A novel technique to study pore-forming peptides in a natural membrane

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na⁺:Ca²⁺,K⁺ exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K⁺, Na⁺ or Ca²⁺), the OS membrane resistance (R _m ) was typically larger than 1 GΩ in the presence of 1 mM external Ca²⁺. Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in ∼50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.     

Alveolar macrophage cytotoxic activity is inhibited by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic component of cigarette smoke

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic compound of cigarette smoke that generates electrophilic intermediates capable of damaging DNA. Recently, we have shown that NNK can modulate mediator production by alveolar macrophages (AM) and bronchial and alveolar epithelial cells, suggesting that cigarette smoke can alter lung immune response. Thus, we investigated the effect of NNK and cigarette smoke extract (CSE) on AM capacity to eliminate tumoral cells. Rat AM cell line, NR8383, was treated with NNK (500 μM) or CSE (3%) and stimulated with lipopolysaccharide (10 ng/ml). The release of cytotoxic mediators, tumor necrosis factor (TNF) and reactive oxygen species (ROS), was measured in cell-free supernatants using ELISA and superoxide anion production. TNF- and ROS-dependent cytotoxicity were studied using a ⁵¹Chromium-release assay and WEHI-164 and P-815 cell lines. Treatment of AM with NNK and CSE for 18 h significantly inhibited AM TNF release. CSE exposure resulted in a significant increase of ROS production, whereas NNK did not. TNF-dependent cytotoxic activity of NR8383 and freshly isolated rat AM was significantly inhibited after treatment with NNK and CSE. Interestingly, although ROS production was stimulated by CSE and not affected by NNK, CSE inhibited AM ROS-dependent cytotoxicity. These results suggest that NNK may be one of the cigarette smoke components responsible for the reduction of pulmonary cytotoxicity. Thus, NNK may have a double pro-carcinogenic effect by contributing to DNA adduct formation and inhibiting AM cytotoxicity against tumoral cells.     

Territory occupancy rate of goshawk and gyrfalcon: no evidence of delayed numerical response to grouse numbers

Two recent studies on territory occupancy rates of goshawk Accipiter gentilis and gyrfalcon Falco rusticolus report a 2–3-year-delayed numerical response to grouse numbers, which is a requirement for a hypothesis of predator-generated grouse cycles. The time lags were assumed to reflect the average age of sexual maturity in the raptor species. In southern Norway, however, subadult (two-year-old) goshawk hens occupied only 18–25% of territories where occupancy was not recorded in the preceding year, and there was no significant relationship between the proportion of subadults among recruits and grouse indices two years earlier. We argue that territory occupancy rates are not appropriate indices of total raptor population levels, but rather reflect the proportion of territorial pairs that attempt to nest. Because this depends on the body condition of the hens, fluctuations in other important winter resident prey species (most important for the goshawk) and winter weather (most important for the gyrfalcon) should also be addressed. During 1988–2006, the annual proportion of goshawk territories with recorded nesting attempts in southern Norway was most closely related to the preceding autumn’s population indices of black grouse Tetrao tetrix and mountain hare Lepus timidus, whereas the annual proportion of gyrfalcon territories with observations of falcons or with confirmed breeding attempts in central Norway were best explained by population indices of willow grouse Lagopus lagopus and ptarmigan L. mutus from the previous autumn, and by December temperatures. Hence, our studies do not support the predation hypothesis for grouse cycles.     

Life history response of <Emphasis Type="Italic">Daphnia galeata</Emphasis> to heterogeneous conditions within a reservoir as determined in a cross-designed laboratory experiment

Growth and reproduction were studied in the laboratory in a cross-designed experimental set-up in four Daphnia galeata subpopulations collected from different locations (with respect to water characteristics) in a reservoir (epilimnion, metalimnion and hypolimnion in the deepest part of the reservoir near the dam and epilimnion of the upstream part of the reservoir) and in a laboratory clone of the same species. The results of two-way ANOVA revealed significant effects of the two parameters manipulated – source of water used for cultures and Daphnia subpopulation – on the life history characteristics of growth and reproduction. The water from the upstream part of the reservoir was the most favourable culture medium for all characteristics of the Daphnia groups studied (the largest primiparae, clutches and eggs, the shortest postembryonic development time and filtering setae). The poorest performances were recorded in the downstream, epilimnetic and metalimnetic waters. The primiparae in the hypolimnetic water were smaller but had relatively larger clutches of smaller eggs and slightly longer postembryonic development times. The Daphnia subpopulation originating from the hypolimnion had the smallest primiparae, the largest clutches, the smallest eggs and the shortest postembryonic development, whereas the opposite was found in animals from the epilmnion. These differences in ecologically relevant traits were supported by analysis of the quasi-neutral genetic markers that indicated significant site-dependent differences in clonal structure between the subpopulations. There was no consistent trend to higher within-group variance in the life history traits in the genetically heterogeneous subpopulations from the reservoir compared to the laboratory clone.     

Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part I: processes with isolated enzymes

Traditional cultivation-based methods to quantify microbial abundance are not suitable for analyses of microbial communities in environmental or medical samples, which consist mainly of uncultured microorganisms. Recently, different cultivation-independent quantification approaches have been developed to overcome this problem. Some of these techniques use specific fluorescence markers, for example ribosomal ribonucleic acid targeted oligonucleotide probes, to label the respective target organisms. Subsequently, the detected cells are visualized by fluorescence microscopy and are quantified by direct visual cell counting or by digital image analysis. This article provides an overview of these methods and some of their applications with emphasis on (semi-)automated image analysis solutions.     

Effect of pyruvate dehydrogenase complex deficiency on <Emphasis Type="SmallCaps">l</Emphasis>-lysine production with <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on l-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the l-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and l-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific l-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific l-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific l-lysine yield by 6 and 56%, respectively. In addition to l-lysine, significant amounts of pyruvate, l-alanine and l-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve l-lysine production by engineering the l-lysine biosynthetic pathway. This study is dedicated to Prof. Dr. Hermann Sahm on the occasion of his 65th birthday.     

Indirect food web effects of <Emphasis Type="Italic">Bythotrephes</Emphasis> invasion: responses by the rotifer <Emphasis Type="Italic">Conochilus</Emphasis> in Harp Lake,Canada

As a recent invader of North American lakes, Bythotrephes longimanus has induced large changes in crustacean zooplankton communities through direct predation effects. Here we demonstrate that Bythotrephes can also have indirect food web effects, specifically on rotifer fauna. In historical time series data, the densities of the colonial rotifer Conochilus unicornis significantly increased after Bythotrephes invasion in Harp Lake, Ontario. No such changes were observed in a non-invaded reference lake, the nearby Red Chalk Lake. Evidence for two mechanisms explaining the Conochilus increase was examined based on changes to the crustacean zooplankton community over time. Rapid and severe declines in several herbivorous species of cladoceran zooplankton after Bythotrephes detection indicated a decrease in exploitative competition pressure on Conochilus. Secondly, a later and significant decline to virtual extinction of native invertebrate predators (Mesocyclops and Leptodora) could account for the observed Conochilus increase which also began 1–2 years after invasion by Bythotrephes. Ultimately, it appears that both reduced competition followed by a loss of native invertebrate predators were necessary to lead to the large Conochilus densities observed following invader establishment. From this analysis of long-term community data, it appears that Bythotrephes has important indirect, as well as direct, food web effects in newly invaded North American lakes with implications for trophic relationships.     

A trypsin inhibitor from <Emphasis Type="Italic">Cassia obtusifolia</Emphasis> seeds: isolation,characterization and activity against <Emphasis Type="Italic">Pieris rapae</Emphasis>

A trypsin inhibitor was isolated from Cassia obtusifolia by ammonium sulfate precipitation, Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography. The inhibitor consisted of a single polypeptide chain with a molecular mass of 19, 812.55 Da. It was stable from pH 2 to 12 for 24 h, whereas it was unstable either above 70°C for 10 min or under reduced conditions. The inhibitor, which inhibited trypsin activity with an apparent Ki of 0.3 μM, had one reactive site involving a lysine residue. The native inhibitor was resistant to pepsin digestion, whereas the heated inhibitor produced 40% degree of susceptibility. The disulfide linkage and lysine residue were important in maintaining its conformation. Partial amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz inhibitor family. Moreover, the inhibitor showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Pieris rapae and could suppress the growth of larvae.     

Evidence for statistical epistasis between catechol-<Emphasis Type="Italic">O</Emphasis>-methyltransferase (COMT) and polymorphisms in RGS4, G72 (DAOA), GRM3, and DISC1: influence on risk of schizophrenia

Catechol-O-methyltransferase (COMT) regulates dopamine degradation and is located in a genomic region that is deleted in a syndrome associated with psychosis, making it a promising candidate gene for schizophrenia. COMT also has been shown to influence prefrontal cortex processing efficiency. Prefrontal processing dysfunction is a common finding in schizophrenia, and a background of inefficient processing may modulate the effect of other candidate genes. Using the NIMH sibling study (SS), a non-independent case-control set, and an independent German (G) case-control set, we performed conditional/unconditional logistic regression to test for epistasis between SNPs in COMT (rs2097603, Val158Met (rs4680), rs165599) and polymorphisms in other schizophrenia susceptibility genes. Evidence for interaction was evaluated using a likelihood ratio test (LRT) between nested models. SNPs in RGS4, G72, GRM3, and DISC1 showed evidence for significant statistical epistasis with COMT. A striking result was found in RGS4: three of five SNPs showed a significant increase in risk [LRT P-values: 90387 = 0.05 (SS); SNP4 = 0.02 (SS), 0.02 (G); SNP18 = 0.04 (SS), 0.008 (G)] in interaction with COMT; main effects for RGS4 SNPs were null. Significant results for SNP4 and SNP18 were also found in the German study. We were able to detect statistical interaction between COMT and polymorphisms in candidate genes for schizophrenia, many of which had no significant main effect. In addition, we were able to replicate other studies, including allelic directionality. The use of epistatic models may improve replication of psychiatric candidate gene studies.