMR 1899, UMR 1116 occupied more than 50% of the nodules at all inoculant ratios tested, though increasing the proportion of UMR 1899 in the inoculant did enhance the number and percentage of effective nodules and plant dry weight. Sixty two strains of bean rhizobia were tested in competition with UMR 1116. An inoculant ratio of 1:1 was used, with all strains applied to the soil rather than to seeds. Strains varied in the number and percentage of effective nodules produced in competition with UMR 1116, and in plant dry weight, and there was a strong correlation between variation in each of these traits and plant N accumulation. Seven of the strains (UMR 1073, 1084, 1102, 1125, 1165, 1378 and 1384) were identified as both superior in competitive ability and active in N₂ fixation. Site of placement of the inoculant and ambient temperature influenced strain response.Journal paper 16736, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

The morphology of suboesophageal ganglion cells innervating the nervus corporis cardiaci III of the locust

Summary The nervus corporis cardiaci III (NCC III) of the locust Locust migratoria was investigated with intracellular and extracellular cobalt staining techniques in order to elucidate the morphology of neurons within the suboesophageal ganglion, which send axons into this nerve. Six neurons have many features in common with the dorsal, unpaired, median (DUM) neurons of thoracic and abdominal ganglia. Three other cells have cell bodies contralateral to their axons (contralateral neuron 1–3; CN 1–3). Two of these neurons (CN2 and CN3) appear to degenerate after imaginal ecdysis. CN3 innervates pharyngeal dilator muscles via its anterior axon in the NCC III, and a neck muscle via an additional posterior axon within the intersegmental nerve between the suboesophageal and prothoracic ganglia. A large cell with a ventral posterior cell body is located close to the sagittal plane of the ganglion (ventral, posterior, median neuron; VPMN). Staining of the NCC III towards the periphery reveals that the branching pattern of this nerve is extremely variable. It innervates the retrocerebral glandular complex, the antennal heart and pharyngeal dilator muscles, and has a connection to the frontal ganglion.Abbreviations AH antennal heart - AN antennal nerves - AO aorta - AV antennal vessel - CA corpus allatum - CC corpus cardiacum - CN1, CN2, CN3 contralateral neuron 1–3 - DIT dorsal intermediate tract - DMT dorsal median tract - DUM dorsal, unpaired, median - FC frontal connective - FG frontal ganglion - HG hypocerebral ganglion - LDT lateral dorsal tract - LMN, LSN labral motor and sensory nerves - LN+FC common root of labral nerves and frontal connective - LO lateral ocellus - MDT median dorsal tract - MDVR ventral root of mandibular nerve - MVT median ventral tract - NCA I, II nervus corporis allati I, II - NCC I, II, III nervus corporis cardiaci I, III - NR nervus recurrens - NTD nervus tegumentarius dorsalis - N8 nerve 8 of SOG - OE oesophagus - OEN oesophageal nerve - PH pharynx - SOG suboesophageal ganglion - T tentorium - TVN tritocerebral ventral nerve - VLT ventral lateral tract - VIT ventral intermediate tract - VMT ventral median tract - VPMN ventral, posterior, median neuron - 1–7 peripheral nerves of the SOG - 36, 37, 40–45 pharyngeal dilator muscles