Microbial metabolism of chlorosalicylates: effect of prolonged subcultivation on constructed strains

The hybrid strain Pseudomonas sp. WR4016 was subcultivated with increasing concentrations of 5-chlorosalicylate (510 mM) as sole carbon source over a period of 9 months. At intervals of approximately 3 months derivative strains WR4017, WR4018 and WR4019 were isolated which exhibited higher growth rates and increased substrate tolerance. Comparative analysis of the turnover rates of the key enzymes in chlorosalicylate degradation showed that the adaptation process did not result from structural modifications of these proteins. Instead, balanced over-production of the salicylate hydroxylase and catechol 1,2-dioxygenase prevented the accumulation of toxic chlorocatechols and accounted for the reduction of the doubling times with 4- or 5-chlorosalicylate. A comparative analysis of a genetically engineered chlorosalicylate degrader PL300-1 showed similar regulatory patterns as the most advanced isolate WR4019 from the adaptation series.     

Elektronenmikroskopische Untersuchungen über die Innenversilberung der Sehnenfibrillen

Zusammenfassung 1. Die Behandlung der nativen und formolfixierten Sehnenfibrillen mit einer ammoniakalischen Silberlösung führt immer zu einer Einlagerung von Silberpartikeln in den D-Teilen der Fibrillen.2. Bei den nativen Fibrillen liegen die Silberkörner in einem, zwei oder drei Streifen im D-Teil.3. In den formolfixierten Fibrillen ist das Silber nur in einem Streifen vorhanden.4. Die Behandlung der nativen und formolfixierten Sehnenfibrillen mit anderen Silbersalzen führt zu keiner Versilberung der Fibrillen.5. Die Behandlung der nativen Sehnenfibrillen mit neutraler Kochsalzlösung oder Trypsin und anschließender Versilberung führt zu keiner wesentlichen Änderung des Silberbildes.6. Hyaluronidase-, Citratpuffer- und Perjodateinwirkung auf native Sehnenfibrillen mit anschließender Versilberung führt zu keiner Innenversilberung der D-Teile.7. Acetylierung und Behandlung mit Bisulfit der nativen Fibrillen und anschließender Versilberung mit ammoniakalischer Silberlösung verhindert eine Innenversilberung der D-Teile.8. Die formolfixierten Fibrillen zeigen eine Innenversilberung der D-Teile nach einer Vorbehandlung mit einer neutralen Kochsalzlösung, Citratpuffer, Hyaluronidase, Trypsin und Perjodat. Nur die Acetylierung und die Behandlung mit Bisulfit verhindert eine Innenversilberung.9. Die Innenversilberung der Sehnenfibrillen durch eine ammoniakalische Silberlösung wird weder durch Licht noch durch Chloride oder lichtempfindliche Silbereiweißverbindungen hervorgerufen.10. Die Versilberung in den D-Teilen wird durch Stoffe in den Fibrillen bewirkt, die Silber aus einer ammoniakalischen Silberlösung ausfällen können.11. Die reduzierenden Stoffe haben enge Beziehungen zur citratlöslichen Fraktion und sind perjodat- und hyaluronidaseempfindlich. Formalinfixierung beeinflußt diesen Versilberungsmodus durch ein vermehrtes Auftreten von Querbindungen.12. Die Sonderstellung der ammoniakalischen Silberlösung für die Innenversilberung wird diskutiert. Sie kann stereochemische Gründe haben oder durch die große Beständigkeitskonstante erklärt werden.13. Das Ausfallen von metallischem Silber in den D-Teilen der Sehnenfibrillen kann nicht mit dem photographischen Prozeß in Verbindung gebracht werden. Das gilt auch für die Bindegewebsversilberung nachGömöri.14. Die Silberorte in den D-Teilen lassen sich nur teilweise mit den bekannten Querstreifungsbildern nach Osmium- oder Phosphorwolframsäurefixierung in Beziehung setzen.

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Summary 1. After treatment of native or formalin-fixed tendon fibrils with an ammoniacal silver solution, silver particles are deposited in the D-bands of the fibrils. In the native fibrils these are arranged in one, two or three striae per band, but after formalin fixation they lie in one stria only.2. No external reducing agent is necessary for the production of the particles.3. Pretreatment of native fibrils with neutral salt solution or with trypsin has no effect on subsequent silvering. On the other hand, silvering is abolished by treatment with hyaluronidase, citrate buffer or periodate and also by acetylation and bisulphite.4. Formalin-fixed fibrils show the silvering effect after all these procedures except acetylation or bisulphite treatment.5. It is postulated that silvering of the D-bands is due to reducing substances which can precipitate silver from ammonical solutions and that formalin influences the process by the production of cross linkages.

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Mit 6 Textabbildungen

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Durchgeführt mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Formalin fixation strongly influences biomechanical properties of the spine

As fresh human cadaveric spine specimens for in vitro testing are hard to obtain and carry a potential risk of infection, the possibility of using embalmed spine specimens has been considered. The cross-linking effect of formalin fixation, however, raises uncertainties regarding the biomechanical likeness of preserved specimens. They have been reported to be stiffer, but no quantitative data exist.

The purpose of this study was to determine the biomechanical differences between fresh and formalin-fixed spine specimens, using L1–2 motion segments from six 16-week-old calf spines. The range of motion and neutral zone were determined in flexion-/extension, left/right axial rotation, and right/left lateral bending.

The range of motion decreased in the formalin fixed specimens by as much as 80%, and the neutral zone by as much as 96%. The results of this study therefore imply that, for biomechanical testing, formalin-fixed specimens are not representative of the in vivo conditions.     

Octopamine-like immunoreactive neurones in locust genital abdominal ganglia

Using a well characterized anti-serum, the distribution of octopamine-like immunoreactive neurones is described in the locust seventh abdominal (A7) and terminal ganglia (TG), which are associated with genital organs. Apart from 4 paired ventral somata occasionally observed in the TG, all labelled cells could be identified as efferent dorsal- and ventral unpaired median (DUM/VUM) neurones by virtue of the characteristic large size and position of their somata, projections of their primary neurites in DUM-cell tracts, and bifurcating axons which arise from dorsal T-junctions and enter peripheral nerves. For the examined ganglia our data indicate that the whole population of efferent DUM and VUM-cells, defined here as progeny of the segment specific unpaired median neuroblast with peripheral axons, are octopaminergic, and that equal numbers of these cells occur in both sexes: 8 in A7 and 11 in TG. Sex-specific differences are probably restricted to the axonal projections of 5 octopamine-like immunoreactive DUM-somata in A7, and 5 in TG, which in females project into their segment specific sternal nerves, but in males into the genital nerve of the TG. Numerous intersegmentally projecting octopamine-like immunoreactive fibres traverse both ganglia. The majority probably stem from previously described octopamine-like immunoreactive neurones in the thoracic and suboesophageal ganglia.     

Glycosylation in cardenolide biosynthesis

The glycosylation and deglycosylation of cardiac glycosides was investigated using cell suspension cultures and shoot cultures, both established from Digitalis lanata EHRH. plants, as well as isolated enzymes. Shoots were capable of glucosylating digitoxigenin, evatromonoside, digiproside, glucodigitoxigenin and digitoxin. Suspension cultured Digitalis cells glucosylated all the substrates mentioned but digiproside, whereas the UDP-glucosedependent cardinolide glucosyltransferase isolated from that source did not accept digitoxigenin and digiproside as substrates. It is concluded that at least three different glucosyltransferases are involved in cardiac glycoside formation in Digitalis. Similar experiments carried out with glucosylated cardenolides which were administered to cultured cells, shoots and a cardenolide -glucosidase isolated from young leaves revealed that at least two different glucosidases occur in Digitalis lanata, albeit in different tissues or during different phases of development. The biotransformation of glucoevatromonoside was investigated using unlabelled compound and [¹⁴C-glucose]-glucoevatromonoside synthesized enzymatically. After 7 d of incubation almost no radioactivity could be recovered from the cardenolide fraction, indicating that the terminal glucose of glucoevatromonoside was now incorporated into volatile, hydrophilic and insoluble compounds. Since, on the other hand, large amounts of cardenolides were found in the experiments with unlabelled glucoevatromonoside it is assumed that steady state or pool size regulation is achieved by the coordinated action of a cardenolide glucosidase and a glucosyltransferase.Abbreviations Acdox D-acetyldigitoxose - dgen digoxigenin - dox D-digitoxose - dten digitoxigenin - dtl D-digitalose - fuc D-fucose - gten gitoxigenin - qun D-quinovose - CGH cardenolide 16-O-glucohydrolase - DFT UDP-fucose:digitoxigenin 3-O-fucosyltransferase - DGT UDP-glucose:Digitoxin 16-O-glucosyltransferase - DQT UDP-quinovose:digitoxigenin 3-O-quinovosyltransferase     

Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 1341. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis, cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harbonring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.     

Motor learning

Bilateral damage of the medial temporal lobe system prevents the formation of new declarative memories but leaves intact knowledge that was acquired before damage. For motor learning, no structure has been identified that plays a comparable role for the consolidation of motor memories. The deficits of motor learning are focal and show a similar allocation to the various of motor learning are focal and show a similar allocation to the various sensorimotor subsystems, as do the corresponding non-mnemonic functions. The involvement of sensorimotor circuitries changes during motor learning so that association areas are preferentially activated in the early stages, and cerebello- and striato-motor-cortical loops are preferentially activated in the late stages of motor learning. Recent neuroanatomical and neurophysiological findings on the effects of brain lesions in human and non-human primates are discussed.     

Homologous npdGI Genes in 2,4-Dinitrophenol- and 4-Nitrophenol-Degrading Rhodococcus spp.

Rhodococcus (opacus) erythropolis HL PM-1 grows on 2,4,6-trinitrophenol or 2,4-dinitrophenol (2,4-DNP) as a sole nitrogen source. The NADPH-dependent F₄₂₀ reductase (NDFR; encoded by npdG) and the hydride transferase II (HTII; encoded by npdI) of the strain were previously shown to convert both nitrophenols to their respective hydride Meisenheimer complexes. In the present study, npdG and npdI were amplified from six 2,4-DNP degrading Rhodococcus spp. The genes showed sequence similarities of 86 to 99% to the respective npd genes of strain HL PM-1. Heterologous expression of the npdG and npdI genes showed that they were involved in 2,4-DNP degradation. Sequence analyses of both the NDFRs and the HTIIs revealed conserved domains which may be involved in binding of NADPH or F₄₂₀. Phylogenetic analyses of the NDFRs showed that they represent a new group in the family of F₄₂₀-dependent NADPH reductases. Phylogenetic analyses of the HTIIs revealed that they form an additional group in the family of F₄₂₀-dependent glucose-6-phosphate dehydrogenases and F₄₂₀-dependent N⁵,N¹⁰-methylenetetrahydromethanopterin reductases. Thus, the NDFRs and the HTIIs may each represent a novel group of F₄₂₀-dependent enzymes involved in catabolism.