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In filamentous fungi, arginine methylation has been implicated in morphogenesis, mycotoxin biosynthesis, pathogenicity, and stress response although the exact role of this posttranslational modification in these processes remains obscure. Here, we present the first genome-wide transcriptome analysis in filamentous fungi that compared expression levels of genes regulated by type I and type II protein arginine methyltransferases (PRMTs). In Aspergillus nidulans, three conserved type I and II PRMTs are present that catalyze asymmetric or symmetric dimethylation of arginines. We generated a double type I mutant (ΔrmtA/rmtB) and a combined type I and type II mutant (ΔrmtB/rmtC) to perform genome-wide comparison of their effects on gene expression, but also to monitor putative overlapping activities and reciprocal regulations of type I and type II PRMTs in Aspergillus. Our study demonstrates, that rmtA and rmtC as type I and type II representatives act together as repressors of proteins that are secreted into the extracellular region as the majority of up-regulated genes are mainly involved in catabolic pathways that constitute the secretome of Aspergillus. In addition to a strong up-regulation of secretory genes we found a significant enrichment of down-regulated genes involved in processes related to oxidation-reduction, transmembrane transport and secondary metabolite biosynthesis. Strikingly, nearly 50% of down-regulated genes in both double mutants correspond to redox reaction/oxidoreductase processes, a remarkable finding in light of our recently observed oxidative stress phenotypes of ΔrmtA and ΔrmtC. Finally, analysis of nuclear and cytoplasmic extracts for mono-methylated proteins revealed the presence of both, common and specific substrates of RmtA and RmtC. Thus, our data indicate that type I and II PRMTs in Aspergillus seem to co-regulate the same biological processes but also specifically affect other pathways in a non-redundant fashion.

Copper containing particles are of high interest to provide antibacterial activity to textiles for medical products, hygiene application or where odor formation as result of bacterial activity has to be controlled. Cu(i)oxide microparticles with a rather uniform diameter between 1.5 and 2 μm can be prepared by controlled reduction of alkaline Cu(ii)-tartaric acid complexes. Such particles can be bound to textile surfaces by means of a pigment binder system used in pigment dyeing. By a simple pad-dry process textile fabrics with a Cu-content of 250-270 mg Cu per kg fabric could be prepared. The samples (fabrics) exhibited a reduction in viability of 100% for Staphylococcus aureus and 84% for Klebsiella pneumonia as estimated by the ASTM E2149 antimicrobial test. Simulated wash procedures led to a reduction in Cu-content to 60-50% of the initial value. Reduction in viability remained at 99% for Staphylococcus aureus and 78% for Klebsiella pneumoniae. The new process is of high value to impart antimicrobial properties to textile products because an antimicrobial product with good wash permanence can be delivered using rather simple processing and ordinary chemicals.

Fabrics with antibacterial properties are of interest as medical textiles and also in hygiene applications where odor formation through bacterial activity has to be prevented. In this work Cu-containing lyocell fibers and fabric were prepared and characterized. Insertion of copper was obtained by treatment of cellulose fabrics with copper– d -gluconate complexes. The stable Cu(II)– d -gluconate complex was used to achieve controlled insertion of copper ions into the cellulose matrix. Secondly, the reduction of copper ions was achieved by use of sodium borohydride. Copper content in treated fabrics was determined by AAS; values ranged between 18.5 and 23.7 mmol Cu/kg cellulose. After five hand-washing cycles, 30% of the initial copper amount was left on the cellulose fabrics. Copper containing particles were identified with electron microscopy and by energy dispersive X-ray (EDX) analysis. The antibacterial properties of copper containing fabrics were tested against Staphylococcus aureus . Even with a rather low copper content (2.6 mmol Cu/kg cellulose fiber) 90% reduction of bacterial viability was achieved after 3 h of contact time.

Indigenous chemolithotrophic nitrifying and sulfur-oxidizing culture enrichments mobilized metals from pristine mineral soil under conditions of ammonium or thiosulfate supplementation in a laboratory experiment carried out over a period of 40 days. The average mineralogical composition of the mineral soil was quartz (62%), feldspar (20%), muscovite (6%), chlorite (2%), hornblende (2%), dolomite (4%), and calcium carbonate (4%). The leaching efficiency of the nitrifying enrichment was calcium (27%), magnesium (15%), zinc (5.4%), manganese (0.6%), and cobalt (1.4%) after 40 days of incubation. In case of sulfur-oxidizing enrichment, leaching efficiency was calcium (56%), magnesium (36%), iron (0.8%), zinc (12%), manganese (2.1%), and cobalt (12%). The impact of these organisms on pristine mineral soil could be important in understanding primary colonization and the early stages of soil formation.

β-Lactoglobulin (BLG) is a member of the lipocalin protein family and a major food-borne allergen in humans. Numerous in vitro studies have suggested a role for BLG in molecular transport processes; however, its physiological role remains enigmatic. A cellular receptor for BLG has been proposed, but has not yet been identified. Here we show that human LIMR, known to act as an endocytic receptor for lipocalin-1, also binds bovine BLG and mediates its cellular uptake. The specificity of this interaction is corroborated by a complete block of cellular uptake of BLG in the presence of LIMR antibodies or LIMR downregulation by antisense RNA. Furthermore, heterologous expression of human LIMR in insect cells mediates cellular internalization of FITC-BLG. Since LIMR is highly expressed in the human intestine, it might also function in the uptake of food-borne BLG.

Multiangle laser light scattering and fluorescence anisotropy decay measurements clarified the oligomeric states of native and recombinant tear lipocalin (lipocalin-1, TL). Native TL is monomeric. Recombinant TL (5-68 microM) with or without the histidine tag shows less than 7% dimer formation that is not in equilibrium with the monomeric form. Fluorescence anisotropy decay showed a correlation time of 9-10 ns for TL (10 microM-1 mM). Hydrodynamic calculations based on the crystallographic structure of a monomeric TL mutant closely concur with the observed correlation time. The solution properties calculated with HYDROPRO and SOLPRO programs from the available crystallographic structure of a monomeric TL mutant concur closely with the observed fluorescence anisotropy decay. The resulting model shows that protein topology is the major determinant of rotational correlation time and accounts for deviation from the Stokes-Einstein relation. The data challenge previous gel filtration studies to show that native TL exists predominantly as a monomer in solution rather than as a dimer. Delipidation of TL results in a formation of a complex oligomeric state (up to 25%). These findings are important as the dynamic processes in the tear film are limited by diffusional, translational as well as rotational, properties of the protein.

There is a lack of relevant methods to assess the colonization of textiles by skin bacteria because present methods are mainly culture-based procedures. Therefore, the goal of this study was to develop a fast and sensitive culture-independent procedure for the quantification of microbial colonization and growth on textiles. We have established a suitable protocol to use DNA quantification as a reliable method for in vitroand in vivoinvestigations of textiles. For DNA extraction, a two-step procedure comprising treatment of the textile with a solution containing Triton X-100 and lysozyme for 1 h and a successive treatment by SDS and proteinase K for 2 h turned out to be most efficient. DNA extracted from textiles and fabrics was than quantified with the highly sensitive PicoGreen fluorescent dye. In vitrochallenge tests demonstrated a strong correlation between numbers of bacteria on textiles and amount of DNA extracted from textiles. Therefore, this method was used to compare different materials after in vivotrials for assessment of their susceptibility for microbial colonization and growth.

Human tear lipocalin (TL; also known as Lcn1) is a secretory protein present in large amounts in fluids that cover epithelial surfaces such as tears and respiratory secretions. It is supposed to act as a physiological scavenger of hydrophobic, potentially harmful molecules, but there is evidence that it also inhibits bacterial growth. In the present study, we reconsidered the possibility that TL might interfere with microbial growth by scavenging of siderophores, as described for human neutrophil gelatinase-associated lipocalin (NGAL). Indeed, our experiments revealed that TL binds to microbial siderophores with high affinities. In contrast to NGAL, which was shown to have some specificity for bacterial catecholate-type siderophores, TL binds to a broad array of siderophores, including bacterial catecholate-type enterobactin and hydroxamate-type desferrioxamine B, and all major classes of fungal siderophores. By adding exogenous TL, bacterial and fungal growth could be inhibited under iron-limiting conditions. Thus, TL might be a novel member of the innate immune system especially involved in mucosal defense against fungal infections.

Human lipocalin-1 (Lcn-1, also called tear lipocalin), a member of the lipocalin structural superfamily, is produced by a number of glands and tissues and is known to bind an unusually large array of hydrophobic ligands. Apart from its specific function in stabilizing the lipid film of human tear fluid, it is suggested to act as a physiological scavenger of potentially harmful lipophilic compounds, in general. To characterize proteins involved in the reception, detoxification, or degradation of these ligands, a cDNA phage-display library from human pituitary gland was constructed and screened for proteins interacting with Lcn-1. Using this method an Lcn-1 interacting phage was isolated that expressed a novel human protein. Molecular cloning and analysis of the entire cDNA indicated that it encodes a 55-kDa protein, lipocalin-1 interacting membrane receptor (LIMR), with nine putative transmembrane domains. The cell membrane location of this protein was confirmed by immunocytochemistry and Western blot analysis of membrane fractions of human NT2 cells. Independent biochemical investigations using a recombinant N-terminal fragment of LIMR also demonstrated a specific interaction with Lcn-1 in vitro. Based on these data, we suggest LIMR to be a receptor of Lcn-1 ligands. These findings constitute the first report of cloning of a lipocalin interacting, plasma membrane-located receptor, in general. In addition, a sequence comparison supports the biological relevance of this novel membrane protein, because genes with significant nucleotide sequence similarity are present in Takifugu rubripes, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Bos taurus, and Sus scrofa. According to data derived from the human genome sequencing project, the LIMR-encoding gene has to be mapped on human chromosome 12, and its intron/exon organization could be established. The entire LIMR-encoding gene consists of about 13.7 kilobases in length and contains 16 introns with a length between 91 and 3438 base pairs.

We have cloned and sequenced the entire genomic fragment of the porcine LCN1 gene which encodes Tear lipocalin/von Ebner's gland protein, a member of the lipocalin superfamily highly expressed in porcine lachrymal and lingual glands. The porcine LCN1 gene is approximately 4.6 kb in size and contains six protein-coding exons and a 3′-nontranslated exon. The structure of this porcine gene is highly similar, in terms of numbers of exons/introns, in size of exons and in intron phasing, to that of the human LCN1 and rat VEGP genes, thus supporting a very close evolutionary relationship of these genes. Within the promoter region of the porcine LCN1 a putative TATA box, a CAAT box and two MRE motifs are found. The same MRE motifs are conserved in the human LCN1 promoter, suggesting that they might be of relevance for LCN1 gene expression. However, additional motifs present in the human LCN1 promoter, such as AP-1 and AP-2 sites, a NF-κB site, and a cAMP-responsive element, could not be detected in the porcine LCN1 promoter.

Northern analysis of human multiple tissue blots containing poly A+ RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes revealed that a prostate specific transcript hybridizes to a tear lipocalin/von Ebner's gland protein (TL/VEGP) gene probe. To characterize this transcript, the corresponding cDNA was amplified by reverse transcription (RT)-PCR. Cloning and sequence analysis showed that it was identical to the tear lipocalin cDNA isolated from human lachrymal glands. Immunohistochemical analysis on thin layer sections of human prostate using a tear lipocalin specific antiserum confirmed the expression of this cDNA in prostate. Thus, our results clearly argue against a unique function of TL/VEGP in human tear fluid or saliva. The human cDNA was expressed in E. coli using the pQE system yielding a recombinant protein which shows biochemical properties identical to the native TL/VEGP.

Apolipoprotein D (apoD), a glycoprotein originally characterized as a component of the high density lipoprotein fraction of human plasma and known to be a member of the lipocalin protein superfamily, has been found in human tear fluid by Western blot analysis. Unlike serum it seems that in the tear fluid apoD exists mainly as a disulphide linked homodimer which is not associated with lecithin/cholesterol acyltransferase (LCAT) or apolipoprotein A-I (apo A-I). By reverse-transcription-PCR (RT-PCR) of mRNA extracted from a human lacrimal gland and use of specific primers we could demonstrate expression of the apoD gene in this tissue. The amplified cDNA was cloned and a subsequent sequence analysis confirmed the identity of apoD mRNA in the human lacrimal gland. These investigations indicate that the lacrimal gland is the site of synthesis of the tear fluid apoD. Although the physiological function of apoD is unknown, it has the ability to bind phospholipids, cholesterol and other small hydrophobic molecules. Therefore, this protein might interact with meibomian lipids present in human tear fluid and probably contribute to the surface spreading of these lipids or it may function as a clearance factor, protecting the cornea from harmful lipophilic molecules.