Enrichment strategies for nitrile-hydrolysing bacteria

A series of enrichments with different nitriles as sole source of nitrogen was performed in order to obtain a relationship between the selective nitrogen source and (i) the enzyme systems that are synthesized by the isolates and (ii) the enzyme specificities for the utilization of the nitriles. Bacteria were enriched with 2-phenylpropionitrile, 2-(2-methoxyphenyl)propionitrile, 2-phenylbutyronitrile, ibuprofen nitrile, naproxen nitrile, ketoprofen nitrile, ketoprofen amide, benzonitrile, or naphthalenecarbonitrile as sole nitrogen source and succinate as sole source of carbon and energy. 2-Phenylpropionitrile as nitrogen source resulted predominantly in the enrichment of gram-negative bacteria, which harboured nitrilase and in some cases also amidase activity. In contrast, with the other nitriles used, a substantial majority of gram-positive strains, mainly of the genus Rhodococcus, were isolated. These strains contained predominantly a nitrile hydratase/amidase system. The nitrilases and nitrile hydratases showed R or S selectivity with generally poor optical yields. In contrast, the amidases were almost exclusively S-selective, often forming the optically pure acids with an enantiomeric excess above 99%. The conversion of different nitriles by the isolates was compared. The nitrile-hydrolysing systems of the new isolates usually showed high activity against those nitriles that were used for the enrichment of the bacteria. Received: 13 November 1996 / Received revision: 4 February 1997 / Accepted: 10 February 1997     

Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells

Glutathione reductase (GR) is an essential enzyme for the glutathione-mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000-fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double-labeling immunocytochemical staining in astroglia-rich and neuron-rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astroglia-rich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron-rich primary cultures, GAP43-positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.     

Maternal prenatal depressive symptoms predict infant NR3C1 1F and BDNF IV DNA methylation

Prenatal maternal psychological distress increases risk for adverse infant outcomes. However, the biological mechanisms underlying this association remain unclear. Prenatal stress can impact fetal epigenetic regulation that could underlie changes in infant stress responses. It has been suggested that maternal glucocorticoids may mediate this epigenetic effect. We examined this hypothesis by determining the impact of maternal cortisol and depressive symptoms during pregnancy on infant NR3C1 and BDNF DNA methylation. Fifty-seven pregnant women were recruited during the second or third trimester. Participants self-reported depressive symptoms and salivary cortisol samples were collected diurnally and in response to a stressor. Buccal swabs for DNA extraction and DNA methylation analysis were collected from each infant at 2 months of age, and mothers were assessed for postnatal depressive symptoms. Prenatal depressive symptoms significantly predicted increased NR3C1 1F DNA methylation in male infants (β = 2.147, P = 0.044). Prenatal depressive symptoms also significantly predicted decreased BDNF IV DNA methylation in both male and female infants (β = −3.244, P = 0.013). No measure of maternal cortisol during pregnancy predicted infant NR3C1 1F or BDNF promoter IV DNA methylation. Our findings highlight the susceptibility of males to changes in NR3C1 DNA methylation and present novel evidence for altered BDNF IV DNA methylation in response to maternal depression during pregnancy. The lack of association between maternal cortisol and infant DNA methylation suggests that effects of maternal depression may not be mediated directly by glucocorticoids. Future studies should consider other potential mediating mechanisms in the link between maternal mood and infant outcomes.     

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca²⁺-imaging using the superior green Ca²⁺-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.     

Relevance of exocytotic glutamate release from retinal glia

Glial cells release molecules that influence brain?development, function, and disease. Calcium-dependent exocytosis has been proposed as potential release mechanism in astroglia, but the physiological relevance of "gliotransmission" in?vivo remains controversial. We focused on the impact of glial exocytosis on sensory transduction in the retina.?To this end, we generated transgenic mice to block exocytosis by Cre recombinase-dependent expression of the clostridial botulinum neurotoxin serotype?B light chain, which cleaves vesicle-associated membrane protein 1-3. Ubiquitous and neuronal toxin expression caused perinatal lethality and?a reduction of synaptic transmission thus validating transgene function. Toxin expression in Müller cells inhibited vesicular glutamate release and impaired glial volume regulation but left retinal histology and visual processing unaffected. Our model to study gliotransmission in?vivo reveals specific functions of exocytotic glutamate release in retinal glia.     

The human ubiquitin C promoter drives selective expression in principal neurons in the brain of a transgenic mouse line

The specificity of promoters used to drive the expression of proteins of interest is a crucial determinant of transgenesis. Numerous strategies have been developed to restrict expression on a certain cell population. On the other hand it has also remained challenging to obtain ubiquitous expression of transgenes which is needed for example to generate recombination reporter mice or to induce expression by recombination mediated excision of STOP-cassettes. We have generated transgenic mice with the expression of nuclear β-galactosidase driven by the human ubiquitin C promoter thought to mediate ubiquitous expression. However, in the brains of these transgenic mice the expression of the transgene was strikingly limited to principal neurons, while no expression was detected in interneurons or glial cells. These results indicate that the human ubiquitin C promoter might be useful to selectively target projections neurons of the brain.     

The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities

We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × –2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H₂-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H₂ production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.