Advanced characterization of natural biofilm on nanofiber scaffold.

Nanofiber scaffolds provide numerous advantages over common carriers engineered for microorganisms. The most important advantage is an increased speed of primary surface colonization (up to four times faster), which shortens the time required for the areal biofilm formation and optimum performance of attached microorganisms (higher efficiency of biological activity of up to twice as fast). Image analysis predicts early formation of biofilm even in beginning stages; analysis of biofilm reveals the different structures of bacterial colonies on both scaffolds (higher porosity, size, and number of bacterial colonies on nanofiber's surface). The image analysis correlates well with determinations of dry matter (linear correlation of 0.96) and proteins (linear correlation of 0.89).

Joint analysis of the NACP-REP1 marker within the alpha synuclein gene concludes association with alcohol dependence.

Various studies have linked alcohol dependence phenotypes to chromosome 4. One candidate gene is NACP (non-amyloid component of plaques), coding for alpha synuclein. Recently, it has been shown that alpha synuclein mRNA is increased in alcohol-dependent patients within withdrawal state. This increase is significantly associated with craving, especially obsessive craving. On the basis of these observations, the present study analysed two polymorphic repeats within the NACP gene. We found highly significant longer alleles of NACP-REP1 in alcohol-dependent patients compared with healthy controls (Kruskal-Wallis test, chi(2)=99.5; df=3, P<0.001). In addition, these lengths significantly correlate with levels of expressed alpha synuclein mRNA (chi(2)=8.83; df=2, P=0.012). The present results point to a novel approach for a genetic determination of craving, a key factor in the genesis and maintenance not only of alcoholism but also of addiction in general.

Development of a 13-locus PCR multiplex system for paternity testing.

In this study the development of a 13-locus multiplex-PCR system fitting the updated demands for paternity testing in Germany is described. For this purpose an existing multiplex PCR system that allows the simultaneous amplification of eight different STR loci together with the sex-specific locus amelogenin ( genRESMPX-2, Serac, Germany) was extended. Whereas some of the primers were taken from the underlying multiplex system, suitable primer sequences were chosen for the STR loci D19S433, TPOX, TH01, D16S539, D5S818, D2S1338 and FGA. Primers of loci resulting in potentially overlapping fragment sizes were labelled with the fluorescent dyes 6-FAM, JOE and NED. Reaction conditions, such as annealing temperature, concentrations of primers and polymerase or buffer conditions were optimised to obtain a robust amplification and reproducible genotype analysis for various sample sources. Full DNA profiles from single source samples were reliably typed from template DNA amounts of as low as 120 pg, suggesting a potential use of this system also in forensic casework analysis. With a mean exclusion chance (MEC) of 99.9989% and a power of discrimination (P(D)) of about 1x10(14) (Caucasians), the new multiplex PCR system provides a significant and sensitive system for forensic DNA analysis. On the basis of these studies, a commercial kit system is now provided by Serac (Bad Homburg, Germany, genRESMPX-3).

Validation of the multiplex kit genRESMPX-2 for forensic casework analysis.

Validation studies were carried out using the commercially available PCR multiplex system genRESMPX-2. In addition to amelogenin, this system comprises the complete set of eight STR systems which are components of the German DNA database established in 1998 by the Federal Criminal Office of Germany (BKA). The minimum amount of template DNA which gave a complete DNA pattern ranged between 100 pg and 200 pg. Mixed samples could clearly be assigned from ratios between 1:5 (ACTBP2) and 1:20 (VWA, FGA). Experimental investigations with different forensic materials, environmental studies, reproducibility and precision data as well as practical casework analysis revealed that the genRESMPX-2 kit can be regarded as a sensitive, reliable and robust multiplex system even in the case of samples containing limited amounts or degraded DNA.

Purification of STR-multiplex-amplified microsamples can enhance signal intensity in capillary electrophoresis.

In this study, the effect of sample purification on total signal intensities of samples amplified with genRES MPX-2 (nine-locus multiplex) prior to capillary electrophoretic analysis has been investigated. Sample purification with the Qiaquick PCR purification kit led to an increase of the relative fluorescent signal intensity by a factor of 3.8 +/- 0.8. In contrast, the application of larger sample volumes led to a decrease of signal intensities from 20% to 80%, depending on whether the samples were purified or not. In addition, increase of injection time showed a linear increase of signal intensity between 3 s and 10 s. Increasing the number of PCR cycles from 30 to 33 also led to a significant increase of signal intensities. Nevertheless, this increase greatly depended on the fragment lengths and was sometimes accompanied by the appearance of non-specific signals. In combination, optimisation of sample preparation and increase of injection time may intensify signals up to 12-fold, thereby increasing the overall sensitivity of the assay. This may be of special interest for forensic analysis of microspecimens containing limited amounts of DNA.

Biodegradability of ethylenediamine-based complexing agents.

Biological degradability of ethylenediamine derivatives depends on the type and number of substituents. The susceptibility to biodegradation decreases in the sequence of substituents -COCH3, -CH3, -C2H5, -CH2CH2OH, -CH2COOH and with polysubstitution. The biodegradability depends also on the kind and number of nitrogen atoms. Complexing agents with a single-nitrogen atom in the molecule (e.g. NTA) succumb relatively readily to biodegradation whereas, compounds with two or more tertiary amino groups are biologically highly stable and do not undergo biodegradation even in experiments with activated sludge adapted at an age of up to 30 days (EDTA, DTPA, PDTA, HEDTA). A lowering of the degree of substitution brings about an increased susceptibility to biodegradation. This holds, e.g., for replacement of tertiary amino groups with secondary ones; thus the symmetrically disubstituted ethylenediamine-N,N'-diacetic acid (EDDA) possesses still sufficient complexing ability while belonging already to the group of potentially degradable substances.

DNA analysis of fingernail debris using different multiplex systems: a case report.

A 55-year-old male nurse was accused of having introduced his fingers by force into the anus of a 20-year-old female patient. Debris from the fingernails of the suspect recovered 2 days after the incident was analysed with the VNTR locus D1S80, the triplex PCR system AmpFlSTR Blue kit, the AmpFlSTR Profiler kit and the pentaplex system genRES MPX. The D1S80 singleplex reaction revealed indications of DNA from the victim in the fingernail debris of the left hand. Using the AmpFlSTR Blue kit and AmpFlSTR Profiler, DNA alleles of the victim were found at four additional loci, while allelic drop-out was observed at five other loci. Only the pentaplex kit genRES MPX revealed alleles at all loci which could be assigned to the victim. Calculation of likelihood ratios resulted in a value of 1.4 x 10(5) using the combination of the multiplex systems AmpF1STR Blue kit and AmpFlSTR Profiler and 2.8 x 10(8) for the genRES MPX kit. This case demonstrates the high sensitivity of the new genRES MPX kit and that DNA profiling of fingernail debris is possible despite a time lapse of 2 days between the incident and recovery of the evidential material.

A new pentaplex PCR system for forensic casework analysis.

In 1998 the Federal Criminal Police Office of Germany (BKA) established a central genetic database of offenders and suspects to facilitate comparisons with biological samples from future criminal offences. The five obligatory short tandem repeat (STR) loci in this database (TH01, SE33, vWA, FGA and D21S11) were co-amplified in a new PCR pentaplex analysing system together with the sex-specific locus amelogenin. Due to overlapping fragment sizes, amplification products were fluorescent dye-labelled with different colours, separated by electrophoresis and detected directly using the ABI PRISM 310 Genetic Analyzer. Reproducible and reliable results were obtained from as low as 125 pg template DNA, indicating high specificity and sensitivity of the assay. Environmental studies and enzymatic digest with DNase I revealed an excellent stability of the pentaplex system with typeable results even in cases of partially degraded DNA. Complete and reproducible DNA typing was possible in blood-stain mixtures with the minor component as low as 10%. Mean stutter peak intensities were analysed for all loci and ranged from 2.7 +/- 0.8% (TH01) to 10.6 +/- 1.6% (vWA) of the main signal intensity. Allele frequencies were determined in a North Bavarian population sample (n = 121). The combination of five systems resulted in a mean exclusion chance of 99.86% and a power of discrimination of 99.999996%. No deviation from Hardy-Weinberg equilibrium could be found.

Population genetics of nine short tandem repeat (STR) loci - DNA typing using the AmpFlSTR profiler PCR amplification kit.

Frequency data for nine short tandem repeat (STR) loci were collected from 130 unrelated Caucasians from North Bavaria using the AmpFlSTR Profiler multiplex system. The loci D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820 and the sex test amelogenin were investigated. Allele frequencies, rates of heterozygosity and the discrimination power of the combined systems were calculated by statistical analysis. Except for D5S818 all loci met Hardy-Weinberg expectations.

Tetracycline-inducible expression systems with reduced basal activity in mammalian cells.

We describe a modification of the tetracycline-inducible eukaryotic gene expression system with decreased basal levels of expression in HeLa cells. It employs the tetracycline-inducible transactivator and a tetracycline-regulated repressor fusion acting on the same promoter. To avoid heterodimerization or competition for the same DNA site, each was provided with different DNA recognition and/or protein dimerization specificities. We achieved active silencing in the uninduced state resulting in approximately 6-fold reduced levels of basal transcription and several hundred-fold activation of gene expression upon addition of tetracycline.

Intragenic suppressors of induction-deficient TetR mutants: localization and potential mechanism of action.

Eight Tn10 Tet repressor mutants with an induction-deficient phenotype and with primary mutations located at or close to the dimer interface were mutagenized and screened for inducibility in the presence of tetracycline. The second-site suppressors with wild-type-like operator binding activity that were obtained act, except for one, at a distance, suggesting that they contribute to conformational changes in the Tet repressor. Many of these long-range suppressors occur along the dimer interface, indicating that interactions between the monomers play an important role in Tet repressor induction.

Tetracycline analogs affecting binding to Tn10-Encoded Tet repressor trigger the same mechanism of induction.

We examined the influence of substituents in tetracycline (tc) analogs modified at positions 2 and 4-9 and anhydrotetracycline (atc) on induction of the Tn10-encoded Tet repressor (TetR) by a quantitative in vitro induction assay. The equilibrium association constants of the modified tc to TetR were independently determined to distinguish effects on binding from those on induction. We found a correlation between the binding affinity and induction of TetR for most tc analogs. While a substitution at position 5 revealed only minor effects, changes at position 6 increased binding and induction efficiencies up to 20-fold. A chlorine at position 7 or 8 enhanced binding and induction about 4- and 9-fold, respectively. Substituents at position 9 decreased binding up to 5-fold. Epimerization of the dimethylamino function at position 4 in 4-epi-tc resulted in about 300-fold-reduced binding and 80-fold-reduced induction. Substitution of this grouping by hydrogen in 4-de(dimethylamino)-tc resulted in no binding and no induction. The respective atc analog failed to induce as well, although binding was still observed. The dimethylamino function may, thus, play a role in triggering the conformational change of TetR necessary for induction. Substitution of the 2-carboxamido by a nitrilo function did not influence binding and induction efficiencies. Atc showed about 30-fold increased binding and induction, being the most effective inducer tested in this study. The equilibrium association constants of most TetR-[Mg-tc]+ and TetR-([Mg-tc]+)2 analog complexes with tet operator are decreased about 10(2)- and 10(8)-fold, respectively, as compared to those of free TetR. This suggests that these tc analogs share the same molecular mechanism of TetR induction.

Thermodynamic analysis of tetracycline-mediated induction of Tet repressor by a quantitative methylation protection assay.

We describe a method for quantitative detection and thermodynamic interpretation of tetracycline (tc)-mediated induction of the Tn10 encoded Tet repressor (TetR). Binding of dimeric TetR to the tet operator (tetO) was quantitated by protection of DNA from methylation as a function of te concentration. A thermodynamic scheme covering all single reactions relevant for TetR induction was used to interpret the data. The equilibrium association constants of the TetR-[Mg-tc]+ and TetR-[Mg-tc]2+ complexes to tetO were determined at different NaCl and TetR concentrations. Variation of total TetR concentration from 0.2 to 1.1 x 10(-7) M yielded identical results. A strong salt dependency of TetR-tetO binding was verified between 2.5 and 100 mM NaCl, whereas [Mg-tc]+ binding to TetR is independent of the ionic strength. The TetR-tetO binding constant drops 10(2)- to 10(3)-fold upon binding of the first and further 10(4)- to 10(7)-fold by binding of the second [Mg-tc]+. This apparent cooperativity of tc-mediated induction indicates that each [Mg-tc]+ interacts with both TetR monomers.

Proximity mapping of the Tet repressor-tetracycline-Fe2+ complex by hydrogen peroxide mediated protein cleavage.

We demonstrate in a quantitative in vitro induction assay that tetracycline-Fe2+ is a more than 1000-fold stronger inducer of Tet repressor compared to tetracycline-Mg2+. Oxidative cleavage of the Tet repressor-tetracycline-Fe2+ complex with H2O2 and ascorbate results in an Fe(2+)-dependent specific fragmentation of the protein. The maximal yield of about 15% and a reaction time of less than 30 s are only observed in the presence of the drug, whereas about 1% cleavage is obtained after 30 min in the presence of Fe2+ without tetracycline. Cleavage is not inhibited by several radical scavengers, suggesting a highly localized reactivity of the redox-active oxo intermediates in the proximity of the Fe(2+)-tc chelater where they are generated. The products can be separated by HPLC only after denaturation, indicating that the complex is not disrupted by cleavage. Residues at which the cleavage takes place are identified using the masses of the fragments determined by electrospray mass spectrometry and their N-terminal sequences. The major cleavage site maps to residues 104 and 105 of Tet repressor. Less efficient cleavages occur at residues 56 and 136, and the least efficiently cleaved sites are around residues 144 and 147. The cleavage efficiencies correlate to the distances and orientations of the respective peptide bonds to Mg2+ in the crystal structure of the Tet repressor-tetracycline-Mg2+ complex. We discuss potential reaction mechanisms leading to protein cleavage.

Matrix effects suggest an important influence of DNA-polyacrylamide interactions on the electrophoretic mobility of DNA.

The electrophoretic mobilities of 34 DNA fragments with a varied phasing of two A-tract bends have been determined in different polyacrylamide gels. They are perfectly described by a cosine function. A systematic variation of the polyacrylamide gel matrix revealed that not only the absolute electrophoretic mobilities of these distance variants, but also their amplitudes and the positions of their extrema, depend on the polyacrylamide content. The displacement of the extrema with increasing polyacrylamide content is attributed to gel-induced unwinding of the DNA. None of these matrix effects can be explained by the change of the pore sizes, which is generally regarded as the central parameter determining electrophoretic mobility of DNA in gels. Instead, we hypothesize an interaction between the electrophoresed DNA and the polyacrylamide matrix and discuss a new qualitative model for electrophoresis of DNA in polyacrylamide gels which accounts for these observations.