The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c₅₅₂, is similar to a number of c-type cytochromes from the α-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c₅₅₂ revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

Cell death in leukemia: passenger protein regulation by topoisomerase inhibitors

Etoposide is a potent inducer of mitotic catastrophe; a type of cell death resulting from aberrant mitosis. It is important in p53 negative cells where p53 dependent apoptosis and events at the G1 and G2 cell cycle checkpoints are compromised. Passenger proteins regulate many aspects of mitosis and siRNA interference or direct inhibition of Aurora B kinase results in mitotic catastrophe. However, there is little available data of clinical relevance in leukaemia models. Here, in p53 negative K562 myeloid leukemia cells, etoposide-induced mitotic catastrophe is shown to be time and/or concentration dependent. Survivin and Aurora remained bound to chromosomes. Survivin and Aurora were also associated with Cdk1 and were shown to form complexes, which in pull down experiments, included INCENP. There was no evidence of Aurora B kinase suppression. These data suggests etoposide will complement Aurora B kinase inhibitors currently in clinical trials for cancer.     

The role of tryptophan residues in the function and stability of the mechanosensitive channel MscS from Escherichia coli

Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels. Three Trp residues occur naturally in the Escherichia coli MscS (MscS-Ec) protein: W16 lies in the periplasm, immediately before the first transmembrane span (TM1), whereas W240 and W251 lie at the subunit interfaces that create the cytoplasmic vestibule portals. The role of these residues in MscS function and stability were investigated using site-directed mutagenesis. Functional channels with altered properties were created when any of the Trp residues were replaced by another amino acid, with the greatest retention of function associated with phenylalanine (Phe) substitutions. Analysis of the fluorescence properties of purified mutant MscS proteins containing single Trp residues revealed that W16 and W251 are relatively inaccessible, whereas W240 is accessible to quenching agents. The data point to a significant role for W16 in the gating of MscS, and an essential role for W240 in MscS oligomer stability.     

Cardiac effects of postconditioning depend critically on the duration of index ischemia

Postconditioning (POC) is known as the phenomenon whereby brief intermittent ischemia applied at the onset of reperfusion following index ischemia limits myocardial infarct size. Whereas there is evidence that the algorithm of the POC stimulus is an important determinant of the protective efficacy, the importance of the duration of index ischemia on the outcome of the effects of POC has received little attention. Pentobarbital sodium-anesthetized Wistar rats were therefore subjected to index ischemia produced by coronary artery occlusions (CAO) of varying duration (15-120 min) followed by reperfusion, without or with postconditioning produced by three cycles of 30-s reperfusion and reocclusion (3POC30). 3POC30 limited infarct size produced by 45-min CAO (CAO45) from 45 +/- 3% to 31 +/- 5%, and CAO60 from 60 +/- 3% to 47 +/- 6% (both P < or = 0.05). In contrast, 3POC30 increased infarct size produced by CAO15 from 3 +/- 1% to 19 +/- 6% and CAO30 from 36 +/- 6 to 48 +/- 4% (both P < or = 0.05). This deleterious effect of 3POC30 was not stimulus sensitive because postconditioning with 3POC5 and 3POC15 after CAO30 also increased infarct size. The cardioprotection by 3POC30 after CAO60 was accompanied by an increased stimulation of Akt phosphorylation at 7 min of reperfusion and a 36% lower superoxide production, measured by dihydroethidium fluorescence, after 2 h of reperfusion. Consistent with these results, cardioprotection by 3POC30 was abolished by phosphatidylinositol-3-OH-kinase inhibition, as well as nitric oxide (NO) synthase inhibition. The deleterious effect of 3POC30 after CAO15 was accompanied by an increased superoxide production with no change in Akt phosphorylation and was not affected by NO synthase inhibition. In conclusion, the effect of cardiac POC depends critically on the duration of the index ischemia and can be either beneficial or detrimental. These paradoxical effects of POC may be related to the divergent effects on Akt phosphorylation and superoxide production.     

Liver glyconeogenesis: a pathway to cope with postprandial amino acid excess in high-protein fed rats?

This paper provides molecular evidence for a liver glyconeogenic pathway, that is, a concomitant activation of hepatic gluconeogenesis and glycogenesis, which could participate in the mechanisms that cope with amino acid excess in high-protein (HP) fed rats. This evidence is based on the concomitant upregulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression, downregulation of glucose 6-phosphatase catalytic subunit (G6PC1) gene expression, an absence of glucose release from isolated hepatocytes and restored hepatic glycogen stores in the fed state in HP fed rats. These effects are mainly due to the ability of high physiological concentrations of portal blood amino acids to counteract glucagon-induced liver G6PC1 but not PEPCK gene expression. These results agree with the idea that the metabolic pathway involved in glycogen synthesis is dependent upon the pattern of nutrient availability. This nonoxidative glyconeogenic disposal pathway of gluconeogenic substrates copes with amino excess and participates in adjusting both amino acid and glucose homeostasis. In addition, the pattern of PEPCK and G6PC1 gene expression provides evidence that neither the kidney nor the small intestine participated in gluconeogenic glucose production under our experimental conditions. Moreover, the main glucose-6-phosphatase (G6Pase) isoform expressed in the small intestine is the ubiquitous isoform of G6Pase (G6PC3) rather than the G6PC1 isoform expressed in gluconeogenic organs.     

Plant cytokinesis requires de novo secretory trafficking but not endocytosis

During plant cytokinesis membrane vesicles are efficiently delivered to the cell-division plane, where they fuse with one another to form a laterally expanding cell plate. These membrane vesicles were generally believed to originate from Golgi stacks. Recently, however, it was proposed that endocytosis contributes substantially to cell-plate formation. To determine the relative contributions of secretory and endocytic traffic to cytokinesis, we specifically inhibited either or both trafficking pathways in Arabidopsis. Blocking traffic to the division plane after the two pathways had converged at the trans-Golgi network disrupted cytokinesis and resulted in binucleate cells, whereas impairment of endocytosis alone did not interfere with cytokinesis. By contrast, inhibiting ER-Golgi traffic by eliminating the relevant BFA-resistant ARF-GEF caused retention of newly synthesized proteins, such as the cytokinesis-specific syntaxin KNOLLE in the ER, and prevented the formation of the partitioning membrane. Our results suggest that during plant cytokinesis, unlike animal cytokinesis, protein secretion is absolutely essential, whereas endocytosis is not.     

Actin is not required for nanotubular protrusions of primary astrocytes grown on metal nano-lawn

We used sub-micron metal rod decorated surfaces, 'nano-lawn' structures, as a substrate to study cell-to-cell and cell-to-surface interactions of primary murine astrocytes. These cells form thin membranous tubes with diameters of less than 100 nm and a length of several microns, which make contact to neighboring cells and the substrate during differentiation. While membrane protrusions grow on top of the nano-lawn pillars, nuclei sink to the bottom of the substrate. We observed gondola-like structures along those tubes, suggestive of their function as transport vehicles. Elements of the cytoskeleton such as actin fibers are commonly believed to be essential for triggering the onset and growth of tubular membrane protrusions. A rope-pulling mechanism along actin fibers has recently been proposed to account for the transport or exchange of cellular material between cells. We present evidence for a complementary mechanism that promotes growth and stabilization of the observed tubular protrusions of cell membranes. This mechanism does not require active involvement of actin fibers as the formation of membrane protrusions could not be prevented by suppressing polymerization of actin by latrunculin B. Also theoretically, actin fibers are not essential for the growing and stability of nanotubes since curvature-driven self-assembly of interacting anisotropic raft elements is sufficient for the spontaneous formation of thin nano-tubular membrane protrusions.     

Optically responsive nanoparticle layers for the label-free analysis of biospecific interactions in array formats

A novel nanocomposite surface is prepared by coating surface-adsorbed dielectric colloidal particles with a contiguous layer of gold nanoparticles. The resulting surface shows pronounced optical extinction in reflection with the extinction peaks located in the UV–Vis and NIR region of the electromagnetic spectrum. The peak positions of these maxima change very sensitively with the adsorption of organic molecules onto the surface. For the adsorption of a monolayer of octadecanethiol, we observe a peak shift of 55 nm on average, which is about five times that of established label-free sensing methods based on propagating and localized surface plasmons. In a first proof-of-principle experiment, the interaction of peptides with specific antibodies has been detected without labeling by means of a fiber-optical set-up with microscopic lateral resolution. To avoid crosstalk in high-density arrays, the optically responsive surface areas can be locally separated on a micro- or even nanometer scale. Accordingly, the newly developed optically responsive surfaces are well suited for integration into high-density peptide or DNA arrays as demanded in genomics, proteomics, and biomedical research in general.     

HPLC-photodiode array detection analysis of curcuminoids in Curcuma species indigenous to Indonesia

An optimized HPLC method with photodiode array detection was developed and applied to analyse the curcuminoids curcumin, demethoxycurcumin, and bis-demethoxycurcumin in rhizomes of Curcuma mangga Val &. v. Zijp, C. heyneana Val. & v. Zijp, C. aeruginosa Roxb. and C. soloensis Val. (Zingiberaceae), indigenous to Indonesia. The method was validated with an isocratic system, a short run time of 10 min and a baseline separation. The curcuminoid content was 0.18-0.47% for C. mangga, 0.98-3.21% for C. heyneana, 0.02-0.03% for C. aeruginosa and 0.40% for C. soloensis.