Substrate specificity of α‐humulene synthase from Zingiber zerumbet Smith and determination of kinetic constants by a spectrophotometric assay

Terpene synthases are the key enzymes in terpene biosynthesis that provide a structurally complex and highly diverse product spectrum. A suitable and reliable analytical assay is indispensable to measure terpene synthase activity accurately and precisely. In this study, a malachite green assay (MG) was adapted to rapidly assay terpene synthase activity and was validated in comparison to an already established gas chromatography assay. A linear correlation between both assays was observed. Kinetic properties for the previously described sesquiterpene synthase α‐humulene synthase (HUM) from Zingiber zerumbet Smith were investigated for the bioconversion of the monoterpene precursors geranyl pyrophosphate (2E‐GPP) and neryl pyrophosphate (2Z‐NPP) as well as for the sesquiterpene precursor farnesyl pyrophosphate (2E,6E‐FPP). Also, gas chromatography mass spectrometry (GS‐MS) was carried out to identify the products of the bioconversion of (2E)‐GPP and (2Z)‐NPP.     

Wildlife detector dogs and camera traps: a comparison of techniques for detecting feral cats

A major challenge in controlling overabundant wildlife is monitoring their populations, particularly as they decline to very low density. Camera traps and wildlife detector dogs are increasingly being used for this purpose. We compared the cost-effectiveness of these two approaches for detecting feral cats (Felis catus) on two pastoral properties in Hawke's Bay, North Island, New Zealand. One property was subject to intensive pest removal, while the other had no recent history of pest control. Camera traps and wildlife detector dogs detected cats at similar rates at both sites. The operating costs of each method were also comparable. We identify a number of advantages and disadvantages of each technique, and suggest priorities for further research.     

Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.     

Synthetic access to spacer-linked 3,6-diamino-2,3,6-trideoxy-alpha-D-glucopyranosides--potential aminoglycoside mimics for the inhibition of the HIV-1 TAR-RNA/Tat-peptide complex

The synthesis of spacer-linked neoaminoglycoside 5 is described. Key steps of the synthesis are the introduction of nitrogen functionalities at C-3 and C-6 and the olefin cross metathesis of allyl glycoside 16. Although it is known that Grubbs catalysts tolerate nitrogen functionalities, difficulties were encountered in the cross metathesis reaction. Factors that govern this dimerization are the steric and electronic demands of the catalyst and the substrate. Preliminary biological evaluation of homodimer 5, by studying the inhibition of HIV-1 TAR-RNA/Tat-peptide complex using a method based on fluorescence titration, revealed an inhibitory effect of 5.     

Synthesis and biological evaluation of a polysialic acid-based hydrogel as enzymatically degradable scaffold material for tissue engineering

Restorative medicine has a constant need for improved scaffold materials. Degradable biopolymers often suffer from uncontrolled chemical or enzymatic hydrolysis by the host. The need for a second surgery on the other hand is a major drawback for nondegradable scaffold materials. In this paper we report the design and synthesis of a novel polysialic acid-based hydrogel with promising properties. Hydrogel synthesis was optimized and enzymatic degradation was studied using a phage-born endosialidase. After addition of endosialidase, hydrogels readily degraded depending on the amount of initially used cross-linker within 2 to 11 days. This polysialic acid hydrogel is not cytotoxic, completely stable under physiological conditions, and could be evaluated as growth support for PC12 cells. Here, additional coating with collagen I, poly-L-lysine or matrigel is mandatory to improve the properties of the material.     

Bacterial networking

A report of the ESF-EMBO Symposium Bacterial Networks (BacNet08), Sant Feliu de Guixols, Spain, 13-18 September 2008.     

Complement activation by phospholipids: the interplay of factor H and C1q

Complement proteins in blood recognize charged particles. The anionic phospholipid (aPL) cardiolipin binds both complement proteins C1q and factor H. C1q is an activator of the complement classical pathway, while factor H is an inhibitor of the alternative pathway. To examine opposing effects of C1q and factor H on complement activation by aPL, we surveyed C1q and factor H binding, and complement activation by aPL, either coated on microtitre plates or in liposomes. Both C1q and factor H bound to all aPL tested, and competed directly with each other for binding. All the aPL activated the complement classical pathway, but negligibly the alternative pathway, consistent with accepted roles of C1q and factor H. However, in this system, factor H, by competing directly with C1q for binding to aPL, acts as a direct regulator of the complement classical pathway. This regulatory mechanism is distinct from its action on the alternative pathway. Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range. Thus factor H, which is regarded as a down-regulator only of the alternative pathway, has a distinct role in downregulating activation of the classical complement pathway by aPL. A factor H homologue, β2-glycoprotein-1, also strongly inhibits C1q binding to cardiolipin. Recombinant globular domains of C1q A, B and C chains bound aPL similarly to native C1q, confirming that C1q binds aPL via its globular heads.