A new, sensitive method for enzyme kinetic studies of scarce glucosides

The maize β-glucosidase Zm-p60.1 is important for the regulation of plant development through its role in the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. Enzyme kinetics studies using these scarce substrates close to physiological concentrations are difficult due to two reasons: (a) Available methods are mainly suited for end-point kinetics. (b) These methods are not sufficiently sensitive when using scarce glucoside substrates.We developed a glucose assay using a system comprising three enzymes β-glucosidase, glucose oxidase and horseradish peroxidase, with the new substrate N-acetyl-3,7-dihydroxyphenoxazine-Amplex Ultra Red reagent (Molecular Probes). A calibration curve was constructed for resorufin and validation was carried out by comparing our method with the standard spectrophotometric method using p-nitrophenyl-β-d-glucopyranoside. In comparison with the other methods, this method is more sensitive, precise and accurate. The assay is rapid and hence suited for continuous kinetics, it is readily adapted to suit automated procedures, and potential applications include its use in studying the physiological role(s) of enzymes that cleave scarce glucoside substrates.     

Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na i + /K i + -independent signaling

Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na⁺,K⁺-ATPase α-subunit but not the result of inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 μM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na⁺]_i/[K⁺]_i ratio triggered by Na⁺,K⁺-ATPase inhibition in K⁺-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Na_i⁺,K_i⁺—independent activation of p38 MAPK.     

The drivers of avian‐haemosporidian prevalence in tropical lowland forests of New Guinea in three dimensions

Haemosporidians are among the most common parasites of birds and often negatively impact host fitness. A multitude of biotic and abiotic factors influence these associations, but the magnitude of these factors can differ by spatial scales (i.e., local, regional and global). Consequently, to better understand global and regional drivers of avian‐haemosporidian associations, it is key to investigate these associations at smaller (local) spatial scales. Thus, here, we explore the effect of abiotic variables (e.g., temperature, forest structure, and anthropogenic disturbances) on haemosporidian prevalence and host–parasite networks on a horizontal spatial scale, comparing four fragmented forests and five localities within a continuous forest in Papua New Guinea. Additionally, we investigate if prevalence and host–parasite networks differ between the canopy and the understory (vertical stratification) in one forest patch. We found that the majority of Haemosporidian infections were caused by the genus Haemoproteus and that avian‐haemosporidian networks were more specialized in continuous forests. At the community level, only forest greenness was negatively associated with Haemoproteus infections, while the effects of abiotic variables on parasite prevalence differed between bird species. Haemoproteus prevalence levels were significantly higher in the canopy, and an opposite trend was observed for Plasmodium. This implies that birds experience distinct parasite pressures depending on the stratum they inhabit, likely driven by vector community differences. These three‐dimensional spatial analyses of avian‐haemosporidians at horizontal and vertical scales suggest that the effect of abiotic variables on haemosporidian infections are species specific, so that factors influencing community‐level infections are primarily driven by host community composition.     

Gallbladder carcinoma cells in cerebrospinal fluid as the first manifestation of a tumor. A case report

BACKGROUND: Meningeal carcinomatosis (MC) rarely occurs as the first evidence of a tumor. In such cases cytology of the cerebrospinal fluid is crucial to the diagnosis. The most frequent primary MCs are lung and breast cancers. MC from a gallbladder carcinoma is uncommon. CASE: A 58-year-old woman presented with paroxysmal headaches, seizures and coma. Analysis of the cerebrospinal fluid revealed carcinoma cells and a low protein concentration. Only postmortem examination discovered gallbladder adenocarcinoma to be the source of the tumor cells. CONCLUSION: A case with the onset of MC secondary to rare mucinous adenocarcinoma of the gallbladder is presented. Cytology of the cerebrospinal fluid was the only examination that uncovered malignancy. Nine similar cases were found in the literature. Low cerebrospinal fluid protein seems to be of diagnostic value.     

Coenzyme Q releases the inhibitory effect of free fatty acids on mitochondrial glycerophosphate dehydrogenase

Data presented in this paper show that the size of the endogenous coenzyme Q (CoQ) pool is not a limiting factor in the activation of mitochondrial glycerophosphate-dependent respiration by exogenous CoQ(3), since successive additions of succinate and NADH to brown adipose tissue mitochondria further increase the rate of oxygen uptake. Because the inhibition of glycerophosphate-dependent respiration by oleate was eliminated by added CoQ(3), our data indicate that the activating effect of CoQ(3) is related to the release of the inhibitory effect of endogenous free fatty acids (FFA). Both the inhibitory effect of FFA and the activating effect of CoQ(3) could be demonstrated only for glycerophosphate-dependent respiration, while succinate- or NADH-dependent respiration was not affected. The presented data suggest differences between mitochondrial glycerophosphate dehydrogenase and succinate or NADH dehydrogenases in the transfer of reducing equivalents to the CoQ pool.     

HER-2/neu mediated down-regulation of MHC class I antigen processing prevents CTL-mediated tumor recognition upon DNA vaccination in HLA-A2 transgenic mice

To study DNA vaccination directed against human HER-2 in the HHD mouse Tg strain, we created a novel HER-2-expressing syngeneic tumor transplantation model. We found that a DNA vaccine encoding the full length HER-2 DNA protected HHD mice from HER-2⁺ tumor challenge by a CTL independent mechanism. A more efficient approach to induce HLA-A2 restricted CTLs, through immunization with a multi-epitope DNA vaccine expressing the HLA-A2 restricted HER-2 369–377, 435–443 and 689–697 epitopes, resulted in high numbers of peptide specific T cells but failed to induce tumor protection. Subsequently we discovered that HER-2 transfected tumor cells down-regulated MHC class I antigen expression and exhibited a series of defects in the antigen processing pathway which impaired the capacity to produce and display MHC class I peptide-ligands to specific CTLs. Our data demonstrate that HER-2 transfection is associated with defects in the MHC class I presentation pathway, which may be the underlying mechanism behind the inability of CTLs to recognize tumors in this HLA-A2 transgenic model. As defective MHC class I presentation may be a common characteristic of HER-2 expressing tumors, vaccines targeting HER-2 should aim at inducing an integrated immune response where also CD4⁺ T cells and antibodies are important components. S. Vertuani and C. Triulzi contributed equally to this work.     

Excitation-contraction coupling in resistance mesenteric arteries: Evidence for NKCC1-mediated pathway

Bumetanide and other high-ceiling diuretics (HCD) attenuate myogenic tone and contractions of vascular smooth muscle cells (VSMC) triggered by diverse stimuli. HCD outcome may be mediated by their interaction with NKCC1, the only isoform of Na⁺, K⁺, 2Cl⁻ cotransporter expressed in VSMC as well as with targets distinct from this carrier. To examine these hypotheses, we compared the effect of bumetanide on contractions of mesenteric arteries from wild-type and NKCC1 knockout mice. In mesenteric arteries from wild-type controls, 100 μM bumetanide evoked a decrease of up to 4-fold in myogenic tone and contractions triggered by modest [K⁺]_o-induced depolarization, phenylephrine and UTP. These actions of bumetanide were preserved after inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester, but were absent in mesenteric arteries from NKCC1^-/- mice. The data show that bumetanide inhibits VSMC contractile responses via its interaction with NKCC1 and independently of nitric oxide production by endothelial cells.