Effects of metal ions on fibroblasts and spiral ganglion cells.

Degeneration of spiral ganglion cells (SGC) after deafness and fibrous tissue growth around the electrode carrier after cochlear implantation are two of the major challenges in current cochlear implant research. Metal ions are known to possess antimicrobial and antiproliferative potential. The use of metal ions could therefore provide a way to reduce tissue growth around the electrode array after cochlear implantation. Here, we report on in vitro experiments with different concentrations of metal salts with antiproliferative and toxic effects on fibroblasts, PC-12 cells, and freshly isolated spiral ganglion cells, the target cells for electrical stimulation by a cochlear implant. Standard cell lines (NIH/3T3 and L-929 fibroblasts and PC-12 cells) and freshly isolated SGC were incubated with concentrations of metal ions between 0.3 µmol/liter and 10 mmol/liter for 48 hr. Cell survival was investigated by neutral red uptake, CellQuantiBlue assay, or counting of stained surviving neurons. Silver ions exhibited distinct thresholds for proliferating and confluent cells. For zinc ions, the effective concentration was lower for fibroblasts than for PC-12 cells. SGC showed comparable thresholds for reduced cell survival not only for silver and zinc ions but also for copper(II) ions, indicating that these ions might be promising for reducing tissue growth on the surface of CI electrode arrays. These effects were also observed when combinations of two of these ions were investigated.

[Concept of a pressure-controlled microstent for glaucoma therapy]. / Konzept eines druckgesteuerten Mikrostents für die Glaukomtherapie.

A pressure-controlled microstent could permanently normalise the intraocular pressure (IOP) for open-angle glaucoma therapy by drainage into the suprachoroidal space. The complex requirements demand new technical solutions as well as an improved understanding of specific cell biological processes at the implant's surface to develop effective local drug delivery (LDD) concepts and surface modifications. Fluid mechanical requirements were derived from physiological data and the analysis of commercial glaucoma implants. The technological basics for the production of suitable structures are refined ultra-short pulse laser technology and 2-photon polymerisation (2PP). All known glaucoma implants induce unwanted cell proliferation resulting in a loss of function. It is assumed that the activity of fibroblasts is low in the suprachoroidal space. However, it was seen that LDD concepts are required to control cell proliferation. Fibroblasts from sclera and choroidea were isolated und cultured as the most relevant cell types for in vitro investigation. Potential materials and drugs were investigated by cell viability tests for biocompatibility or suppression of cell viability. The fluid mechanical analysis leads to smallest stent lumina (ID = 50 µm) at anatomically suitable implant lengths (7 - 10 mm). Only pressure control can manage the individual conditions with changing IOP. Finite element analysis of valves showed the need for highly flexible structures. This can be achieved by combining basic structures with micromechanically active valves added by 2PP. The potential materials show perfect in vitro and in vivo biocompatibility. Ormocers which are best suited for 2PP are also highly biocompatible. The selected drugs paclitaxel and triamcinolon acetonide open a wide therapeutic window to impair fibroblast growth. The surgical procedure was established by implantation of prototypes in rabbit eyes, connecting the anterior chamber with the suprachoroidal space. Highly flexible implants are required for correct placement within the eye. The new concept of the microstent combines biomechanical approaches, technologies for microfabrication and current LDD concepts and opens new perspectives for glaucoma therapy.

Protective effect of endotoxin preconditioning in pancreas: analysis with DNA chip arrays.

Recently we demonstrated a protective effect of endotoxin preconditioning 24 hours before pancreatic ischemia-reperfusion injury, which has also been described for other organs. The mechanisms underlying this phenomenon, such as differential gene expression, are poorly investigated. We chose to approach this question by investigating differential gene expression in the rat pancreas over the time course of endotoxin pretreatment. Male Wistar rats (5 groups, 5 animals per group) were pretreated with endotoxin intraperitoneally (1 mg/kg of body weight). After treatment at 30 minutes, and at 3 and 24 hours the pancreas was removed. Untreated animals and animals with injection of saline solution served as controls. After RNA isolation, RNA was pooled and hybridized to Affymetrix chips to measure the relative mRNA levels of 7000 genes and 1000 expression sequence tags. Three hours after administration of endotoxin there was an activation of proinflammatory transcription factors and other proinflammatory genes. After 24 hours there was a clear decrease of these proinflammatory genes, but a remaining and increasing upregulation of important antiapoptotic genes, antiproteases, and other probably protective genes. There was also a significant upregulation of complement factors. It was surprising that heat-shock proteins and other typical immediate early genes of the AP-1 complex were not upregulated. Our data show that 24 hours after endotoxin stress there is a regulation of a network of genes that represents a multifaceted preconditioning. As most important factors, inhibition of apoptosis and antiproteatic strategies are identified. Heat-shock proteins seem to play no important role in the mechanism of endotoxin preconditioning.

Two phosphoglycerate kinase cDNAs from Arabidopsis thaliana.

The deduced amino acid sequences of two Arabidopsis thaliana cDNAs share 94% sequence identity among each other. They are 90 to 93% identical to chloroplast phosphoglycerate kinases (chl-PGK) from other plant species.

An Arabidopsis thaliana cDNA homologous to cellulase.

A cDNA containing an ORF encoding a protein homologous to cellulase has been isolated from an Arabidopsis cDNA library.

Genomic sequence and mapping of a methyljasmonate-induced O-methyltransferase from barley (Hordeum vulgare L.).

We have isolated a genomic clone corresponding to a caffeic acid O-methyltransferase (COMT) from barley (Hordeum vulgare L.) using a cDNA for a previously described jasmonate-regulated mRNA showing homology to COMT. Primer extension was used to characterize the 5' end of the mRNA while the 3' end, intron/exon structure and other features of the sequence were deduced by comparison to the cDNA sequence and/or conserved motifs. The gene is mapped to chromosome five and is absent in the barley cultivar Morex. Southern and northern analyses suggest that no differences in genomic structure and jasmonate inducibility exist between the barley cultivar Salome (source of the cDNA clone) and Igri (source of the genomic clone). This genomic clone is thus suitable for promoter studies with respect to jasmonate induction.

Three-dimensional laser micro- and nano-structuring of acrylated poly(ethylene glycol) materials and evaluation of their cytoxicity for tissue engineering applications.

The natural cell environment is characterized by complex three-dimensional structures, which contain features at multiple length scales. Many in vitro studies of cell behavior in three dimensions rely on the availability of artificial scaffolds with controlled three-dimensional topologies. In this paper, we demonstrate fabrication of three-dimensional scaffolds for tissue engineering out of poly(ethylene glycol) diacrylate (PEGda) materials by means of two-photon polymerization (2PP). This laser nanostructuring approach offers unique possibilities for rapid manufacturing of three-dimensional structures with arbitrary geometries. The spatial resolution dependence on the applied irradiation parameters is investigated for two PEGda formulations, which are characterized by molecular weights of 302 and 742. We demonstrate that minimum feature sizes of 200nm are obtained in both materials. In addition, an extensive study of the cytotoxicity of the material formulations with respect to photoinitiator type and photoinitiator concentration is undertaken. Aqueous extracts from photopolymerized PEGda samples indicate the presence of water-soluble molecules, which are toxic to fibroblasts. It is shown that sample aging in aqueous medium reduces the cytotoxicity of these extracts; this mechanism provides a route for biomedical applications of structures generated by 2PP microfabrication and photopolymerization technologies in general. Finally, a fully biocompatible combination of PEGda and a photoinitiator is identified. Fabrication of reproducible scaffold structures is very important for systematic investigation of cellular processes in three dimensions and for better understanding of in vitro tissue formation. The results of this work suggest that 2PP may be used to polymerize poly(ethylene glycol)-based materials into three-dimensional structures with well-defined geometries that mimic the physical and biological properties of native cell environments.

Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). I. Purification by immunological methods and characterization.

The purification of a putative auxin receptor is one possibility to elucidate the first event in the mechanism of auxin action. By affinity chromatography of membrane proteins on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration on Ultrogel a fraction enriched in auxin-binding protein (ABP) was obtained and used for rabbit immunization. From the immunoglobulin G (IgG) fraction of the antisera IgGs against proteins not binding auxin (nonABP) could be obtained which were used to eliminate the nonABP from the eluates of the 2-OH-3,5-diiodobenzoic acid-Sepharose. The remainder fraction was further purified and concentrated on IgG-Sepharose which retained the ABP that could be eluted without loss of binding activity. A 600-fold purification with a yield of 42% was achieved. The ABP could be identified as the site I "receptor" described by Dohrmann et al. (Dohrmann, U., Hertel, R., and Kowalik, H. (1978) Planta (Berl.) 140, 97-106). It is shown that the competitors tested reduce [14C]1-naphthylacetic acid-(NAA) binding in the following order of effectiveness: NAA greater than 2-naphthylacetic acid greater than 1-phenylacetic acid greater than 2,3,5-triiodobenzoic acid greater than 3-indolylacetic acid greater than 2,4-dichlorophenoxyacetic acid. The ABP has a sharp binding optimum at pH 5.5, and the KD was calculated to be 5.7 X 10(-8) M to [14C]NAA. The binding activity of the ABP linearly decreased with increasing temperature but could partially be restored upon chilling in the presence of auxin. The ABP seems to be a 40-kDa dimer in its native form without disulfide bonds between its monomers.

Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). II. Localization of a putative auxin receptor.

With the aid of affinity chromatography on auxin-binding protein-Sepharose (ABP-Sepharose) monospecific IgGanti-ABP from rabbit antisera were isolated as judged by immuno-double diffusion test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this IgGanti-ABP the ABP is localized within the outer epidermal cells of coleoptiles using indirect immunofluorescence labeling. Auxin-induced growth of coleoptile segments can be inhibited by IgGanti-ABP, and the auxin response of split coleoptile sections is also strongly reduced by IgGanti-ABP. The ABP, therefore, is referred to as an auxin receptor. This auxin receptor is localized at the plasmalemma of the outer epidermal cells of the coleoptile.

An alfalfa (Medicago sativa L.) cDNA encoding an acidic leghemoglobin (MsLb3).

We have found an alfalfa cDNA clone that encodes an acidic leghemoglobin. To date, 14 alfalfa leghemoglobin clones have been identified. Five different leghemoglobin 'components' have been biochemically defined on the basis of their pI. A higher-resolution comparison, provided by sequence data analysis, identifies six leghemoglobin 'classes'. All 14 leghemoglobins are assigned to the six 'classes', which can be distributed among the five leghemoglobin 'components'. The newly identified leghemoglobin is the only member of a sixth 'class' of leghemoglobins, and it also is the only member of one of the acidic leghemoglobin 'components' IV or V.

Jasmonate signalling can be uncoupled from abscisic acid signalling in barley: identification of jasmonate-regulated transcripts which are not induced by abscisic acid.

Jasmonate and abscisic acid induce several identical mRNAs and proteins in barley. In order to study whether both hormones act through the same signalling pathway, we identified four transcripts induced by jasmonic acid methylester (JM) in leaf segments of barley (Hordeum vulgare L. cv. Salome). These newly identified transcripts were not induced by abscisic acid within the tested times of 2-72 h. This finding supports the conclusion that jasmonate signalling in barley is independent of abscisic acid, in contrast to the wound-induction signal cascade of proteinase-inhibitor II in tomato and potato. Of the four isolated cDNAs, the putative translation frame of one was homologous to caffeic acid methyltransferase, another was homologous to chalcone synthase, and the C-terminus of the third showed homology to two proteins from rice (a salt-induced protein and a root-specific protein); the last cDNA was not homologous to any sequences in the databases. The new cDNAs will be valuable tools for studying jasmonate signal transduction in barley.

A gene that encodes a proline-rich nodulin with limited homology to PsENOD12 is expressed in the invasion zone of Rhizobium meliloti-induced alfalfa root nodules.

To define the early stages of the interaction between Rhizobium and host legumes, we have cloned and characterized three early nodulin-encoding sequences from an alfalfa (Medicago sativa L.) cDNA library by probing with a fragment of a cDNA clone for PsENOD12, an infection-related nodulin from pea (Pisum sativum L.). Although the coding regions of the three clones are 95 to 98% homologous to each other, they are only 43% homologous to the pea clone. However, the putative signal peptide encoded by the alfalfa cDNA clones is 100% homologous to the PsENDO12 signal peptide. The spatial and temporal expression patterns of PsENOD12 and the alfalfa clones were compared. In situ hybridization experiments detected RNA transcripts in the invasion zone of mature nitrogen-fixing nodules, the same site where PsENOD12 mRNAs are found. Transcripts were also found by in situ hybridization in cells of Rhizobium meliloti exoH mutant-induced nodules penetrated by infection threads, but northern analysis did not detect transcripts in inf- (infection thread minus) nodules elicited by R. meliloti exoB nodules or in pseudonodules elicited by treatment with the auxin transport inhibitor N-1-(naphthyl)phthalamic acid. In addition, the alfalfa gene represented by these cDNA clones exhibited a temporal expression pattern that differed from that of PsENOD12, which is transiently expressed. These data, plus information derived from Southern blot analysis, indicate that we have isolated cDNA clones for a novel early nodulin, which we have designated MsENOD10 (Medicago sativa Early Nodulin 10).

Methyl jasmonate induces an O-methyltransferase in barley.

We have previously described a truncated cDNA clone for a barley (Hordeum vulgare L. cv. Salome) jasmonate regulated gene, JRG5, which shows homology to caffeic acid O-methyltransferase (COMT). A cDNA encompassing the coding region was amplified by PCR and cloned for overexpression in E. coli. Western blot analyses indicate that the recombinant protein crossreacts with the antibodies directed against the tobacco class II OMT and only weakly with the antibodies for the tobacco class I OMT. An immunoreactive band in the protein extract of jasmonate-treated leaf segments suggests that JRG5 transcripts that accumulate after jasmonate treatment are also translated. Specific methylating activities on caffeic acid and catechol were obtained from the recombinant protein through renaturation of protein extracted from inclusion bodies or from bacteria grown and induced at low temperature. On Northern blots, the JRG5 transcripts were detected in the leaf sheath but not the leaf lamina; stem, root or inflorescence and accumulated in leaf segments after jasmonate application. Several hormone or stress treatments did not induce JRG5 mRNA accumulation. This includes sorbitol stress which is known to lead to enhanced endogenous jasmonate levels and the implications for jasmonate signaling are discussed. Based on quantitative measurements and fluorescence microscopy, jasmonate-induced accumulation of ferulic acid and phenolic polymers in the cell wall were detected and the possibility of cell wall strengthening mediated through phenolic crosslinks is discussed.

The PsENOD12 Gene Is Expressed at Two Different Sites in Afghanistan Pea Pseudonodules Induced by Auxin Transport Inhibitors.

A number of early nodulin genes are expressed in specific cell types as pea (Pisum sativum) root nodules develop. The Pisum sativum early nodulin PsENOD2 is detected only in the uninfected cells of the nodule parenchyma, whereas PsENOD12 is expressed at two spatially removed sites: in root hairs and adjacent cortical cells, both of which can be invaded by Rhizobium entering through infection threads, and in derivatives of newly divided root inner cortical cells that establish the nodule primordium. We tested whether Rhizobium infection is required for triggering PsENOD12 gene expression by inducing nodule-like structures on Afghanistan pea roots with the auxin transport inhibitor N-(1-naphthyl)phthalamic acid (NPA). These nodule-like structures lack infection threads but resemble Rhizobium-induced nodules in other aspects. For one, both PsENOD2 and PsENOD12 transcripts were detected in these structures. PsENOD2 mRNA was localized by in situ hybridization to a zone equivalent to the nodule parenchyma of Rhizobium-induced nodules, whereas PsENOD12 transcripts were detected in a group of cells comparable to the nodule primordium of developing nodules. In addition, PsENOD12 mRNA was detected in uninfected root hairs 48 h after NPA treatment. These results indicate that infection is not a trigger for PsENOD12 gene expression in Afghanistan pea and rather suggest that the expression of the PsENOD2 and PsENOD12 genes is correlated with the differentiation of specific cell types in the developing nodule.

Differential gene expression after implantation of biomaterials into rat gastrointestine.

Two biodegradable materials, polyglycolide-copoly(L)-lactide/poly-dioxanone composite and poly(3-hydroxybutyrate), have been employed for surgery in the gastrointestine of laboratory rats. The tissue response was analyzed at distinct time intervals up to 2 months by differential mRNA display. Two specific PCR fragments were transiently present 7 and 14 days after contact with poly(3-hydroxybutyrate). Both fragments represent distinct rat lipases which might play a role in poly(3-hydroxybutyrate) degradation.

cDNA clones of the auxin-binding protein from corn coleoptiles (Zea mays L.): isolation and characterization by immunological methods.

An auxin-binding protein (ABP) cDNA clone was selected from a lambda gt11 cDNA library from corn coleoptiles with highly purified IgGanti ABP. The sequence of 794 bp contains an open reading frame (ORF) of 603 bp, coding for a 22 kd protein. There are indications of a signal peptide of 38 amino acids (von Heijne, G. 1983, Eur. J. Biochem., 133, 17-21). A N-glycosylation site can be deduced and a C-terminal KDEL amino acid sequence is detected. An EcoRI fragment containing the beginning portion of the cDNA with about three quarters of the ORF was used to select cDNA clones from an independently produced lambda gt11 cDNA library of corn coleoptiles. Northern blot analysis with in vitro transcribed biotinylated RNA showed a single band of not more than 850 bases. The full-length in vitro transcript directed the in vitro synthesis of a protein which is precipitated by IgGanti ABP. Rabbit antibodies raised against a fusion protein detect the ABP as a double band on Western blots. Only the smaller of the two ABP bands is labeled by two different KDEL-specific IgG preparations.

Characterization of a methyljasmonate-inducible lipoxygenase from barley (Hordeum vulgare cv. Salome) leaves.

We found three methyl jasmonate-induced lipoxygenases with molecular masses of 92 kDa, 98 kDa, and 100 kDa (LOX-92, -98 and -100) [Feussner, I., Hause, B., Vörös, K., Parthier, B. & Wasternack, C. (1995) Plant J. 7, 949-957]. At least two of them (LOX-92 and LOX-100), were shown to be localized within chloroplasts of barley leaves. Here, we describe the isolation of a cDNA (3073 bp) coding for LOX-100, a protein of 936 amino acid residues and a molecular mass of 106 kDa. By sequence comparison this lipoxygenase could be identified as LOX2-type lipoxygenase and was therefore designated LOX2:Hv:1. The recombinant lipoxygenase was expressed in Escherichia coli and characterized as linoleate 13-LOX and arachidonate 15-LOX, respectively. The enzyme exhibited a pH optimum around pH 7.0 and a moderate substrate preference for linoleic acid. The gene was transiently expressed after exogenous application of jasmonic acid methyl ester with a maximum between 12 h and 18 h. Its expression was not affected by exogenous application of abscisic acid. Also a rise of endogenous jasmonic acid resulting from sorbitol stress did not induce LOX2:Hv:1, suggesting a separate signalling pathway compared with other jasmonate-induced proteins of barley. The properties of LOX2:Hv:1 are discussed in relation to its possible involvement in jasmonic acid biosynthesis and other LOX forms of barley identified so far.