Oxidative metabolism of dopamine: a colour reaction from human midbrain analysed by mass spectrometry

In order to identify the protein responsible for a dopamine peroxidizing activity, previously described in human normal and parkinsonian substantia nigra by our group, we developed non-denaturing polyacrylamide gel electrophoresis conditions, mimicking the characteristic colour in vitro reaction, resulting from cyclic oxidation of dopamine (DA). After separating protein mixtures from human normal midbrain homogenates on two sets of identical native gels, one gel set was subjected to specific activity staining by using DA and hydrogen peroxide. An activity red/orange band appeared in midbrain tissue lanes, similarly to the lane where commercial horseradish peroxidase (HRP) was present as control of peroxidative activity. The second set of gels, stained with Coomassie Blue, showed other, not enzymatically active protein bands. Mass spectrometry analysis of the bands containing the activity and the corresponding Coomassie Blue bands revealed the presence of proteins that may play a role in neurodegenerative disease, highlighting a possible functional link among dopamine/dopaminochrome redox cycle and protein metabolism.     

Disruption of seasonality in growth hormone-transgenic coho salmon (Oncorhynchus kisutch) and the role of cholecystokinin in seasonal feeding behavior

Seasonal variation in daily food intake is a well-documented phenomenon in many organisms including wild-type coho salmon where the appetite is noticeably reduced during periods of decreased day length and low water temperature. This reduction may in part be explained by altered production of cholecystokinin (CCK) and growth hormone (GH). CCK is a hormone produced in the brain and gut that mediates a feeling of satiety and thus has an inhibitory effect on food intake and foraging behaviour. Growth hormone (GH) enhances feeding behaviour and consequently growth, but its production is reduced during winter. The objectives of this study were: first, to compare the seasonal feeding behaviour of wild and GH-transgenic coho salmon; second, to determine the behavioural effect of blocking the action of CCK (by using devazepide) on the seasonal food intake; and third, to measure CCK expression in brain and gut tissues between the two genotypes across seasons. We found that, in contrast to wild salmon, food intake in transgenic salmon was not reduced during winter indicating that seasonal control of appetite regulation has been disrupted by constitutive production of GH in transgenic animals. Blocking of CCK increased food intake in both genotypes in all seasons. The increase was stronger in wild genotypes than transgenic fish; however blocking CCK in wild-type fish in winter did not elevate appetites to levels observed in the summer. The response to devazepide was generally faster in transgenic than in wild salmon with more rapid effects observed during summer than during winter, possibly due to a higher temperature in summer. Overall, a seasonal effect on CCK mRNA levels was observed in telencephalon with levels during winter being higher compared to the summer in wild fish, but with no seasonal effect in transgenic fish. No differences in seasonal CCK expression were found in hypothalamus. Higher levels of CCK were detected in the gut of both genotypes in winter compared to summer. Thus, CCK appears to mediate food intake among seasons in both wild-type and GH-transgenic salmon, and an altered CCK regulation may be responsible at least in part for the seasonal regulation of food intake.     

Interface of biotechnology and ecology for environmental risk assessments of transgenic fish

Genetically engineered fish with enhanced phenotypic traits have yet to be implemented into commercial applications. This is partly because of the difficulties in reliably predicting the ecological risk of transgenic fish should they escape into the wild. The ecological consequences of the phenotypic differences between transgenic and wild-type fish, as determined in the laboratory, can be uncertain because of genotype-by-environment effects (GXE). Additionally, we are limited in our ability to extrapolate simple phenotypes to the complex ecological interactions that occur in nature. Genetic background can also shape the phenotypic effects of transgenes, which, over time and among different wild populations, can make risk assessments a continuously evolving target. These uncertainties suggest that assessments of transgenic fish in contained facilities need to be conducted under as wide a range of conditions as possible, and that efficacious physical and biological containment strategies remain as crucial approaches to ensure the safe application of transgenic fish technology.     

Mitochondrial regulation of flower development

Flower development in plants depends not only on a set of nuclear genes but also on the coordinate action of the mitochondrion. Certain mitochondrial genomes in combination with certain nuclear genomes lead to the expression of cytoplasmic male-sterility (CMS). Both mitochondrial genes that determine male-sterility and nuclear Restorer-of-fertility genes that suppress the male-sterile phenotype have been cloned. Lately, the interactions between mitochondrial and nuclear genes through retrograde signalling in CMS-systems have been dissected. Of special interest are the altered expression patterns of floral homeotic genes in certain CMS-systems. Here, we review the mitochondrial influence on flower development and give examples from CMS-systems developed in Brassica, Daucus carota, Nicotiana tabacum and Triticum aestivum.     

Software sensors for fermentation processes

Four software sensors based on standard on-line data from fermentation processes and simple mathematical models were used to monitor a number of state variables in Escherichia coli fed-batch processes: the biomass concentration, the specific growth rate, the oxygen transfer capacity of the bioreactor, and the new R _O/S sensor which is the ratio between oxygen and energy substrate consumption. The R _O/S variable grows continuously in a fed-batch culture with constant glucose feed, which reflects the increasing maintenance demand at declining specific growth rate. The R _O/S sensor also responded to rapid pH shift-downs reflecting the increasing demand for maintenance energy. It is suggested that this sensor may be used to monitor the extent of physiological stress that demands energy for survival.     

Science in drug control: the alkaloid content of afghan opium

Opium samples from Afghanistan were analyzed by HPLC for their content of morphine and three further alkaloids (codeine, thebaine, and papaverine). To our knowledge, this is the largest set of authentic opium samples analyzed in one study until now. The purpose was to assess possible correlations between samples and selected external factors, such as region of origin within Afghanistan, year of harvest, or intra-batch variation. In the investigated samples, a trend towards higher morphine concentrations in opium from the North-Eastern parts of Afghanistan was observed in the period from 2003 to 2005. More than 75% of the samples contained above 10% of morphine, the overall average was 14.4%.     

HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children

Background

HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5^broad viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression.

Methodology/Principal Findings

Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5^narrow phenotype (n = 20), but R5^broad and R5X4 viruses were also found in seven and one case, respectively. The presence of R5^broad and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5^broad phenotype, however, the presence of the R5^broad virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5^broad viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5^broad phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn''s viral variant.

Conclusions/Significance

Our results show that R5^broad viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5^narrow phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.     

Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements

Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.     

Reproductive sharing among queens in the ant Formica fusca

Reproductive sharing among cobreeders, in which reproductiveshares may vary from equal contribution (low reproductive skew)to reproductive dominance by one individual (high reproductiveskew), is a fundamental feature of animal societies. Recenttheoretical work, the reproductive skew models, has focusedon factors affecting the degree to which reproduction is skewedwithin a society. We used the parameters provided by skew modelsas a guideline to study determinants of reproductive sharingin polygyne ants. As a model system we used two-queen laboratorycolonies of the ant Formica fusca in which the reproductiveshares of each queen was assessed from offspring by using allozymesand DNA microsatellites. We tested how the different variablesincluded in reproductive skew models (queen-queen relatedness,potential fighting ability, productivity, and worker relatednessreflected by queen number in the colony of origin) affect reproductivesharing among queens. The results showed that the relatednessamong queens explained 26% of the variation in reproductiveskew. The size difference between queens (reflecting potentialfighting ability), colony productivity, and worker relatednessdid not have an effect on reproductive partitioning among cobreeders.To our knowledge, this is the first study to test for the effectsof various determinants of skew in an experimental setting.     

Lep d 2 polymorphisms in wild and cultured Lepidoglyphus destructor mites.

We have previously cloned, expressed and characterized two variants of the major allergen Lep d 2 from cultured Lepidoglyphus destructor mites. These variants, Lep d 2.0101 and Lep d 2.0201, differ at 13 amino acid positions. In this study we investigated Lep d 2 sequence diversity between wild and cultured mites. PCR, Southern blot and DNA sequence analysis revealed the presence of two different Lep d 2 genes, one with and one without an intron. In addition, two new variants of Lep d 2, Lep d 2.0102 and Lep d 2.0202, were found at different frequencies in wild and cultured mites. When we expressed the Lep d 2 variants and compared their IgE binding properties by ELISA inhibition, we found that Lep d 2.0102 was a more potent inhibitor than Lep d 2.0101, and to a lesser extent Lep d 2.0202 was more potent than Lep d 2.0201. Long-term cultures of peripheral blood mononuclear cells were used to assess the ability of the expressed Lep d 2 variants to induce cytokine release. Although cells from different individuals released different amounts of interferon-gamma and interleukin-5, no consistent cytokine release pattern could be linked to any specific Lep d 2 variant. In conclusion, we show that both cultured and wild Lepidoglyphus destructor mites contain the same pattern of polymorphism. Furthermore, this Lep d 2 sequence diversity seems not to have any significant impact on the allergens IgE binding or its ability to induce T cell cytokine release.