A novel epitope-presenting thermostable scaffold for the development of highly specific insulin-like growth factor-1/2 antibodies.

High sequence and structural homology between mature human insulin-like growth factors IGF-1 and IGF-2 makes serological discrimination by immunodiagnostic IGF tests a challenging task. There is an urgent need for highly specific IGF-1 and IGF-2 antibodies, yet only a short sequence element, i.e. the IGF loop, provides enough difference in sequence to discriminate between the two molecules. We sought to address this unmet demand by investigating novel chimeric immunogens as carriers for recombinant peptide motif grafting. We found Thermus thermophilus sensitive to lysis D (SlyD) and Thermococcus gammatolerans SlyD FK-506-binding protein (FKBP) domains suitable for presentation of the predefined epitopes, namely the IGF-1 and IGF-2 loops. Chimeric SlyD-IGF proteins allowed for the development of exceptionally specific IGF-1 and IGF-2 monoclonal antibodies. The selected antibodies bound with high affinity to the distinct IGF epitopes displayed on the protein scaffolds, as well as on the mature human IGF isoforms. The respective SlyD scaffolds display favorable engineering properties in that they are small, monomeric, and cysteine-free and can be produced in high yields in a prokaryotic host, such as Escherichia coli In conclusion, FKBP domains from thermostable SlyD proteins are highly suitable as a generic scaffold platform for epitope grafting.

Validation of a quantitative PCR based detection system for indoor mold exposure assessment in bioaerosols.

Determination and assessment of airborne fungal particles is complex and results of different sampling and analytical strategies are hard to compare due to limitations of each of the techniques. Here, an indoor mold detection system based on quantitative polymerase chain reaction (qPCR) is described and validated for its reliability and stability to identify airborne fungal particles collected. Data obtained from testing the system with fungal DNA, spore suspensions and bioaerosols indicated a need for spiking and normalization of measurements due to material loss and assay specific bias. Considering the loss of material during sample processing, detection limits defined for suspensions of Tritirachium oryzae spores were roughly 18 spores per sample. Detection of fungal spore mixtures nebulized under controlled conditions in a bioaerosol chamber showed generally 2-3 times higher normalized values measured with the molecular system compared to cultivation. Data obtained from a mold infested indoor sampling site and its corresponding outdoor reference measurement showed good correlations between qPCR and high-throughput sequencing (rho = 0.83, p < 0.01), if Cladosporium species were excluded. Taking necessary data normalization into account, the described qPCR detection system shows great potential to complement commonly used culture based approaches with the aim to improve the precision of indoor mold assessments. In contrast to already available qPCR assays that detect certain molds on a species level, this system covers a broad range of relevant fungal communities, serving as a promising alternative to high-throughput sequencing to identify indoor molds.

Deciphering the stepwise binding mode of HRG1ß to HER3 by surface plasmon resonance and interaction map.

For the development of efficient anti-cancer therapeutics against the HER receptor family it is indispensable to understand the mechanistic model of the HER receptor activation upon ligand binding. Due to its high complexity the binding mode of Heregulin 1 beta (HRG1ß) with its receptor HER3 is so far not understood. Analysis of the interaction of HRG1ß with surface immobilized HER3 extracellular domain by time-resolved Surface Plasmon Resonance (SPR) was so far not interpretable using any regular analysis method as the interaction was highly complex. Here, we show that Interaction Map (IM) made it possible to shed light on this interaction. IM allowed deciphering the rate limiting kinetic contributions from complex SPR sensorgrams and thereby enabling the extraction of discrete kinetic rate components from the apparently heterogeneous interactions. We could resolve details from the complex avidity-driven binding mode of HRG1ß with HER3 by using a combination of SPR and IM data. Our findings contribute to the general understanding that a major conformational change of HER3 during its activation is induced by a complex sequential HRG1ß docking mode.

The GATA factor AreA regulates localization and in vivo binding site occupancy of the nitrate activator NirA.

The GATA factor AreA is a wide-domain regulator in Aspergillus nidulans with transcriptional activation and chromatin remodelling functions. AreA interacts with the nitrate-specific Zn(2)-C(6) cluster protein NirA and both proteins cooperate to synergistically activate nitrate-responsive genes. We have previously established that NirA in vivo DNA binding site occupancy is AreA dependent and in this report we provide a mechanistic explanation for our previous findings. We now show that AreA regulates NirA at two levels: (i) through the regulation of nitrate transporters, AreA affects indirectly the subcellular distribution of NirA which rapidly accumulates in the nucleus following induction; (ii) AreA directly stimulates NirA in vivo target DNA occupancy and does not act indirectly by chromatin remodelling. Simultaneous overexpression of NirA and the nitrate transporter CrnA bypasses the AreA requirement for NirA binding, permits utilization of nitrate and nitrite as sole N-sources in an areA null strain and leads to an AreA-independent nucleosome loss of positioning.

Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions.

The major laccase isoenzyme LAP2 secreted by the white-rot basidiomycete Trametes pubescens in response to high copper concentrations was purified to apparent electrophoretic homogeneity using anion-exchange chromatography and gel filtration. The monomeric protein has a molecular mass of 65 kDa, of which 18% is glycosylation, and a pI value of 2.6. The pH optima of the laccase depend on the substrates oxidized and show bell-shaped pH activity profiles with an optimum of 3-4.5 for phenolic substrates such as 2,6-dimethoxyphenol or syringaldazine, while the non-phenolic substrates ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] and ferrocyanide show a monotonic pH profile with a rate increasing with decreasing pH. The catalytic efficiencies k(cat)/K(m) determined for some of its substrates were 48 x 10(6), 47 x 10(6), 20 x 10(6) and 7 x 10(6) M(-1) s(-1) for ABTS, syringaldazine, ferrocyanide and oxygen, respectively. Furthermore, the gene lap2 encoding the purified laccase was cloned and its nucleotide sequence determined. The gene consists of 1997 bp, with the coding sequence interrupted by eight introns and flanked by an upstream region in which putative CAAT, TATA, MRE and CreA consensus sequences were identified. Based on Northern analysis containing total RNA from both induced and uninduced cultures, expression of lap2 is highly induced by copper, which is also corroborated by an increase in laccase activity in response to copper. A stimulating effect of various other heavy metal ions on laccase synthesis was also observed. In addition to induction, a second regulatory mechanism seems to be repression of lap2 transcription by glucose.

Nitrate and the GATA factor AreA are necessary for in vivo binding of NirA, the pathway-specific transcriptional activator of Aspergillus nidulans.

In Aspergillus nidulans, the genes coding for nitrate reductase (niaD) and nitrite reductase (niiA), are transcribed divergently from a common promoter region of 1200 basepairs. We have previously characterized the relevant cis-acting elements for the two synergistically acting transcriptional activators NirA and AreA. We have further shown that AreA is constitutively bound to a central cluster of four GATA sites, and is involved in opening the chromatin structure over the promoter region thus making additional cis-acting binding sites accessible. Here we show that the asymmetric mode of NirA-DNA interaction determined in vitro is also found in vivo. Binding of the NirA transactivator is not constitutive as in other binuclear C6-Zn2+-cluster proteins but depends on nitrate induction and, additionally, on the presence of a wild-type areA allele. Dissecting the role of AreA further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly exert its effect on NirA via inducer exclusion. We have tested this possibility in a strain accumulating nitrate in the absence of areA. We found that in such a strain the intracellular presence of inducer is not sufficient to promote either chromatin rearrangement or NirA binding, implying that both processes are directly dependent on AreA.