Export,purification, and activities of affinity tagged <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> levansucrase produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His₆- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His₆-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru. R. Biedendieck and R. Beine contributed equally to this work.     

Frequent frameshift and point mutations in the SH gene of human metapneumovirus passaged in vitro

During the preparation of recombinant derivatives of the CAN97-83 clinical isolate of human metapneumovirus (HMPV), consensus nucleotide sequencing of the recovered RNA genomes provided evidence of frequent sequence heterogeneity at a number of genome positions. This heterogeneity was suggestive of sizable subpopulations containing mutations. An analysis of molecularly cloned cDNAs confirmed the presence of mixed populations. The biologically derived virus on which the recombinant system is based also contained sizeable mutant subpopulations, whose presence was confirmed by biological cloning and nucleotide sequencing. Most of the mutations occurred in the SH gene. For example, partial consensus sequencing of 40 independent preparations of recombinant HMPV (wild-type and various derivatives) showed that 31 of these preparations contained a total of 41 instances of small insertions in the SH gene and a total of five small insertions elsewhere. In each of these 31 preparations, there was at least one insert in SH that changed the reading frame and would yield a truncated protein. Nearly all of these insertions involved adding one or more A residues to various tracks of four or more A residues, with the most frequent site being a tract of seven A residues. There were also two instances of nucleotide deletions and numerous instances of nucleotide substitution point mutations, mostly in the SH gene. The occurrence of mutant subpopulations was greatly reduced by the replacement of the SH gene with a synthetic version in which these oligonucleotide tracts were eliminated by silent nucleotide changes. We suggest that we frequently detected subpopulations in which the expression of full-length SH protein was ablated because it provided a modest selective advantage to this clinical isolate in vitro. Adaptation involving the functional loss of a gene is unusual for an RNA virus.     

Mapping and characterization of the primary and anamnestic H-2(d)-restricted cytotoxic T-lymphocyte response in mice against human metapneumovirus

Cytotoxic T lymphocytes (CTLs) are important for the control of virus replication during respiratory infections. For human metapneumovirus (hMPV), an H-2(d)-restricted CTL epitope in the M2-2 protein has been described. In this study, we screened the hMPV F, G, N, M, M2-1, and M2-2 proteins using three independent algorithms to predict H-2(d) CTL epitopes in BALB/c mice. A dominant epitope (GYIDDNQSI) in positions 81 to 89 of the antitermination factor M2-1 and a subdominant epitope (SPKAGLLSL) in N(307-315) were detected during the anti-hMPV CTL response. Passive transfer of CD8(+) T-cell lines against M2-1(81-89) and N(307-315) protected Rag1(-/-) mice against hMPV challenge. Interestingly, diversification of CTL targets to include multiple epitopes was observed after repetitive infections. A subdominant response against the previously described M2-2 epitope was detected after the third infection. An understanding of the CTL response against hMPV is important for developing preventive and therapeutic strategies against the virus.     

Cell type-specific functions of the lysosomal protease cathepsin L in the heart

Deficiency of the lysosomal cysteine protease cathepsin L (Ctsl) in mice results in a phenotype affecting multiple tissues, including thymus, epidermis, and hair follicles, and in the heart develops as a progressive dilated cardiomyopathy (DCM). To understand the role of Ctsl in the maintenance of regular heart morphology and function, it is critical to determine whether the DCM in Ctsl-/- mice is primarily because of the lack of Ctsl expression and activity in the cardiomyocytes or is caused by the additional extracardiac pathologies. Cardiomyocyte-specific expression of Ctsl in Ctsl-/- mice, using an alpha-myosin heavy chain promoter-Ctsl transgene, results in improved cardiac contraction, normal mRNA expression of atrionatriuretic peptide, normal heart weight, and regular ultrastructure of cardiomyocytes. Epithelial expression of cathepsin L2 (CTSL2) by a K14 promoter-CTSL2-transgene resulted in rescue of the Ctsl-/- hair loss phenotype. In these mice, cardiac atrionatriuretic peptide expression and end systolic heart dimensions were also significantly attenuated. However, cardiac contraction was not improved, and increased heart weight as well as the typical changes in lysosomal ultrastructure of Ctsl-/- hearts persisted. Myocardial fibrosis was detected in all Ctsl-/- mice irrespective of transgene-mediated cardiac Ctsl expression or extracardiac CTSL2 expression. Expression of collagen 1 was not enhanced in Ctsl-/- hearts, but a reduced collagenolytic activity suggests a role for Ctsl in collagen turnover by cardiac fibroblasts. We conclude that the DCM of Ctsl-/- mice is primarily caused by absence of the protease in cardiomyocytes, whereas the complex gross phenotype of Ctsl-deficient mice, i.e. the fur defect, results in additional stress to the heart.     

Biodegradation Processes in a Laboratory-Scale Groundwater Contaminant Plume Assessed by Fluorescence Imaging and Microbial Analysis

Flow reactors containing quartz sand colonized with biofilm were set up as physical model aquifers to allow degrading plumes of acetate or phenol to be formed from a point source. A noninvasive fluorescent tracer technique was combined with chemical and biological sampling in order to quantify transport and biodegradation processes. Chemical analysis of samples showed a substantial decrease in carbon concentration between the injection and outflow resulting primarily from dilution but also from biodegradation. Two-dimensional imaging of the aqueous oxygen [O₂(aq)] concentration field quantified the depletion of O₂(aq) within the contaminant plume and provided evidence for microbial respiration associated with biodegradation of the carbon source. Combined microbiological, chemical, and O₂(aq) imaging data indicated that biodegradation was greatest at the plume fringe. DNA profiles of bacterial communities were assessed by temperature gradient gel electrophoresis, which revealed that diversity was limited and that community changes observed depended on the carbon source used. Spatial variation in activity within the plume could be quantitatively accounted for by the changes observed in active cell numbers rather than differences in community structure, the total biomass present, or the increased enzyme activity of individual cells. Numerical simulations and comparisons with the experimental data were used to test conceptual models of plume processes. Results demonstrated that plume behavior was best described by growth and decay of active biomass as a single functional group of organisms represented by active cell counts.     

Intersexual niche segregation in Cepero’s Ground-hopper, <Emphasis Type="Italic">Tetrix ceperoi</Emphasis>

Sexual differences in habitat preferences have been reported from a variety of animal taxa. However, the ultimate causes for this intersexual niche segregation remain poorly understood. It has been suggested that sexual dimorphism is a consequence of dimorphic niches based upon different reproductive costs and activities of the sexes. Here we provide evidence from field data to examine this hypothesis by studying the behavioral background of niche segregation in Tetrix ceperoi. Our data revealed distinct sexual differences in the substrates on which the insects perched and in the solar radiation of these locations. Males were found at brighter locations and more often on bare ground than females. Incorporation of behavioral data in our analysis showed that patches of bare ground were mainly utilized during mating behavior, in which males invested more time than females. In contrast, females spent more time resting and feeding in the vegetation. Intersexual differences in the proportion of autotomized individuals indicate that males might suffer higher predation risks. These patterns support the dimorphic niches hypothesis, which suggests that differential habitat utilization is caused by differences in the life history strategies of males and females, since males should accept a higher predation risk due to the benefits of multiple matings. Females should invest more time in gaining nutrients and energy for egg production and survival, whereas males should spend more time with searching for mates. We suggest that behavioral covariates should more often be implemented in ecological analyses, since these might have a strong explanatory power.     

Poly(ADP-ribosyl)ation in mammalian ageing

Poly(ADP-ribose) polymerases (PARPs) catalyze the post-translational modification of proteins with poly(ADP-ribose). Two PARP isoforms, PARP-1 and PARP-2, display catalytic activity by contact with DNA-strand breaks and are involved in DNA base-excision repair and other repair pathways. A body of correlative data suggests a link between DNA damage-induced poly(ADP-ribosyl)ation and mammalian longevity. Recent research on PARPs and poly(ADP-ribose) yielded several candidate mechanisms through which poly(ADP-ribosyl)ation might act as a factor that limits the rate of ageing.     

Insensitivity to Abeta42-lowering nonsteroidal anti-inflammatory drugs and gamma-secretase inhibitors is common among aggressive presenilin-1 mutations

Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of gamma-secretase modulators that avoid side effects of pan-gamma-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free gamma-secretase assays indicating that they directly target the gamma-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to Abeta42-lowering NSAIDs. Of particular interest is the PS1-DeltaExon9 mutation, which provokes a pathogenic increase in the Abeta42/Abeta40 ratio and dramatically reduces the cellular response to the Abeta42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr(291)-Ser(319) including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-DeltaExon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the Abeta42/Abeta40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to Abeta42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to gamma-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent gamma-secretase inhibitor LY-411575 failed to reduce brain levels of soluble Abeta42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at gamma-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of gamma-secretase modulators and inhibitors.