Oxidative bioconversion of toluene to 1,3-butadiene-1,4-dicarboxylic acid (cis,cis-muconic acid)

Pseudomonas sp. strains, isolated from soil, utilized toluene as their sole carbon source through ameta cleavage pathway. Strains metabolizing toluene through anortho cleavage pathway were selected from the wild typemeta strain. Theortho pathway strains were subjected to chemostat selection to obtain a fast-growing strain with doubling time reduced from 14 to 1.2 h. Benzoale and antibiotics enrichment selection procedures were utilized to select a blocked mutant. The blocked mutant grew on acetate as its sole carbon source and oxidatively converted toluene tocis, cis-muconic acid. Double-blocked and muconate-permeable mutants were also selected to reduce reversion frequency and to enhance muconic acid production. In shake-flask experiments, muconic acid at 3.5 g/l was obtained after 2 days of fermentation. In a 14 l fermenter, muconic acid was produced at 45 g/l in 4 days of controlled fed-batch fermentation. The oxidative bioconversion process was also demonstrated in a 1500 l fermenter.

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Résumé Des souches dePseudomonas sp., isolées du sol, ont utilisé le toluène comme seule source de carbone par la vole de la rupture de cycle enmeta. On a sélectionné des souches métabolisant le toluène par la voie de la rupture de cycle enortho, à partir de la souche sauvage de typemeta. Les souches de la voieortho ont été soumises à la sélection en chémostat pour obtenir des souches à croissance rapide dont le temps de doublement est rédult de 14 à 1.2 h. Les procédures de sélection par enrichissement sur benzoale et antibiotiques ont été utilisées pour sélectionner un mutant bloqué. Le mutant bloqué croît sur acétate comme seule source de carbone et convertit le toluène par voie oxydative en acidecis,cis-muconique. On a également sélectionné des mutants doublement bloqués et perméables au muconate pour réduire la lréquence de réversion et pour augmenter la production d'acide muconique. En expérimentation en flacons agités, on a obtenu 3.5 g/l d'acide muconique après 2 jours de fermentation. En fermentuer de 14 l, on a produit 45 g/l d'acide muconique en 4 jours de fermentation contrôlée en milieu non renouvelé à allmentation étagée. Le processus de bioconversion oxydative a également été démontré en fermenteur de 1500 l.

     

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.     

Neurite Outgrowth Stimulated by the Tyrosine Kinase Inhibitor Herbimycin A Requires Activation of Tyrosine Kinases and Protein Kinase C

Abstract: Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events downstream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N-and L-type calcium channels.     

A comparison of the binding of methylated cap analogues to wheat germ protein synthesis initiation factors 4F and (iso)4F

The binding of the 5'-terminal cap analogues m7GpppG and m7GTP to wheat germ protein synthesis initiation factors eIF-4F and eIF-(iso)4F as a function of pH, ionic strength, and temperature is described. Equilibrium binding data indicate that eIF-4F and eIF-(iso)4F have different mechanisms for interacting with the 5'-cap structure, but the complexes formed between m7GpppG and wheat germ factor eIF-(iso)4F more closely resemble complexes formed between this cap analogue and either mammalian eIF-4E or eIF-4F. The binding of these initiation factors to the hypermethylated cap analogues m2,7GMP, m2,7GpppG, and m2,2,7GpppG is also investigated. The differences in affinity of eIF-4F and eIF-(iso)4F for the hypermethylated 5'-terminal cap structures suggest that these factors may have discriminatory activity.     

A fluorescence study of the interaction of protein synthesis initiation factors 4A, 4E, and 4F with mRNA and oligonucleotide analogs

The initial interaction of mRNA with the protein synthesis machinery presumably involves recognition of the 5'-cap (m7GpppN), although it is not clear at the present time whether this recognition is by eIF-4E or eIF-4F. This process has been studied by direct fluorescence titration experiments. The equilibrium constants for the formation of the binary protein: m7GpppG, protein:mRNA, and protein:protein complexes as well as the ternary mRNA:eIF-4E:eIF-4A complexes were measured. These studies show, for the first time, direct evidence for an eIF-4A:eIF-4E interaction. In contrast to earlier studies, we show that the affinity of eIF-4E and eIF-4F for globin mRNA is similar. Furthermore, the relative affinities of mRNA analogs (capped oligonucleotides) for these initiation factors indicate that the cap is the predominant feature recognized for binding, but other features also contribute to the eIF-4E:mRNA interaction.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.     

Invasive interactions: can Argentine ants indirectly increase the reproductive output of a weed?

The direct and indirect interactions of invasive ants with plants, insect herbivores, and Hemiptera are complex. While ant and Hemiptera interactions with native plants have been well studied, the effects of invasive ant–scale insect mutualisms on the reproductive output of invasive weeds have not. The study system consisted of Argentine ants (Linepithema humile), boneseed (Chrysanthemoides monilifera monilifera), and sap-sucking scale insects (Hemiptera: Saissetia oleae and Parasaissetia nigra), all of which are invasive in New Zealand. We examined the direct and indirect effects of Argentine ants on scale insects and other invertebrates (especially herbivores) and on plant reproductive output. Argentine ants spent one-third of their time specifically associated with scale insects in tending behaviours. The invertebrate community was significantly different between uninfested and infested plants, with fewer predators and herbivores on ant-infested plants. Herbivore damage was significantly reduced on plants with Argentine ants, but sooty mould colonisation was greater where ants were present. Herbivore damage increased when ants were excluded from plants. Boneseed plants infested with Argentine ants produced significantly more fruits than plants without ants. The increase in reproductive output in the presence of ants may be due to increased pollination as the result of pollinators being forced to relocate frequently to avoid attack by ants, resulting in an increase in pollen transfer and higher fruit/seed set. The consequences of Argentine ant invasion can be varied; not only does their invasion have consequences for maintaining biodiversity, ant invasion may also affect weed and pest management strategies.