Copper(i)oxide microparticles - synthesis and antimicrobial finishing of textiles.

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.

The N-terminal part of recombinant human tear lipocalin/von Ebner's gland protein confers cysteine proteinase inhibition depending on the presence of the entire cystatin-like sequence motifs.

Human Tear Lipocalin/von Ebner's gland protein (TL) is a member of the lipocalin superfamily. The protein is secreted by a number of serous glands and tissues and is overproduced under conditions of stress, infection and inflammation. In addition to its typical affinity for lipophilic ligands it was recently found to be able to inhibit cysteine proteinases [van't Hof et al., J. Biol. Chem. 272 (1997), 1837-1841], probably due to the presence of amino acid motifs resembling the papain binding domains of family 2 cystatins. In this work we have used a recombinant protein to confirm the results obtained with native TL. The inhibitory activity of the recombinant protein against papain was dependent on the ratio of papain and TL. At higher papain concentrations, the N-terminal sequence of TL was cleaved off by the protease, indicating that it can act in an inhibitor- or a substrate-like mode. This behaviour resembles that observed with certain chicken cystatin mutants. Using a recombinant TL mutant we found that the two Leu residues (Leu4-Leu5) contained within the first cystatin-like motif are absolutely essential for the inhibitory activity. These results were supported by experiments using a recombinant form of the corresponding pig von Ebner's gland protein (VEGp). This protein, which does not possess a fully conserved first cystatin-like motif, is unable to inhibit papain.

Molecular cloning of a novel lipocalin-1 interacting human cell membrane receptor using phage display.

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.

Phage display reveals a novel interaction of human tear lipocalin and thioredoxin which is relevant for ligand binding.

Human tear lipocalin (TL) is an unusual member of the lipocalin protein family, since it is known to bind a large variety of lipophilic ligands in vivo and acts as a cysteine proteinase inhibitor in vitro. It is suggested to function as a physiological protection factor by scavenging lipophilic potentially harmful compounds. Since protein-protein interaction or macromolecular complexation is a common feature of many lipocalins, we applied phage display technology to identify TL interacting proteins. By panning of a human prostate cDNA phagemid library against purified TL we isolated a thioredoxin (Trx) encoding phage clone. Biochemical analysis revealed that TL indeed interacts with Trx and is reduced by this redox protein. Reduction of the TL-specific disulfide bond is of functional relevance, since the reduced protein shows a nine-fold increase in ligand affinity when tested with retinoic acid as ligand.

Complement component C8gamma is expressed in human fetal and adult kidney independent of C8alpha.

Human complement component C8gamma is an unusual complement factor since it shows no homology to other complement proteins but is a member of the lipocalin superfamily. So far, it has been found exclusively in plasma, covalently linked to C8alpha by disulfide bridging. We have used dot blot and Northern blot analyses of a large number of different human tissues to survey systematically the expression pattern of C8gamma. Our experiments clearly showed that besides in liver, this gene is also expressed in fetal and adult kidney. Renal expression of C8gamma is not dependent on C8alpha expression, since we could not detect C8alpha expression in kidney. Thus its physiological function is not restricted to a specific action in association with complement components. As a prerequisite for further characterization of the structure and binding activities of the uncomplexed C8gamma, we have expressed the encoding cDNA in Escherichia coli. To increase the probability for proper folding of the characteristic intramolecular disulfide bridge the recombinant protein was produced by secretion to the periplasm.

Structure and organization of the porcine LCN1 gene encoding Tear lipocalin/von Ebner's gland protein.

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-kappaB site, and a cAMP-responsive element, could not be detected in the porcine LCN1 promoter.

Expression of the gene for tear lipocalin/von Ebner's gland protein in human prostate.

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.

The human lacrimal gland synthesizes apolipoprotein D mRNA in addition to tear prealbumin mRNA, both species encoding members of the lipocalin superfamily.

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.