Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone.

The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50+/-10%), reconstitutable dimers (20+/-10%) and irreversibly aggregated oligomers (30+/-20%). Incubation of monomeric apoGOx with FAD or N(6)-(6-aminohexyl)-FAD (ahFAD) restored glucose oxidase (GOx) activity and induced dimerization with stoichiometric incorporation of FAD. N(6)-(6-aminohexyl)-FAD progesterone conjugates also induced dimerization. However, holoenzyme reconstitution required relatively high concentrations of apoprotein and was dependent on the type of conjugate. Restoration to 25-50% of the original enzyme activity was obtained. Binding of the FAD-progesterone conjugates might hinder the closure of a protein lid needed for dimer formation. Our results illustrate the prospects of FAD conjugates in sensitive detection of progesterone in biological matrices in a biosensor based on the recombination of apoGOx with progesterone-conjugated FAD.

Antifungal activity of synthetic peptides derived from Impatiens balsamina antimicrobial peptides Ib-AMP1 and Ib-AMP4.

Seeds of Impatiens balsamina contain a set of related antimicrobial peptides (Ib-AMPs). We have produced a synthetic variant of Ib-AMP1, oxidized to the bicyclic native conformation, which was fully active on yeast and fungal strains; and four linear 20-mer Ib-AMP variants, including two all-D forms. We show that the all-D variants are as active on yeast and fungal strains as native peptides. In addition, fungal growth inhibition nor salt-dependency of Ib-AMP4 could be improved by more than two-fold via replacement of amino acid residues by arginine or tryptophan. Native Ib-AMPs showed no hemolytic nor toxic activity up to a concentration of 100 microM. All these data demonstrate the potential of the native Ib-AMPs to combat fungal infections.

Discovery and in vivo evaluation of new melanocortin-4 receptor-selective peptides.

The melanocortin-4 receptor (MC4R) is involved in several physiological processes, including body weight regulation and grooming behaviour in rats. It has also been suggested that the MC4R mediates the effects of melanocortin ligands on neuropathic pain. Selective compounds are needed to study the exact role of the MC4R in these different processes. We describe here the development and evaluation of new melanocortin compounds that are selective for the MC4R as compared with the other centrally expressed receptors, MC3R and MC5R. First, a library of 18 peptides, in which a melanocortin-based sequence was systematically point-mutated, was screened for binding to and activity on the MC3R, MC4R and MC5R. Compound Ac-Nle-Gly-Lys-D-Phe-Arg-Trp-Gly-NH(2) (JK1) appeared to be the most selective MC4R compound, based on affinity. This compound is 90- and 110-fold selective for the MC4R as compared to the MC3R and MC5R, respectively. Subsequent modification of JK1 yielded compound Ac-Nle-Gly-Lys-D-Nal(2)-Arg-Trp-Gly-NH(2) (JK7)(,) a selective MC4R antagonist with 34-fold MC4R/MC3R and 109-fold MC4R/MC5R selectivity. The compounds were active in vivo as determined in a grooming assay and a model for neuropathic pain in rats. Intravenous (i.v.) injections suggested that they were able to pass the blood-brain barrier.The compounds identified here will be useful in further research on the physiological roles of the MC4R.

Active immunization against gonadotrophin-releasing hormone in Chinese male pigs: effects of dose on antibody titer, hormone levels and sexual development.

The objective of this study was to determine the optimal dose of a GnRH vaccine for immunocastration of Chinese male pigs, based on immune, endocrine and testicular responses. Forty-two crossbred (Chinese Yanan x Large White) male pigs were randomly assigned to one of the five treatments as follows: (I) 0 microg (control, n=8); (II) 10 microg (n=8); (III) 62.5 microg (n=8); (IV) 125 microg (n=8); (V) 250 microg (n=10), D-Lys6-GnRH tandem dimer (TDK) peptide equivalent of conjugate (TDK-OVA), using Specol as the adjuvant. Pigs were immunized at 13 and 21 weeks of age and were slaughtered at 31 weeks of age. Blood samples for antibody titer and hormone assays were collected at 13, 21, 24 and 31 weeks of age. At these time-points, testis size was also measured. At slaughter, testis weight was recorded and fat samples were collected for androstenone assay. Four animals, one out of each immunized group, responded poorly to the immunization (non-responders). At slaughter, serum testosterone and LH levels, fat androstenone levels and testis size/weight of these non-responders were similar to those in control animals. Antibody titers of non-responders were substantially lower (P<0.05) than in other immunized pigs. For the animals that responded well to the immunization (immunocastrated pigs), serum testosterone and LH levels, fat androstenone levels and testis size or weight were reduced (P<0.05) as compared to either controls or non-responders, at all doses tested. There was a significant effect of dose of TDK-OVA on antibody titers. The overall mean antibody titers in the 62.5 or 125 microg dose group (53.6 and 50.5% binding, respectively) were significantly higher than in the 10 or 250 microg group (39.2 and 40.24% binding, respectively). At slaughter, there was a significant dose effect on testis size or weight and on serum testosterone levels, but there was no dose effect on serum LH levels and fat androstenone levels. Testis size or weight in the 10 microg group was reduced to a lesser extent (P<0.05) than in the three higher dose groups. At slaughter, in comparison to controls, mean testis size of immunocastrated pigs in treatments II-V was reduced to 55, 21, 33 and 25%, respectively, whereas testis weight was reduced to 39, 12, 18 and 14%, respectively. Reduction of testis size and/or weight is important for visual assessment of castration at the slaughterline, therefore, it is concluded that a dose of 10 microg peptide is not suitable. We conclude that, within the dose-range studied, the 62.5 microg dose is optimal for future GnRH immunization studies or future practical use in immunocastration of Chinese male pigs.

Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins.

We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.

Functional mimicry of a discontinuous antigenic site by a designed synthetic peptide.

Functional reproduction of the discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate each of the three protein loops that define the antigenic site into a single molecule. The site D mimics were designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulfide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194) (VP=viral protein). Guinea pig antisera to the peptides recognized the viral particle and competed with site D-specific monoclonal antibodies, while inoculation with a simple (not covalently joined to one another) admixture of the three VP1-VP3 sequences yielded no detectable virus-specific serum conversion. Similar results have been reproduced in two bovines. Antisera to the peptides also moderately neutralize FMDV in cell cultures and partially protect guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent, peptide-based vaccine against FMDV.