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Abstract: Over the past decade, microfluidic techniques have been established as a versatile platform to perform live cell experiments under well-controlled conditions. To investigate the directional responses of cells, stable concentration profiles of chemotactic factors can be generated in microfluidic gradient mixers that provide a high degree of spatial control. However, the times for built-up and switching of gradient profiles are in general too slow to resolve the intracellular protein translocation events of directional sensing of eukaryotes. Here, we review an example of a conventional microfluidic gradient mixer as well as the novel flow photolysis technique that achieves an increased temporal resolution by combining the photo-activation of caged compounds with the advantages of microfluidic chambers. [Copyright &y& Elsevier]

Advanced microscopy and the corresponding image analysis have been developed in recent years into a powerful tool for studying molecular and morphological events in cells and tissues. Cell-based high-content screening (HCS) is an upcoming methodology for the investigation of cellular processes and their alteration by multiple chemical or genetic perturbations. Multiparametric characterization of responses to such changes can be analyzed using intact live cells as reporter. These disturbances are screened for effects on a variety of molecular and cellular targets, including subcellular localization and redistribution of proteins. In contrast to biochemical screening, they detect the responses within the context of the intercellular structural and functional networks of normal and diseased cells, respectively. As cell-based HCS of small-molecule libraries is applied to identify and characterize new therapeutic lead compounds, large pharmaceutical companies are major drivers of the technology and have already shown image-based screens using more than 100000 compounds. [Copyright &y& Elsevier]

Abstract: Butterflies of the genus Heliconius are well known for their peculiar habits of utilizing pollen as a source of amino acids. Saliva plays a major role in the process of extracting amino acids and proteins from the pollen grains. In this investigation, we obtained samples of saliva from adult Heliconius melpomene by placing pumpkin pollen or fine glass-beads on the proboscis, which stimulates the butterflies to release saliva. Proteolytic activity was determined in the saliva by an insoluble protein-dye that turns blue when cleaved by proteases. Its extinction value was measured with a spectrophotometer at 595nm. Both the saliva sampled with pollen and the saliva obtained from inert glass-beads exhibit proteolytic activity demonstrating that the saliva contains proteases. The proteolytic activity of the pollen/saliva samples was higher than that of the glass-bead/saliva samples, which we attribute to the stimulating effects of pollen, such as taste, smell, and texture, and not to proteases which might have been liberated from the pollen. This is indicated by the fact that pollen samples without saliva showed only a negligible indication for proteolytic activity. In general, females exhibit higher proteolytic activities than males, presumably due to their greater amino acid investment in reproduction. We present here first evidence for the existence of proteases in the saliva of a butterfly species and suggest that these enzymes are crucial for the use of amino acids and proteins from pollen in Heliconius butterflies. [Copyright &y& Elsevier]

Abstract: N→O acyl migrations at serine or threonine residues in peptides or proteins have previously been observed upon treatment with strong acids. Here we show that the extent of such N→O shifts depends on the peptide sequence and even in the presence of moderately acidic trifluoroacetic acid, as during Fmoc or Bsmoc-based solid phase peptide synthesis, may give rise to large amounts of depsipeptide by-products. [Copyright &y& Elsevier]

Novel high-throughput techniques are capable of producing dense time series of many simultaneous in vivo measurements of the expression of genes, concentrations of metabolites, the prevalence and activation states of proteins, and of other biomarkers characterizing the physiological state of a cell or organism. These data contain enormous information, much of which, however, is not explicit but must be extracted with mathematical and computational means. The magnitude of high-throughput data generation and the potential and demands that these data entail will dramatically affect strategies for modeling, analyzing, and optimizing pathways and usher in a new era of metabolic systems analysis. [Copyright &y& Elsevier]

A more detailed characterization of particulate organic matter in wastewater streams is needed to improve solid–liquid separation and biological processes for wastewater treatment. The objective of this paper was to evaluate particle size distributions and the associated chemical composition for municipal, industrial, and agricultural waste streams. Most of the organic matter in these wastewaters was larger than a molecular weight of 103 amu and therefore would require extracellular hydrolysis before any bacterial metabolism. Particle size distributions were significantly different for the studied waste streams. In municipal wastewater, the organic matter was evenly distributed in all eight size fractions ranging from 103 amu up to 63 μm. The industrial and agricultural wastewaters, however, contained mainly soluble organic matter (<103 amu) and larger particles (>1.2 μm for the industrial and >10 μm for the agricultural waste) leaving a gap in the size range of large macromolecules and colloids. The relative protein and carbohydrate concentrations varied for the different size fractions compared to the measured chemical oxygen demand (COD) in the corresponding size fraction. Thus, the design of the solid–liquid separation at a treatment plant could be used to purposefully modify the overall chemical composition of the organic matter before further biological treatment. Particle size distributions will influence design and operation of biological nutrient removal processes such as denitrification or biological phosphorus removal that may be carbon limited if a large fraction of the organic matter is composed of large particles with slow hydrolysis rates. Measured particle size distributions for the different waste streams in this study (municipal, industrial, agricultural) were significantly different requiring specific approaches for treatment plant design. [Copyright &y& Elsevier]

Abstract: In order to keep the anaerobic process stably and uniformly producing biogas it needs to be supplied with either an even amount of substrate of stable quality or varying amounts according to variations in quality. Feeding amounts are usually adjusted manually as a reaction to changing rates of biogas production. Continuous information about the actual substrate quality is not available and feedstuff analyses are costly. Aim of this study was to assess the feasibility of near infrared spectroscopic (NIRS) online monitoring of substrate quality in order to find ways towards more exact control of biogas plant feeding. A NIRS sensor system was designed, constructed and calibrated for continuous monitoring of (RMSECV in brackets) dry matter (DM) (0.75%fresh matter (FM)), volatile solids (0.74%FM), crude fat (0.09%FM), crude protein (0.22%FM), crude fiber (1.50%DM) and nitrogen-free extracts (0.93%FM) of maize silage. [Copyright &y& Elsevier]

We investigated the association of polymorphisms of drug-metabolizing enzymes and susceptibility to oral cavity cancer. Polymerase chain reaction (PCR)-based analyses were performed on genomic DNA of 94 Caucasian patients in Germany and 92 healthy German controls to determine genotypes of polymorphisms in CYP1A1, GSTM1 and NAT2. For CYP1A1, the homozygous mutant genotype Val/Val did not occur. The heterozygous genotype Ile/Val (6.5% cases versus 4.3% controls) and the homozygous wild-type Ile/Ile (95.7% cases versus 93.5% controls) showed no statistically significant differences between groups (X2=0.47; P=0.534, Fisher''s exact test, two-sided). The GSTM1 homozygous null genotype occurred more frequently in cancer patients (59.6%) compared to controls (53.3%) but this difference remained insignificant in X2-analysis (X2=1.07; P=0.587). Almost identical genotype distributions between cases and controls were found for all three NAT2 acetylators. Hence, these three genetic polymorphisms are unlikely to be associated with oral cavity cancer in the population studied. [ABSTRACT FROM AUTHOR]

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome. [ABSTRACT FROM AUTHOR]

Abstract: Background: Exposure to sulfur dioxide (SO2) affects large populations worldwide. Pulmonary effects have been reported at concentrations relevant in the general (<0.5ppm) and working environment (>0.5ppm). SO2 is an irritant but the existing studies often emphasize only pulmonary effects and no clear dose–response relationship has yet been described. Objectives: Using a multi-level, multi-method approach, odor annoyance, sensory irritation and pulmonary effects of SO2 were to be investigated in an experimental exposure study. Methods: Eye blink frequency, rhinomanometry, spirometry and symptom ratings of acute health effects were assessed before, during, and after the exposures. Each session lasted 4h and concentrations of 0.5, 1, and 2ppm were investigated and compared to a control condition using clean air. Sixteen human volunteers (8 females/8 males) participated and during exposure light physical exercise was simulated with bicycle ergometry. Results: Eye blink frequency, nasal airflow, and lung function were not affected by the acute SO2 exposure investigated. These physiological responses to moderate SO2 exposures were not significantly affected by gender. Regarding subjectively measured chemosensory sensations, only odor annoyance ratings increased in a dose-dependent manner, but the average magnitudes were labeled weak to moderate. Conclusions: In healthy volunteers, without hyperresponsiveness to SO2, no dose-dependent effects of acute SO2 exposures up to 2ppm could be measured. Due to olfactory perceptions subjects were aware of the different SO2 exposures but the associated annoyance was relatively low. [Copyright &y& Elsevier]

The role of hepatitis C virus (HCV) protein non-structural (NS) 5A in HCV-associated pathogenesis is still enigmatic. To investigate the in vivo role of NS5A for viral persistence and virus-associated pathogenesis a transgenic (Tg) mouse model was established. Mice with liver-targeted NS5A transgene expression were generated using the albumin promoter. Alterations in the hepatic immune response were determined by Western blot, infection by lymphocytic choriomeningitis virus (LCMV), and using transient NS3/4A Tg mice generated by hydrodynamic injection. Cytotoxic T lymphocyte (CTL) activity was investigated by the Cr-release assay. The stable NS5A Tg mice did not reveal signs of spontaneous liver disease. The intrahepatic immunity was disrupted in the NS5A Tg mice as determined by clearance of LCMV infection or transiently NS3/4A Tg hepatocytes in vivo. This impaired immunity was explained by a reduced induction of interferon β, 2',S'-OAS, and PKR after LCMV infection and an impairment of the CTL-mediated elimination of NS3-expressing hepatocytes, In conclusion, these data indicate that in the present transgenic mouse model, NS5A does not cause spontaneous liver disease. However, we discovered that NS5A could impair both the innate and the adaptive immune response to promote chronic HCV infection. [ABSTRACT FROM AUTHOR]

Abstract: Covalent binding of the acyl glucuronide (AcMPAG) metabolite of the immunosuppressant mycophenolic acid (MPA) to proteins is considered a possible initiating event for organ toxicity. Since the kidney is involved in the formation and excretion of AcMPAG, it can be hypothesized that this tissue may be exposed to relatively high concentrations of this metabolite and would, therefore, be a particularly suitable organ to investigate AcMPAG protein targets. In the present study we identified potential AcMPAG target proteins in kidney tissues from Wistar rats treated with mycophenolate mofetil (40mg/kg/day over 21 days). Proteins were separated by 2-DE and covalent protein adducts were detected by Western blotting with an antibody specific for MPA/AcMPAG. The corresponding coomassie blue stained proteins from parallel gels were subjected to in-gel tryptic digestion and peptides were characterized on a Q-TOF Ultima Global. The protein targets were further verified by immunoprecipitation with anti-MPA/AcMPAG antibody to purify the modified proteins followed by 1-DE and MS analysis. Database searches revealed several AcMPAG target proteins that could be related to ultrastructural abnormalities, metabolic effects, and altered oxidative stress/detoxification responses. Predominately cytosolic proteins such as selenium binding protein, protein disulfide isomerase, aldehyde dehydrogenase, triosephosphate isomerase, and kidney aminoacylase were involved in adduct formation. Two cytoskeletal proteins tropomyosin 1 and 4 as well as the antioxidant proteins peroxiredoxin 3 and 6 were also targets of AcMPAG. Functional consequences from these protein modifications remain to be demonstrated. [Copyright &y& Elsevier]

Abstract: Application of high pressure can be used for gentle pasteurizing of food, minimizing undesirable alterations such as vitamin losses and changes in taste and color. In addition, pressure has become a useful tool for investigating structural changes in proteins. Treatments of proteins with high pressure can reveal conformations that are not obtainable by other physical variables like temperature, since pressure favors structural transitions accompanied with smaller volumes. Here, we discuss both the potential use of high pressure to inactivate infectious TSE material and the application of this thermodynamic parameter for the investigation of prion folding. This review summarizes our findings on the effects of pressure on the structure of native infectious scrapie prions in hamster brain homogenates and on the structure of infectious prion rods isolated from diseased hamsters brains. Native prions were found to be pressure sensitive, whereas isolated prions revealed an extreme pressure-resistant structure. The discussion will be focused on the different pressure behavior of these prion isoforms, which points out differences in the protein structure that have not been taken into consideration before. [Copyright &y& Elsevier]

Background/Aims: This study investigates the application of protein based therapeutic suicide enzyme/prodrug approaches providing novel means for both safe and effective local therapeutic regimes in solid tumors. Methods: Employing a novel cell permeable peptide, known as the translocation motif (TLM) of hepatitis B virus, E. coli cytosine deaminase (BCDase) suicide fusion proteins were generated. Results: TLM fusion proteins formed hexamers (as do parental wtBCDase proteins) and retained the specific enzymatic activity of cytosine conversion to uracil also being comparable to parental wtBCDase proteins. However, only BCDase-TLM fusion proteins, but not TLM-BCDase fusion nor parental wtBCDase proteins were found to be taken up to the cytoplasm of target cells as demonstrated both by confocal laser scanning microscopy and cell fractionation. Uptake of BCDase-TLM worked both efficiently and rapidly and was found to be independent from the endosomal pathway. Since BCDase-TLM fusion proteins completely retained their suicide enzymatic activity in the course of translocation across the plasma membrane their usage as profound inducers of chemo-sensitivity to 5-FC strongly is suggested. Conclusions: Future therapeutic local application of cell-permeable BCDase-TLM fusion proteins together with a systemic 5-FC prodrug application could result in profound antitumor activities without apparent side effects. [Copyright &y& Elsevier]

Crude brain homogenates of terminally diseased hamsters infected with the 263K strain of scrapie (PrPSc) and purified prion fibrils were heated or pressurized at 800 megapascals and 60°C for 2 h in different buffers and in water. Prion proteins (PrP) were analyzed for their proteinase K resistance in immunoblots and for their infectivity in hamster bioassays. A notable decrease in the proteinase K resistance of unpurified prion proteins, probably because of pressure-induced changes in the protein conformation of native PrPSc or the N-truncated PrP-(27–30), could be demonstrated when pressurized at initially neutral conditions in several buffers and in water but not in a slightly acidic pH. A subsequent 6–7 log10 reduction of infectious unitslg in phosphate-buffered saline buffer, pH 7.4, was found. The proteinase K-resistant core was also not detectable after purification of prions extracted from pressurized samples, confirming pressure effects at the level of the secondary structure of prion proteins. However, opposite results were found after pressurizing purified prions, arguing for the existence of pressure-sensitive β-structures (PrPScΔPsen) and extremely pressure-resistant β-structures (PrPScΔPes). Remarkably, after the first centrifugation step at 540,000 × g during isolation, prions remained proteinase K-resistant when pressurized in all tested buffers and in water. It is known that purified fibrils retain infectivity, but the isolated protein (full and N-truncated) behaved differently from native PrPSc under pressure, suggesting a kind of semicrystalline polymer structure. [ABSTRACT FROM AUTHOR]

Homologous recombination between identical stretches of DNA depends on the coordinated action of many tightly regulated proteins. Cellular defects in homologous recombination are strongly associated with increased genomic instability and tumorigenesis. In cells of the cancer-prone syndrome ataxia telangiectasia (A-T), increased intrachromosomal recombination has been demonstrated, while extrachromosomal recombination has been discussed controversially. We constructed a novel, episomally replicating pGrec recombination vector containing two mutated alleles of the enhanced green fluorescent protein (eGFP) gene. Homologous recombination can reconstitute functional wildtype eGFP, thus allowing detection of recombination events based on cellular eGFP fluorescence. Using an isogenic cell pair of A-T fibroblasts and derivatives complemented by an ATM expression vector, we were able to demonstrate in A-T cells high extrachromosomal recombination rates, which are suppressed upon ectopic ATM expression. We thus found that ATM deficiency increases spontaneous recombination not only in intrachromosomal but also in extrachromosomal substrates, suggesting that lack of ATM increases homologous recombination independent of the chromatin structure. [Copyright &y& Elsevier]

Innervation of prostate cancer (CaP) tissue favors tumor progression and metastasis but the regulation of innervation in CaP is unclear. The oncogenic transcription factor ERG is commonly induced by a typical TMPRSS2-ERG (TE) gene fusion in CaP and may affect innervation. Here, we analyzed whether nerve density of CaP tissue is related to TE status or perineural infiltration status of CaP tissue. In parallel, we measured several members of the neuropilin/plexin/semaphorin family (NRP, PLXN, and SEMA) as possible targets mediating innervation. The TE-gene-fusion status was determined at the mRNA level in CaP tissues by nested RT-PCR. Transcript levels were analyzed by quantitative RT-PCR in CaP tissue or cell line homogenate. ERG was analyzed by immunostaining, and the nerve density was evaluated by immunostaining for PGP9.5 and axonal neurofilament. Data were analyzed by correlation (Spearman), linear regression, Mann-Whitney U test, and contingency table analyses. TE-positive (TE-1) vs. TE-negative (TE-0) CaP tissues displayed significantly enhanced ERG-mRNA levels (TE-0: -4.183; TE-1: -2.994, P < 0.001) and ERG immunostaining (Erg-IH score; TE-0: 0.4211; TE-1: 1.391; P < 0.0001). Notably, the nerve density was significantly increased in CaP tissue samples with positive TE status compared to negative TE status (TE-0, ND score = 1.5; TE-1, ND score = 2.0; P <0.01). NRP1, NRP2, PLXNA2, PLXNB1, SEMA3A, and SEMA4B mRNAs were detectable in CaP tissues and CaP cell lines at quite heterogeneous levels. In CaP tissues, we observed significant positive correlations of ERG with NRP2, PLXNA2, PLXNB1, and SEMA4B. TE-positive CaP tissues displayed enhanced nerve density. ERG correlated with some NRP/PLXN/SEMA components suggesting possible regulatory relevance of ERG for CaP innervation. [ABSTRACT FROM AUTHOR]