Evaluation of CuO oxidation parameters for determining the source and stage of lignin degradation in soil

The composition of phenols and other aromatic compounds in organic and mineral soil horizons and their respective source vegetation from different climatic zones of the Canadian Prairies were analyzed using CuO oxidation and gas chromatography-mass spectrometry (GC-MS) to investigate the stage of lignin degradation. Parameters based on the CuO oxidation products were calculated for the soils and corresponding vegetation to determine the lignin sources and to monitor the lignin degradation. In addition to the widely used lignin monomer parameters, parameters resulting from lignin-derived phenolic dimers are used for the first time to assess lignin degradation in soils. The composition of lignin-derived phenols (S/V, C/V) in soil closely matches the composition observed in their respective source plants (grass, Aspen, Pine) reflecting the preservation of characteristic lignin patterns in soils. Degradation parameters based on lignin phenols and benzenes derived from tannins or other phenolic biomolecules indicate a progressive degradation from the vegetation to the soil horizons. In addition to commonly used lignin monomer indicators, parameters based on the lignin dimers are applied. Lignin degradation is found to be lowest in the Pine forest, intermediate in the grassland soils and highest in the Aspen-grassland transition soil. Degradation parameters based on non-lignin aromatic derivatives (3,5-dihydroxybenzoic acid, benzenepolycarboxylic acids) demonstrate a similar trend. The lignin from samples in the cooler climate (Black Chernozems) is observed to be more oxidized than in the soils from the warmer climate (Brown Chernozems) suggesting that abiotic processes may be in involved in the alteration of lignin and other phenolic biomolecules in soils. The results indicate that the comparative analysis of CuO oxidation products of soils and source vegetation is a valuable tool to assess the sources and degradation of lignin in soils.     

Divergent genetic and epigenetic post-zygotic isolation mechanisms in Mus and Peromyscus

Interspecific hybridization in the rodent genera Peromyscus and Mus results in abnormal placentation. In the Peromyscus interspecies hybrids, abnormal allelic interaction between an X-linked locus and the imprinted paternally expressed Peg3 locus was shown to cause the placental defects. In addition, loss-of-imprinting (LOI) of Peg3 was positively correlated with increased placental size. As in extreme cases this placental dysplasia constitutes a post-zygotic barrier against interspecies hybridization, this finding was the first direct proof that imprinted genes may be important in speciation and thus in evolution. In the Mus interspecies hybrids, a strong role of an X-linked locus in placental dysplasia has also been detected. However, here we show by backcross and allele specific expression analyses that neither LOI of Peg3 nor abnormal interactions between Peg3 and an X-linked locus are involved in generating placental dysplasia in Mus hybrids, although the placental phenotypes observed in the two genera seem to be identical. In contrast to this, another dysgenesis effect common to Peromyscus and Mus hybrids, altered foetal growth, is caused at least in part by the same X-chromosomal regions in both genera. These findings first underline the strong involvement of the X-chromosome in the genetics of speciation. Secondly, they indicate that disruption of epigenetic states, such as LOI, at specific loci may be involved in hybrid dysgenesis effects in one group, but not in another. Thus, we conclude that even in closely related groups divergent molecular mechanisms may be involved in the production of phenotypically similar post-zygotic barriers against hybridization.     

Two technetium-99m-labeled cholecystokinin-8 (CCK8) peptides for scintigraphic imaging of CCK receptors

A broad spectrum of radiolabeled peptides with high affinity for receptors expressed on tumor cells is currently under preclinical and clinical investigation for scintigraphic imaging and radionuclide therapy. The present paper evaluates two (99m)Tc-labeled forms of the C-terminal octapeptide of cholecystokinin (CCK8): sulfated (s)CCK8, with high affinity for CCK1 and CCK2 receptors, and nonsulfated (ns)CCK8, with high affinity for CCK2 receptors but low affinity for CCK1 receptors. Peptides were conjugated with the bifunctional chelator N-hydroxysuccinimidyl hydrazino niconitate (s-HYNIC). (99m)Tc-labeling, performed in the presence of nicotinic acid and tricine, was highly efficient (approximately 95%) and yielded products with a high specific activity (approximately 700 Ci/mmol) and good stability (approximately 5% release of radiolabel during 16 h incubation in phosphate buffered saline at 37 degrees C). Chinese hamster ovary cells stably expressing the CCK1 receptor (CHO-CCK1 cells) internalized approximately 3% of added (99m)Tc-sCCK8 per confluent well during 2 h at 37 degrees C. Internalization was effectively blocked by excess unlabeled sCCK8. CHO-CCK1 cells did not internalize (99m)Tc-nsCCK8. Displacement of (99m)Tc-sCCK8 and -nsCCK8 by unlabeled CCK-8 (performed at 0 degrees C to prevent internalization) revealed 50% inhibitory concentrations (IC(50)) of 8 nM and >1 microM, respectively. CHO-CCK2 cells internalized approximately 25% and approximately 5% of added (99m)Tc-sCCK8 and -nsCCK8, respectively. In both cases internalization was blocked by excess unlabeled peptide. IC(50) values for the displacement of (99m)Tc-sCCK8 and -nsCCK8 were 3 nM and 10 nM, respectively. CHO-CCK1 cell-derived tumors present in one flank of athymic mice accumulated 2.0% of injected (99m)Tc-sCCK8 per gram tissue at 1 h postinjection. This value decreased to 0.6% following coinjection with excess unlabeled peptide. Uptake of (99m)Tc-nsCCK8 was low (0.2%) and not did change by excess unlabeled peptide (0.3%). Accumulation of (99m)Tc-sCCK8 and -nsCCK8 by CHO-CCK2 cell-derived tumors (present in the other flank) amounted to 4.2% and 0.6%, respectively. In both cases uptake was significantly reduced by excess unlabeled peptide to 1.0% and 0.4% for sCCK8 and nsCCK8, respectively. Accumulation of (99m)Tc-sCCK8 was also high in pancreas (11.7%), stomach (2.0%), and kidney (2.1%), whereas uptake of (99m)Tc-nsCCK8 was high in stomach (0.7%) and kidney (1.4%). Both radiolabeled peptides showed a rapid blood clearance. In conclusion, these data show that CCK8 analogues can be efficiently labeled with (99m)Tc using s-HYNIC as chelator and nicotinic acid/tricine as coligand system without compromising receptor binding. Furthermore, the present study demonstrates that CCK1 tumors hardly accumulate (99m)Tc-nsCCK8, CCK2 tumors accumulate 2 times more (99m)Tc-sCCK8 than CCK1 tumors, and CCK2 tumors accumulate 15 times more (99m)Tc-sCCK8 than (99m)Tc-nsCCK8. Although accumulation in some nontarget organs was also higher with (99m)Tc-sCCK8, this may not reflect the human situation due to a different receptor expression pattern in humans as compared to mice. Therefore, further studies are warranted to investigate the possible use of (99m)Tc-sCCK8 for scintigraphic imaging of CCK receptor-positive tumors in humans.     

RCPAQAP First Combined Measurement and Reference Interval Survey

Reference intervals are commonly considered to allow for between-laboratory bias. The RCPAQAP Liquid Serum Chemistry Program has collected data on laboratory measurements as well as reference intervals. This allows assessment of the between-laboratory variation in results, reference intervals and the information transmitted by the combination of these factors. For the majority of common chemistry analytes, the between-laboratory variation in reference intervals is greater than the variation in results. Additionally the reference interval variation is generally not related to bias between the results. Use of common reference intervals, either as an average of the current intervals in use, or the intervals proposed by the AACB Harmonisation Group, improved the variation seen in the information produced by different laboratories.     

Oncoprotein CIP2A is stabilized via interaction with tumor suppressor PP2A/B56

Protein phosphatase 2A (PP2A) is a critical human tumor suppressor. Cancerous inhibitor of PP2A (CIP2A) supports the activity of several critical cancer drivers (Akt, MYC, E2F1) and promotes malignancy in most cancer types via PP2A inhibition. However, the 3D structure of CIP2A has not been solved, and it remains enigmatic how it interacts with PP2A. Here, we show by yeast two‐hybrid assays, and subsequent validation experiments, that CIP2A forms homodimers. The homodimerization of CIP2A is confirmed by solving the crystal structure of an N‐terminal CIP2A fragment (amino acids 1–560) at 3.0 Å resolution, and by subsequent structure‐based mutational analyses of the dimerization interface. We further describe that the CIP2A dimer interacts with the PP2A subunits B56α and B56γ. CIP2A binds to the B56 proteins via a conserved N‐terminal region, and dimerization promotes B56 binding. Intriguingly, inhibition of either CIP2A dimerization or B56α/γ expression destabilizes CIP2A, indicating opportunities for controlled degradation. These results provide the first structure–function analysis of the interaction of CIP2A with PP2A/B56 and have direct implications for its targeting in cancer therapy.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Guianin: A neolignan from Aniba guianensis

The wood of Aniba guianensis Aubl. (Lauraceae) contains benzyl benzoate, benzyl salicylate, sitosterol, O-methyleugenol, O-methylisoeugenol and the neolignan guianin for which the structure of 1-allyl-8-hydroxy-3-methoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene (VI) is proposed.     

Neolignans from an Aniba species

A benzene extract of the trunk of an Aniba species (Lauraceae) contained benzyl benzoate, benzyl salicylate, sitosterol and the neolignans (2S,3S,3aR)-3a-allyl-5-methoxy-3-methyl-2-piperonyl-2,3,3a,6-tetrahydro-6-oxobenzofuran (burchellin); (2S,3S,3aR)-3a-allyl-5-methoxy-3-methyl-2-veratryl-2,3,3a,6-tetrahydro-6-oxobenzofuran; (2S,3S,3aR)-3a-allyl-5,7-dimethoxy-3-methyl-2-veratryl-2,3,3a,6-tetrahydro-6-oxobenzofuran; (2S,3S,5S)-5-allyl-5-methoxy-3-methyl-2-veratryl-2,3,5,6-tetrahydro-6-oxo-benzofuran; (2R,3R)-7-methoxy-3-methyl-5-propenyl-2-veratryl-2,3-dihydrobenzofuran; rel-(1R,5R,6R,7R,8S)-1-allyl-8-hydroxy-3-methoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene (guianin); rel-(1S,5S,6S,7R,8R)-1-allyl-8-hydroxy-3,5-dimethoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene; rel-(1S,5S,6S,7R,8R)-8-acetoxy-1-allyl-3-hydroxy-5-methoxy-7-methyl-4-oxo-6-piperonyl-bicyclo[3,2,1]oct-2-ene; rel-1S,5S,6S,7R,8R)-8-acetoxy-3,5-dimethoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene; rel-(1R,5S,6R,7R)-1-allyl-3-methoxy-7-methyl-4,8-dioxo-6-piperonylbicyclo[3,2,1]oct-2-ene.     

Anti-predator adaptations in a neustonic insect (Velia caprai)

Summary The risk of a Velia caprai (Heteroptera: Veliidae) individual to fall victim to brown trout (Salmo trutta) was demonstrated experimentally to be markedly lower when several bugs were simultaneously exposed to the trout. V. caprai was found distasteful to brown trout, and a high proportion was ejected alive after being captured. We suggest that school formation in V. caprai lowers the risk of predation because frequent predator-prey encounters may assist in retention of the avoidance learned by the predator. Individual behavioural responses to attacks were variable and the frequency of expansion skating and thanatosis was temperature dependent. Thanatosis postures were either symmetric with the legs pressed to the body or irregular.