Steroids originating from bacterial bile acid degradation affect Caenorhabditis elegans and indicate potential risks for the fauna of manured soils.

Bile acids are steroid compounds from the digestive tracts of vertebrates that enter agricultural environments in unusual high amounts with manure. Bacteria degrading bile acids can readily be isolated from soils and waters including agricultural areas. Under laboratory conditions, these bacteria transiently release steroid compounds as degradation intermediates into the environment. These compounds include androstadienediones (ADDs), which are C19-steroids with potential hormonal effects. Experiments with Caenorhabditis elegans showed that ADDs derived from bacterial bile acid degradation had effects on its tactile response, reproduction rate, and developmental speed. Additional experiments with a deletion mutant as well as transcriptomic analyses indicated that these effects might be conveyed by the putative testosterone receptor NHR-69. Soil microcosms showed that the natural microflora of agricultural soil is readily induced for bile acid degradation accompanied by the transient release of steroid intermediates. Establishment of a model system with a Pseudomonas strain and C. elegans in sand microcosms indicated transient release of ADDs during the course of bile acid degradation and negative effects on the reproduction rate of the nematode. This proof-of-principle study points at bacterial degradation of manure-derived bile acids as a potential and so-far overlooked risk for invertebrates in agricultural soils.

Cluster binding studies with two anti-Thomsen-Friedenreich (anti-core-1, CD176, TF) antibodies: Evidence for a multiple TF epitope.

Antibodies to carbohydrate epitopes are often of the IgM isotype and require multiple binding for sufficient avidity. Therefore clusters of epitopes are preferred antigenic sites in these cases. We have examined the type of clusters recognized by two anti-Thomsen-Friedenreich (TF, core-1, CD176) IgM antibodies, NM-TF1 and NM-TF2, using several different sets of TF-carrying synthetic glycoconjugates in ELISA experiments. To our surprise, the single most important factor determining binding strength was a close vicinity of several TF glycans at distances of ≤1 nm. Considering the known dimensions of IgM antibodies, our data strongly suggest that a cluster of up to four TF moieties, presenting as a "multiple epitope", is required to attach to a single combining site in order to result in adequate binding strength. This effect can also be achieved by "surrogate-multiple epitopes" consisting of separate TF-carrying molecules in close vicinity. In addition, it was found that serine-linked TFs are stronger bound than threonine-linked TFs by both antibodies. This peculiar type of cluster recognition may contribute to improved avidity and explicit tumor specificity.

Defined sequence segments of the small heat shock proteins HSP25 and alphaB-crystallin inhibit actin polymerization.

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N-terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine alphaB-crystallin showed the same behaviour. The results suggest that both HSP25 and alphaB-crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.

Receptor binding sites and antigenic epitopes on the fiber knob of human adenovirus serotype 3.

The adenovirus fiber knob causes the first step in the interaction of adenovirus with cell membrane receptors. To obtain information on the receptor binding site(s), the interaction of labeled cell membrane proteins to synthetic peptides covering the adenovirus type 3 (Ad3) fiber knob was studied. Peptide P6 (amino acids [aa] 187 to 200), to a lesser extent P14 (aa 281 to 294), and probably P11 (aa 244 to 256) interacted specifically with cell membrane proteins, indicating that these peptides present cell receptor binding sites. Peptides P6, P11, and P14 span the D, G, and I beta-strands of the R-sheet, respectively. The other reactive peptides, P2 (aa 142 to 156), P3 (aa 153 to 167), and P16 (aa 300 to 319), probably do not present real receptor binding sites. The binding to these six peptides was inhibited by Ad3 virion and was independent of divalent cations. We have also screened the antigenic epitopes on the knob with recombinant Ad3 fiber, recombinant Ad3 fiber knob, and Ad3 virion-specific antisera by enzyme-linked immunosorbent assay. The main antigenic epitopes were presented by P3, P6, P12 (aa 254 to 269), P14, and especially the C-terminal P16. Peptides P14 and P16 of the Ad3 fiber knob were able to inhibit Ad3 infection of cells.

T cell response of I-Aq mice to self type II collagen: meshing of the binding motif of the I-Aq molecule with repetitive sequences results in autoreactivity to multiple epitopes.

Type II collagen (CII) is of immunological interest because of its repetitive structure and properties as an autoantigen. The mouse gene has recently been cloned, thus enabling T cell-defined epitopes to be identified. Multiple novel epitopes on mouse CII are here detected in the autoreactive T cell response. The major response is directed to an epitope with residues 707-721 located on the CB10 fragment. Some 25 other epitopes are also recognized, including the autologous homologue of the 256-270 epitope which dominates in the response to foreign collagen. The cells reactive with mouse collagen peptides were of Th1 type, as judged by release of IFN-gamma. No significant reactivity was detected to mouse CII peptides during ongoing disease. Alignment of the mouse epitopes revealed a sequence motif with characteristic side chains at residues P1, P4 and P7, and to a lesser extent at P5, within a nonamer core sequence. Binding of these epitopes was simulated in a computer model of the I-Aq molecule, where peptides with anchor residues at P1, P4 and P7 were indeed found to fit the binding groove best. The spacing of pockets and the fine structure of the binding surface of the I-Aq molecule meshes with the repetitive structure of the collagen (X-Y-Gly), thus providing a likely explanation for the occurrence of multiple epitopes. Comparison with human DR binding motifs showed that the I-Aq motif resembles most closely that of the DR4 subtypes which predispose for rheumatoid arthritis.

RGD-containing peptides of VP1 of foot-and-mouth disease virus (FMDV) prevent virus infection in vitro.

RGD-containing peptides from the immunodominant region of VP1 between amino acids 135-160 from foot-and-mouth disease virus (FMDV) type O1 Kaufbeuren (O1K) prevented virus adsorption to piglet kidney (PK) cells. The highly conserved amino acid RGD sequence (Arg.-Gly.-Asp.) was a prerequisite of this effect. To prevent infection with 100-200 TCID50 in 10(6) PK cells, 20-250 micrograms of each peptide should have been added.

Investigation of the influence of peptide-carrier conjugation on the immunological activity of VP1-peptides of foot-and-mouth disease virus O1-Kaufbeuren.

We found coupling of short sequences of VP1 to keyhole limpet hemocyanin (KLH) by means of glutaraldehyde to be a very complex phenomenon which could only be controlled by strict standardization of components and reaction conditions. Considering the results, we may conclude that big immunogenic proteins, like KLH, are advantageous for achieving sufficient and specific antibody response with neutralizing activity. When using KLH, we did not find simple dependence of immunogenicity or neutralizing activity on the incorporation rate of hapten in KLH in a region between 50 and 200. To develop a synthetic vaccine of good economy, investigations have to be continued, primary with a view to lowering the doses involved.

Synthetic peptides against foot-and-mouth disease--immunization with VP1-peptides of type O1-Kaufbeuren.

Coupled synthetic peptides, representing the sequences of amino acids 130-160, 141-160 and 145-160 of foot-and-mouth disease virus O1K protein VP1, induced virus-binding and virus-neutralizing antibody response in guinea pigs, rabbits, and pigs. We also detected antibody response in guinea pigs after immunization with uncoupled peptides and in cattle with 21 aa-peptide-Keyhole-limpet hemocyanin (-KLH). The best results were obtained from 21 aa-peptide-KLH and 31 aa-peptide with or without KLH or thyroglobulin as carrier. Our preliminary results show the induction of virus-neutralizing antibodies to be obviously influenced by length of the peptide as well as by the kind of carrier and coupling.

[Chemosynthetic peptides against foot-and-mouth disease--immune response to free and carrier bound peptides of the VP1 of O1-Kaufbeuren]. / Chemosynthetische Peptide gegen Maul- und Klauenseuche--Immunantwort gegen freie und trägergebundene Peptide des VP1 von O1-Kaufbeuren.

Three peptides of main epitope of FMD virus O1-Kaufbeuren, VP1 (16, 21, 31), were found to induce in the 130-160 sequence range, in free and/or carrier-bonded form, virus-neutralising antibodies in guinea pig, rabbit, mouse, swine, and cattle. Five carrier proteins were tested, with thyroglobulin, next to keyhole limpet hemocyanin (KLH), being most effective for 16-peptides (145-160) and 21-peptides (141-160 Tyr161). To protect guinea pig from FMD, minimum dosage of 21-peptide was found to be 2 x 8 micrograms. The immunogenic spectrum of peptides and conjugates proved to be broader than that of monovalent vaccines of inactivated virus. Free peptides were found to be also capable in vitro of inhibiting virus infection.