Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Responses of foliage‐living spider assemblage composition and traits to a climatic gradient in Themeda grasslands

For speciose, but poorly known groups, such as terrestrial arthropods, functional traits present a potential avenue to assist in predicting responses to environmental change. Species turnover is common along environmental gradients, but it is unclear how this is reflected in species traits. Community‐level change in arthropod traits, other than body size, has rarely been explored across spatial scales comparable to those examined here. We hypothesized that the composition and morphological traits of spider assemblages would differ across a gradient of climate and habitat structure. We examined foliage‐living spider assemblages associated with Themeda triandra grasslands along a 900 km climatic gradient in south‐eastern Australia. We used sweep‐netting to collect T. triandra‐associated spiders and counted juveniles and identified adults. We also measured morphological traits of adult spiders and noted their hunting mode. Associations with measures of habitat structure were less consistent than relationships with climate. Both juvenile and adult spiders were more abundant in warmer sites, although species richness was not affected by temperature. We found distinct turnover in species composition along the climatic gradient, with hunting spiders, particularly crab spiders (Thomisidae), making up a greater proportion of assemblages in warmer climates. A range of traits of spiders correlated with the climatic gradient. For example, larger spider species and species that were active hunters were more common in warmer climates. Changes in morphological traits across species, rather than within species drove the morphology‐climate relationship. Strong climate‐trait correlations suggest that it may be possible to predict changes in functional traits of assemblages in response to anthropogenic disturbances such as climate change.     

Homologous and Heterologous Overexpression in Clostridium acetobutylicum and Characterization of Purified Clostridial and Algal Fe-Only Hydrogenases with High Specific Activities

Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag II-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 μmol H₂ min⁻¹ mg⁻¹, while HydA from C. acetobutylicum (HydA_Ca) shows the highest activity (5,522 μmol H₂ min⁻¹ mg⁻¹) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydA_Ca protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.     

Cloning and over-expression in Escherichia coli of the gene encoding NADPH group III alcohol dehydrogenase from Thermococcus hydrothermalis. Characterization and comparison of the native and the recombinant enzymes.

A NADP-dependent group III alcohol dehydrogenase (ADH) was purified from the hyperthermophilic strictly anaerobic archaeon Thermococcus hydrothermalis, which grows at an optimum temperature of 85 degrees C and an optimum pH of 6. The gene encoding this enzyme was cloned, sequenced, and over-expressed in Escherichia coli. The recombinant enzyme was purified, characterized and compared with the native form of the enzyme. The enzyme structure is pH-dependent, being a 197-kDa tetramer (subunit of 45 kDa) at pH 10.5, the pH optimum for alcohol oxidation, and a 80.5-kDa dimer at pH 7.5, the pH optimum for aldehyde reduction. The kinetic parameters of the enzyme show that the affinity of the enzyme is greater for the aldehyde substrate and NADPH cofactor, suggesting that the dimeric form of the enzyme is probably the active form in vivo. The ADH of T. hydrothermalis oxidizes a series of primary aliphatic and aromatic alcohols preferentially from C2 to C8 but is also active towards methanol and glycerol and stereospecific for monoterpenes. T. hydrothermalis ADH is the first Thermococcale ADH to be cloned and overproduced in a mesophilic heterologous expression system, and the recombinant and the native forms have identical main characteristics.     

Studien am netz der kreuzspinne (Aranea Diadema L.)

Zusammenfassung Die Herstellung des Kreuzspinnennetzes beginnt oft mit der Anfertigung eines Y-förmigen Fadengerüstes, dessen Zentrum den Mittelpunkt des Netzes bezeichinet. In diesel Gerüst werden Rahmenf=äden und Radialfäden eingespannt. Die Rahmenfäden entstehen dabei in einer einzigen geschlossenen Bewegungsfolge zusammen mit je einem Radialfaden. Auch von einem meter oder weniger vertikalen Faden aus kann dal Netz angelegt werden. Rahmenfäden und Radialfaden werden dann allmählich nach allen Seiten hin angesetzt. Die Reihenfolge der Radialfäden ist von zwei Regeln beherrscht. Erstens gilt die schon bekannte Regel der alternierenden Herstellung nach immer wieder verschiedenen Richtungen. Nach der zweiten Regel werden die auf die allerersten Radialfäden folgenden Speichen von oben nach unten im Abstand eines Sektors an jene erste angesetzt. Das erklärt gewisse Unregelmäßigkeiten in den Speichenabständen. Diese Abstände, die Sektorenwinkel, muß die Spinne als solche wahrnehmen. Das geschieht wohl kinästhetisch, wenn das Tier bei der Herstellung der Radialfäden an den Grenzen der Sektoren entlang läuft. Die Methoden des Speichenziehens sind bei den verschiedenen Radnetzspinnen verschieden; es werden vier Typen unterschieden. Experimente mit Verlagerung von Speichen zeigen, daß die Spinne einen solchen Radialfaden an einer bis dahin noch leeren Stelle wie einen von ihr selbst gezogenen annimmt. Andere Versuche lehren, daß die Spinne die Abstände der Hilfsspirale durch Abtasten des jeweils vorigen Umgangs bestimmt, daß sic aber gleichwohl auch den Kurvenverlauf als solchen unabhängig von der Richtlinie wahrnehmen kann. Schließlich ergeben Experimente, bei denen das in einen Holzrahmen eingebaute Netz während der Herstellung in seiner Ebene um 90° gedreht wind, enge Beziehungen des Netzes zum Schwerefeld als Bezugssystem.     

The secE gene of Helicobacter pylori

Despite extensive annotation by two independent teams, the Helicobacter pylori genome appeared to lack a complete secretion machinery. The use of clinical isolates to substantiate in silico annotation is used here to identify the missing secE component of the major secretion machinery of Helicobacter pylori.