Anaerobic degradation of cyclohexane-1,2-diol by a new Azoarcus species

A bacterium, strain 22Lin, was isolated on cyclohexane-1,2-diol as sole electron donor and carbon source and nitrate as electron acceptor. Cells are motile rods and are facultatively anaerobic. A phylogenetic comparison based on the total 16S rRNA gene sequence allowed the assignment of the isolate to the genus Azoarcus. Cyclohexanol, cyclohexanone, cyclohexane-1,3-diol, and cyclohexane-1,3-dione, which are oxidized by a different denitrifying strain, did not support denitrifying growth of isolate 22Lin. On the contrary, cyclohexanol (I₅₀ = 37 μM) and cyclohexanone (I₅₀ = 28 μM) inhibited growth on cyclohexane-1,2-diol, but not on acetate. NAD was reduced by crude extracts of strain 22Lin in the presence of cyclohexane-1,2-dione, but not in the presence of cyclohexanone or cyclohexane-1,3-dione. The formation of 6-oxohexanoate from cyclohexane-1,2-dione and of adipate during NAD reduction suggests that strain 22Lin possesses a carbon–carbon hydrolase that transforms cyclohexane-1,2-dione into 6-oxohexanoate. This pathway was once observed in an aerobic pseudomonad that was lost and could not be reisolated. Here, the application of strictly anoxic enrichment conditions enabled the reisolation of another strain (22Lin) that uses this pathway. Received: 3 February 1997 / Accepted: 12 May 1997     

Invariant association of ecdysis with increases in cyclic 3′,5′-guanosine monophosphate immunoreactivity in a small network of peptidergic neurons in the hornworm, Manduca sexta

At the end of each molt insects shed their old cuticle by performing the stereotyped behavior of ecdysis. In the moth, Manduca sexta, this behavior is triggered by the neuropeptide eclosion hormone (EH). Insights into the mechanism of action of EH have come from the identification of a small network of peptidergic neurons that shows increased cyclic 3′,5′-guanosine monophosphate (cGMP) immunoreactivity at ecdysis in insects from many different orders. Here we present further evidence that strengthens the association between ecdysis and the occurrence of this cGMP response in Manduca. We found that the cGMP increases occurred at every ecdysis, although some of the neurons that showed a response at larval ecdysis did not participate at pupal and adult ecdysis. Both ecdysis and the cGMP increases only required an intact connection with the brain for the first 30 min after EH injection. Interestingly, ecdysis in debrained animals only occurred if the cGMP response had been initiated, suggesting that the onset of this response marks the time at which the central nervous system is first able to drive ecdysis. Finally, we found that the appearance of sensitivity to EH for triggering the cGMP response coincided with the time at which EH first triggers ecdysis. Accepted: 6 May 1997     

Metabolism of zeatin riboside in a hormone autonomous genetic tumour line of tobacco

The metabolic fate of externally applied [³H]-zeatin riboside ([9R]Z) was studied in a cultured genetic tumour line of Nicotiana glauca (Grah.) × N. langsdorffii (Weinm.), which grows on auxin and cytokinin free medium. Metabolism by 3.5-week-old tissues showed enhanced stability of supplied [9R]Z; unmetabolized [9R]Z accounted for 48.7 and 37.5% of extracted radioactivity following 8 and 24 h incubation, respectively; tissues of different ages (1–10 weeks following subculture) also indicated high cytokinin stability following 8 h incubation (unmetabolized [9R]Z accounted for 32.5–53.0% of extracted radioactivity). All analyses were performed by thin layer chromatography (TLC) and the results subsequently confirmed by high performance liquid chromatography (HPLC). Side-chain cleavage and modification of the purine ring were the major forms of metabolism; metabolites with an intact cytokinin moiety included zeatin (Z), [9R]Z nucleotides and glucosyl derivatives. Detailed analysis of metabolites carried out in the experiments using 3.5-week-old tissues indicated that both dihydro-derivatives as well as cis isomers of Z and [9R]Z were not formed. Adenine, adenosine and its nucleotide(s) were the main degradative metabolites; in 3.5-week-old tissues these metabolites accounted for about 5.9 and 7.8% (of ³H extracted) following 8 and 24 h incubation, respectively. In tissues of different ages (1–10 weeks following subculture), these metabolites accounted for about 7.6–22.9% of the extracted ³H. Some metabolites (zeatin, adenine and adenosine) were also detected in the staled incubation media. The observed high [9R]Z stability in this tissue may reflect low levels of cytokinin oxidase activity and/or some form of compartmentation.     

Coevolution of traits determining migratory tendency: correlated response of a critical enzyme,juvenile hormone esterase,to selection on wing morphology

Migratory tendency in insects is a complex trait, composed of a suite of correlated behavioural, physiological, morphological and life history traits. We investigate the genetic and physiological basis of the coevolution of this suite of traits using laboratory lines of the wing dimorphic cricket, Gryllus firmus, selected for increasing and decreasing incidence of macroptery. Selection on wing morphology has produced strong direct responses in proportion macropterous as well as correlated (indirect) responses in wing muscle histolysis, flight propensity and fecundity. We investigate the hypothesis that these responses have been mediated by changes in the metabolism of juvenile hormone (JH) during the final nymphal stadium (the critical period for wing morph determination). Previous studies of Gryllus sp. have established that JH titre in this period is determined primarily by the activity of the degradative enzyme, juvenile hormone esterase (JHE). Assays of JHE activity in the final nymphal stadium of the replicated control and selected lines demonstrate highly significant differences in both mean activity and the probability of macroptery for a given level of activity (i.e., the threshold activity required to induce wing formation). These correlated responses in JH metabolism support the general hypothesis that the correlations among traits determining migratory tendency result at least in part from the common influence of JH during the final nymphal stadium. We discuss these results in the context of the quantitative genetic model for the evolution of polygenic, dichotomous traits (the threshold model), and present four general predictions concerning the coevolution of traits associated with ecological (i.e., trophic, life history, behavioural) dimorphisms.     

Biochemical and Immunological Relationships Between Endo-β-1,4-Glucanases from Cockroaches

The cellulase from Geoscapheus dilatatus consisted of two major and four minor endo-β-1,4-glucanase components. Two major and one minor component were purified to homogeneity. The major endo-β-1,4-glucanase components, named GD1 and GD2, were similar to EG1 and EG2 from Panesthia cribrata in terms of M_r and kinetic properties. The purified minor component, named GD3, was distinct from GD1 and GD2 because of a lower M_r and a lower specific activity. Polyclonal antibodies raised against the two major endo-β-1,4-glucanase components, EG1 and EG2, of the cellulase from P. cribrata cross-reacted with each other and with pure GD1 and GD2 from the foregut and midgut of the related cockroach G. dilatatus but did not cross-react with GD3. Endo-β-1,4-glucanase components were partially purified from the foregut and midgut of four other cockroaches. These comprised three other Australian cockroaches also from the superfamily Blaberoidea and one American cockroach, Cryptocercus punctulatus, from the superfamily Blattoidea. The endogenous cellulases from all cockroaches examined consisted of either two or three major endo-β-1,4-glucanase components. The amino acid sequence of the N-terminus region of the two major endo-β-1,4-glucanase components from P. cribrata were determined and are homologous with those belonging to glycosyl hydrolase family 9 (cellulase family E).     

THE RELATIONSHIP BETWEEN INSECT GROWTH REGULATORS WITH JUVENILE HORMONE ACTIVITY AND INSECT FEEDING BEHAVIOURAL RESPONSE

Abstract  Five insect growth regulators (IGRs) with juvenile hormone (JH) activity including fenoxy-carb, methoprene, NC-170, NC-184 and NC-196 were selected to study on the relationship between IGRs and feeding behaviour of the fourth instar larvae of Calospilos suspecta (Warren) and the fifth instar larvae of Ostrinia furnacalis Guenee, by topical and dietary applications as well as "diet column" methods. The results indicated that the antifeeding indices of methoprene, fenoxycarb, NC-170, NC-184, NC-196 and toosendanin against the fifth instar larvae of Ostrinia furnacalis in no-choice test were -37. 7, 14. 6, -12. 3, 10. 9, 14. 7 and 62. 2% respectively. Methoprene could stimulate the two species of insects to feed, whereas fenoxycarb had some antifeeding activity, and the effects of all the tested chemicals were different. Therefore, disturbances of IGRs with JH activity on insect feeding behavioural response may be one of the mechanisms of action not to be ignored. Moreover, by comparing "diet column" method with "faeces weight" method, it was suggested that the two methods might be applied in bioassay of antifeeding behaviour.     

Comparative analyses of the pigment-aggregating and -dispersing actions of MCH on fish chromatophores

In melanophores of the peppered catfish and the Nile tilapia, melanin-concentrating hormone (MCH) at low doses (<1 μM) induced pigment aggregation, and the aggregated state was maintained in the presence of MCH. However, at higher MCH concentrations (such as 1 and 10 μM), pigment aggregation was immediately followed by some re-dispersion, even in the continued presence of MCH, which led to an apparent decrease in aggregation. This pigment-dispersing activity at higher concentrations of MCH required extracellular Ca²⁺ ions. By contrast, medaka melanophores responded to MCH only by pigment aggregation, even at the highest concentration employed (10 μM). Since it is known that medaka melanophores possess specific receptors for α-melanophore-stimulating hormone (α-MSH), the possibility that interaction between MSH receptors and MCH at high doses in the presence of Ca²⁺ might cause pigment dispersion is ruled out. Cyclic MCH analogs, MCH (1–14) and MCH (5–17), failed to induce pigment dispersion, whereas they induced aggregation of melanin granules. These results suggest that another type of MCH receptor that mediates pigment dispersion is present in catfish and tilapia melanophores, and that intact MCH may be the only molecule that can bind to these receptors. Determinations of cAMP content in melanophores, which were isolated from the skin of three fish species and treated with 10 nM or 10 μM MCH, indicate that MCH receptors mediating aggregation may be coupled with Gi protein, whereas MCH receptors that mediate dispersion may be linked to Gs. The response of erythrophores, xanthophores and leucophores to MCH at various concentrations was also examined, and the results suggest that the distribution patterns of the two types of MCH receptors may differ among fish species and among types of chromatophore in the same fish.     

Involvement of PKC-β in PTH, TNF-α, and IL-1β Effects on IL-6 Promoter in Osteoblastic Cells and on PTH-Stimulated Bone Resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-β, LY379196, was used to determine the role of the PKC-β isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) induced translocation of PKC-α and -β_I to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-β_I translocation by PTH, TNF-α or IL-1β was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-α-, or IL-1β-stimulated translocation of PKC-α. PTH, TNF-α, and IL-1β increased luciferase expression in UMR-106 cells transiently transfected with a −224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-β_I is a component of the signaling pathway that mediates PTH-, TNF-α-, and IL-1β-stimulated IL-6 expression and PTH-stimulated bone resorption.