Production and processing of a 59-kilodalton exochitinase during growth of Streptomyces lividans carrying pCHIO12 in soil microcosms amended with crab or fungal chitin.

Streptomyces lividans (pCHIO12), which carries the previously cloned Streptomyces olivaceoviridis exo-chiO1 gene on a multicopy vector, secretes a 59-kDa exochitinase, consisting of a catalytic domain (40 kDa), a central fibronectin type III-like module, and a chitin-binding domain (12 kDa). The propagation rate of S. lividans (pCHIO12) was higher in soil microcosms amended with fungal mycelia than in those containing crab chitin. Comparative biochemical and immunological studies allowed the following conclusions to be drawn. Within soil microcosm systems amended with crab shell chitin or chitin-containing Aspergillus proliferans mycelia, the strain expressed the clones exo-chiO1 gene and produced high quantities of a 59-kDa exochitinase. The enzyme was preferentially attached via its binding domain to the pellet from soil or liquid cultures. In contrast, truncated forms of 47, 40, and 25 kDa could be easily extracted from soil. The relative proportions of the 59-kDa enzyme and its truncated forms varied depending on the source of chitin and differed in soil and in liquid cultures.     

Biochemical and Electron Microscopic Studies of the Streptomyces reticuli Cellulase (Avicelase) in Its Mycelium-Associated and Extracellular Forms

Streptomyces reticuli is able to grow efficiently with crystalline cellulose (Avicel) as the sole carbon source. Cultivation in the presence of the nonionic detergent Tween 80 at a concentration of 0.1% led to a 10-fold increase in extracellular cellulolytic activity. Under these conditions, one single 82-kDa cellulase (Avicelase) capable of degrading crystalline and soluble cellulose as well as cellodextrins and p-nitrophenylcellobioside was purified to apparent homogeneity by a procedure which consisted of two consecutive anion-exchange chromatographies followed by chromatofocusing. Aggregation, which was a major problem during protein purification, could be avoided by including Triton X-100 at a concentration of 0.1% in every chromatographic step. The Avicelase was identified in extracellular and mycelium-associated forms, the latter of which could be released efficiently by nonionic detergents. In addition, a 42-kDa truncated form retaining cellulolytic activity was identified which had been generated from the 82-kDa enzyme by a protease. Antibodies raised against the mycelium-associated Avicelase reacted with the 42-kDa derivative and the extracellular form. The mycelial association of the enzyme was confirmed by immunofluorescence and immunoelectron microscopies.     

Simulation of the nutrient supply in fracture healing

The healing process for bone fractures is sensitive to mechanical stability and blood supply at the fracture site. Most currently available mechanobiological algorithms of bone healing are based solely on mechanical stimuli, while the explicit analysis of revascularization and its influences on the healing process have not been thoroughly investigated in the literature. In this paper, revascularization was described by two separate processes: angiogenesis and nutrition supply. The mathematical models for angiogenesis and nutrition supply have been proposed and integrated into an existing fuzzy algorithm of fracture healing. The computational algorithm of fracture healing, consisting of stress analysis, analyses of angiogenesis and nutrient supply, and tissue differentiation, has been tested on and compared with animal experimental results published previously. The simulation results showed that, for a small and medium-sized fracture gap, the nutrient supply is sufficient for bone healing, for a large fracture gap, non-union may be induced either by deficient nutrient supply or inadequate mechanical conditions. The comparisons with experimental results demonstrated that the improved computational algorithm is able to simulate a broad spectrum of fracture healing cases and to predict and explain delayed unions and non-union induced by large gap sizes and different mechanical conditions. The new algorithm will allow the simulation of more realistic clinical fracture healing cases with various fracture gaps and geometries and may be helpful to optimise implants and methods for fracture fixation.     

Salivation into sieve elements in relation to plant chemistry: the case of the aphid Sitobion fragariae and the wheat, Triticum aestivum

Extended sieve element salivation (E1 waveform in the electrical penetration graph) is a characteristic activity during early sieve element punctures, particularly in resistant plants. In order to explore a chemically-mediated mechanism of resistance associated with sieve element salivation, we compared the pattern of feeding behaviour of the aphid, Sitobion fragariae (Walker), on two cultivars of the wheat Triticum aestivum L., with different concentrations of hydroxamic acids (Hx). During 24 h of electronic monitoring, aphids dedicated over 50% of the total time to phloem ingestion from the sieve elements. Total time allocated to E1 in the experiment, time to first E1 within the experiment, time allocated to E1 before a sustained phloem ingestion (E2) and the contribution of sieve element salivation to the phloem phase (E1/[E1+E2]) were significantly higher in the high-Hx cultivar. The increased salivation in plants with higher contents of Hx suggests the existence, at least in this system, of a chemically-mediated sieve element constraint.     

Mikrospektrofluorometrische Untersuchungen der biogenen Amine im Glomus caroticum des Kaninchens nach Reserpin- und PCPA-Applikation

Zusammenfassung Die Glomera carotica von 12 Kaninchen wurden nach viertägiger Applikation von Reserpin (2 mg/kg KG; i.p.) (I), Parachlorphenylalanin-methylester-hydrochlorid (PCPA; 100 mg/kg KG; i.p.) (II) und physiologischer NaCl-Lösung (III) mit der Falckschen Fluoreszenzmethode aufgearbeitet und cytospektrofluorometrisch (Mikrospektrograph Leitz) ausgewertet.An Hand der Fluoreszenzspektren der spezifischen Glomuszellen (Typ I) ließ sich eine Depletion der Catecholamine und des Serotonins verfolgen: hiernach senkt PCPA vor allem das Serotonin, Reserpin hingegen die Catechol- und Indolamine in etwa gleichem Umfang.Durch Zerlegung einiger Gesamtspektren in die Anteile für Catecholamine und das Serotonin mit Hilfe eines Iterationsverfahrens der nichtlinearen Regression konnte gezeigt werden, daß nur eine geringfügige Beeinflussung der Catecholamin-Peakhöhe durch das Serotonin vorliegt. Über die Messung der Peakhöhe im Catecholaminbereich der Gesamtspektren konnte daher die Reduktion der Catecholamine auf 64±22% durch PCPA und auf 13±4% durch Reserpin statistisch gesichert werden.Die Befunde werden im Zusammenhang mit den möglichen Wirkungsmechanismen der applizierten Substanzen diskutiert. Eine ausführliche tabellarische Zusammenstellung der bisherigen Angaben über Gehalt und Zusammensetzung der biogenen Amine des Glomus caroticum ergänzt diese Diskussion.

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Microspectrofluorometric investigation of biogenic amines in the rabbit glomus caroticum after reserpine and PCPA treatment

Summary After the administration for 4 days of (I) reserpine (2 mg/kg body weight daily, i.p.), (II) p-chlorophenylalanine-methylester-hydrochloride (PCPA; 100 mg/kg body weight daily; i.p.) and III physiological NaCl solution, the carotid bodies of 12 rabbits were examined using Falck's fluorescence technique and evaluated cytospectrofluorometrically. Fluorescence spectra of the type I specific glomus cells revealed that a depletion of catecholamines and serotonin took place: PCPA lowered mainly serotonin, whereas reserpine depleted catechol- and indolamines about equally.It was shown by splitting of a few combined spectra with an iteration procedure of nonlinear regression, that serotonin affected slightly the height of the catecholamine peak. Thus, measuring the peak height in the catecholamine region of the total spectrum, the reduction of catecholamines to 64±22% by PCPA and to 13±4% by reserpine was statistically significant.These data are discussed in relation to possible mechanisms of action of the test substances and related to the content and nature of the biogenic amines in the carotid body.

Für technische Hilfe danken wir Fräulein B. Remki und Frau K. Wilp.