Active immunization against gonadotrophin-releasing hormone (GnRH) is successfully applied to prevent boar taint in pork. In men, GnRH immunization could be an alternative to hormone therapy in patients with prostate cancer. In this study, a new GnRH vaccine formulation (a modified GnRH peptide conjugate formulated with CoVaccine adjuvant) was investigated for its pharmacological efficacy and safety in young-adult male pigs. Immunization resulted in castrate-like plasma testosterone levels in all treated pigs from week 8 until the end of the study, 30 weeks after the first immunization. Testosterone depletion retarded testes growth, reduced the relative weight of the testes and accessory sex organs, and reduced sperm counts and motility. There was no clinically relevant toxicity. Typical vaccination-related adverse reactions, such as swelling at the injection site and fever, were considered acceptable. We conclude that this GnRH vaccine efficiently and rapidly reduced serum testosterone levels, without inducing chronic toxic effects, and therefore could be applicable in both veterinary and human medicine.

Summary The immune response upon vaccination and revaccination of cattle with a vaccine against FMD type A was analysed. Neutralizing antibodies were measured and antibodies precipitating with homologous complete virus, trypsin treated virus, 12 S viral sub-units and with heterologous virus type C were assayed by radial immuno diffusion, both in whole sera and in sera fractionated over Sephadex G 200. The results show the development of neutralizing activity and precipitating activity against the complete and the trypsin treated virus in both the 19 S and 7 S serum fractions. Precipitating activity against 12 S viral subunits was found in the 7 S serum fractions, whereas the precipitating activity against the heterologous (type C) virus was only observed in the 19 S serum fractions. After revaccination the neutralizing ability of a given quantitiy of precipitating antibodies increased. In the course of the primary response the proportion of antibodies directed to the 12 S virus sub-units as compared to that directed to the complete virus increased slightly. In contrast the ratio of antibodies directed to trypsin treated virus to antibodies directed to complete virus hardly changed. In the 19 S serum fraction only part of the precipitating activity seems to be due to the vaccination. This also applied to the activity against heterologous type C virus. Zusammenfassung Antikörperreaktion gegen Maul- und Klauenseuche-Virus (FMDV) Teil I: Die Reaktionen in Seren von vakzinierten Tieren mit komplettem Virus, Trypsin-behandeltem Virus 12S-Virusuntereinheiten und heterologem Virus Es wurde die Immunreaktion auf die Vakzination und Revakzination von Rindern mit einer Vakzine gegen MKS-Typ A analysiert. Die neutralisierenden Antikörper wurden gemessen, sowie die mit homologem komplettem Virus, Trypsin-behandeltem Virus, 12 S-Virusuntereinheiten und mit heterologem Typ C-Virus präzipitierendem Antikörper mit Hilfe der Radial-Immunodiffusion, einmal an den Gesamtseren und andererseits an über Sephadex G 200 fraktionierten Seren bestimmt. Die Ergebnisse zeigen die Entwicklung neutralisierender und präzipitierender Aktivität gegen komplettes und Trypsin-behandeltes Virus sowohl in der 19 S- als auch in der 7 S-Serumfraktion. Präzipitierende Aktivität wurde auch in der 7 S-Fraktion gegen die 12 S-Virusuntereinheiten nachgewiesen, wogegen sie gegen heterologes Typ C-Virus nur in der 19 S-Serumfraktion beobachtet werden konnte. Nach der Revakzination stieg die Neutralisationskraft einer vorhandenen Menge präzipitierender Antikörper an. Im Verlauf der primären Antikörperreaktion stiegen die Antikörper gegen die 12 S-Virusuntereinheiten im Verhältnis zu denen gegen das komplette Virus etwas stärker an. Dagegen änderte sich das Verhältnis zwischen den Antikörper gegen Trypsin-behandeltes Virus und denen gegen komplettes Virus kaum. In der 19 S-Serumfraktion scheint nur ein Teil der präzipitierenden Aktivität auf die Vakzination zurückführbar zu sein. Dies trifft auch für die Aktivität gegen heterologes Typ C-Virus zu. Résumé Réaction des anticorps contre virus de la fièvre aphteuse (FMDV) Première partie: Les réactions dans des sérums d'animaux vaccinés avec un virus complet, un virus trypsinisé, de sous-unités virales 12 S et un virus hétérologue La réaction immunitaire à une vaccination et à une revaccination de bovins avec un vaccin anti-aphteux du type A a été analysée. On a mesuré les anticorps neutralisants ainsi que les anticorps précipitants à l'aide de l'immunodiffusion radiale avec des sérums complets et des sérums fractionnés sur Sephadex G 200 pour un virus homologue complet, un virus trypsinisé, des sous-unités virales 12 S et un virus hétérologue du type C. Les résultats montrent le développement d'une activité neutralisante et précipitante contre un virus complet et trypsinisé dans la fraction sérique 19 S et 7 S. Une activité précipitante a également été démontrée dans la fraction 7 S vis-à-vis de sous-unités virales 12 S, mais n'a pu être observée que dans la fraction 19 S pour le virus hétérologue du type C. La force de neutralisation s'est augmentée d'une quantité présente d'anticorps précipitants après le revaccination. Au cours de la réaction primaire des anticorps, les anticorps contre les sous-unités virales 12 S sont montés un peu plus fort que ceux contre l'anticorps complet. Le rapport entre les anticorps anti-virus trypsinisé et antivirus complet fut à peine différent. Une partie seulement de l'activité précipitante dans la fraction sérique 19 S semble être due à la vaccination. Ceci est également valable pour l'activité vis-à-vis du virus hétérologue du type C. Resumen Reacción de los anticuerpos frente al virus aftoso (FMDV) Parte Io: Las reacciones en sueros sanguíneos de animales vacunados con virus completo, virus tratado con tripsina, subunidades virósicas 12 S y virus heterólogo Se analizó la reacción inmunológica a la vacunación y revacunación de bovinos con una vacuna antiaftosa del tipo A. Se midieron los anticuerpos neutralizantes, así como los anticuerpos precipitantes con virus homólogo completo, virus tratado con tripsina, subunidades virósicas 12 S y el virus heterólogo tipo C, con ayuda de la inmunodifusión radial, valorándose una vez en los sueros totales y otra en los sueros fraccionados sobre Sephadex G 200. Los resultados evidencian el desarrollo de la actividad neutralizante y precipitante frente al virus completo y tratado con tripsina tanto en la fracción sérica 19 S como en la 7 S. Actividad precipitante se identificó también en la fracción 7 S frente a las subunidades virósicas 12 S, mientras que frente al virus heterólogo tipo C se pudo apreciar solo en la fracción sérica 19 S. Tras la revacunación ascendió la potencia de neutralización de una cantidad presente de anticuerpos precipitantes. En el curso de la reacción primaria de anticuerpos aumentaron un poco más los anticuerpos frente a las subunidades virósicas 12 S con relación a aquellas frente al virus completo. Por el contrario, apenas se modificó la relación entre los anticuerpos frente al virus tratado con tripsina y los mismos frente al virus completo. Parece ser que en la fracción sérica 19 S solo se puede atribuir una parte de la actividad precipitante a la vacunación. Esto también reza con la actividad frente al virus heterólogo tipo C.

A systemic acute phase reaction may develop during infection and inflammation, due to the action of peripherally liberated proinflammatory cytokines. Hepatic metabolism changes, and negative and positive acute phase proteins (APPs) can be measured in the blood: the APPs therefore represent appropriate analytes to assess health. While they are non-specific markers, their levels change with biological effects and this can be used to assess nutritional deficits and reactive processes, especially when positive and negative acute phase variables are combined in an index. Unfortunately, at present, no comprehensive, easy-to-use and cheap system is available to assess various acute phase proteins in serum or blood samples. Protein micro-array technology may satisfy this need; it will permit simultaneous analysis of numerous analytes in the same small volume sample and enable integration of information derived from systemic reactivity and nutrition with disease-specific variables. Applying such technology may help to address health problems in many countries.

Protein transduction domains (PTDs) have proven to be an invaluable tool to transduce a wide variety of cargoes across the plasma membrane and into intact tissue including brain. The PTDs are able to deliver biologically active molecules both in vitro and in vivo. To exploit the full potency of transport peptides it is important to know the efficiency, what kind of cargoes can be transported and for what application it can be used. Because fixation artifacts and irreversible membrane binding may cause misinterpretation of the amount of internalization of polybasic peptides, we have developed an enzyme transduction assay based on the intracellular loading of a cell permeable substrate. In this assay, a fluorescent signal is generated by internalized enzyme in intact cells and not by membrane-bound or extracellular enzyme. This assay demonstrates the true efficiency of transport peptides. To check whether the efficiency is sufficient for intracellular applications we demonstrated the internalization of an enzyme substrate in the cell and the replacement of an enzyme in a deficient cell. Comparison of many different polybasic PTDs showed that the efficiency of transport was dependent on the type of cargo. Therefore, the unique characteristic of a PTD may only become apparent when it is selected for a particular application. Rules for optimisation of PTDs for particular applications are now emerging and opening the way for a new generation of drug delivery agents.

Immunization against gonadotrophin releasing hormone (GnRH) was studied as an alternative for the commonly used surgical castration in stallions. Two GnRH vaccines comprising non-mineral oil adjuvants were evaluated for their potential to induce high antibody titers directed against GnRH and subsequent effects on reproductive characteristics. Twelve sexually mature male hemicastrated Shetland ponies were assigned to three groups. Group 1 and 2 were injected with 1 mg peptide equivalent of G6k-GnRH-tandem-dimer conjugated to ovalbumin (OVA) in CoVaccine (TM) HT adjuvant (GnRH/CoVaccine) and in Carbopol (GnRH/Carbopol), respectively, and group 3 was injected with CoVaccine (TM) HT adjuvant without antigen (controls).After immunization no adverse effects were observed with respect to the injections sites or general health. Two weeks after the second vaccination antibody titers against GnRH increased rapidly in all animals of the GnRH/CoVaccine group, at the same time reducing serum testosterone levels maximally for the further duration of the experiment. In the GnRH/Carbopol group antibody responses and effects on testosterone levels were intermediate in two stallions and not apparent in the remaining stallions of this group.Semen evaluation showed that from 2 weeks after the second immunization onwards, sperm motility was affected in all stallions treated with GnRH/CoVaccine and one stallion treated with GnRH/Carbopol. Seven weeks after the second immunization, no semen could be collected from two stallions, one of each group, due to suppressed libido. Histological examination of the testes, 15 weeks after the initial immunization, demonstrated reduction in seminiferous tubuli diameters in all stallions of the GnRH/CoVaccine group and one stallion of the GnRH/Carbopol group. Furthermore, spermatogenesis was extremely disorganized in these stallions, as indicated by absence of the lumen in the seminiferous tubules, the absence of spermatozoa and spermatids in the tubular cross-sections and the impossibility to determine the stage of the tubular cross-sections. Testis size was also substantially reduced in three out of four stallions treated with GnRR/CoVaccine.The results demonstrate that two immunizations with G6k-GnRH-tandem-dimer-OVA conjugate in a suitable adjuvant such as CoVaccine (TM) HT caused a rapid and complete reduction of serum testosterone levels in sexually mature stallions, subsequently leading to reduced sperm motility and affected testis function, while no adverse reactions were observed after immunizations. (TM) 2004 Elsevier B.V. All rights reserved.

Gonadotropin releasing hormone (GnRH) occurs in various isoforms in mammals, i.e. GnRH-I (mammalian GnRH), GnRH-II (chicken GnRH-II), GnRH-III (salmon GnRH) and two forms of lamprey GnRH. The function of the latter four molecules have only been partially investigated. Also not much is known about the physiological effects of GnRH-I immunization on the function of these GnRH isoforms. In order to avoid possible harmful side-effects due to undesired neutralization of GnRH isoforms, GnRH-I specificity of antibodies raised against a panel of alternative GnRH antigens was determined. The results show that GnRH antigens can be designed which generate antibodies that specifically bind GnRH-I, without cross-reacting with other GnRH isoforms.

Protein transduction domains (PTDs) have proven to be an invaluable tool to transduce a wide variety of cargo's including peptides across the plasma membrane and into intact tissue. The PTDs are able to deliver biologically active molecules both in vitro and in vivo. This study describes many new polybasic PTDs of which some are just as potent as the PTDs derived from extracellular RNAses or other published PTDs. Large differences in potency became apparent when the PTDs are coupled to particular cargoes. Therefore, the unique characteristic of a PTD may only become apparent when it is selected for a particular application. Rules for optimization of PTDs for particular applications are now emerging and open the way for a new generation of drug delivery agents. Because fixation artifacts and irreversible membrane binding may cause misinterpretation of the amount of internalization of polybasic peptides, we have developed an enzyme transduction assay based on the intracellular loading of a cell permeable substrate. In this assay, a fluorescent signal is generated by internalized enzyme in intact cells and not by membrane-bound or extracellular enzyme.

Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Abeta((1-42)), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 microM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.

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.

Immunocastration by immunisation against gonadotrophin-releasing hormone (GnRH), is a good alternative for surgical castration in male pigs, both to prevent boar taint and for animal welfare reasons. In this study we investigated the effect of immunocastration on growth performance of pigs from 25 to 110 kg fed two diets differing in NEf content. Sixty piglets were divided into 20 blocks. Each block consisted of three littermates: a boar, a surgical castrate and an immunocastrate. Two blocks were housed in each pen. The pigs were fed ad libitum and individual feed intake was recorded with an automatic feed registration device. A high-energy and a low-energy diet were fed (9.70 and 8.30 MJ NEf/kg, respectively, based on tabulated values). The immunocastrates were immunised at 10 and 17 weeks of age with 62.5 μg d-Lys6-GnRH tandem dimer peptide conjugated to ovalbumin in Specol adjuvant. Plasma testosterone levels in immunocastrates decreased after the first injection and reached non-detectable levels at 20 weeks of age and thereafter. Immunocastrates had non-detectable fat androstenone levels at slaughter. The results of the concomitant digestibility experiments showed that the estimated NEf contents of the diets were 9.64 and 9.04 MJ/kg, respectively. No differences were observed in digestibility of proximate nutrients among sexes, but digestibility of Ca and P was higher for boars and immunocastrates as compared to surgical castrates (P<0.05). There was no interaction between type of diet and sex for growth performance characteristics except for energy conversion ratio, that approached significance (P=0.095). Feed intake for immunocastrates and surgical castrates was similar, and was higher than for boars on both diets (P<0.001). There was a tendency towards a better growth rate for immunocastrates compared to surgical castrates, irrespective of the energy level of the diet. When compared to boars, growth rate was higher in both immunocastrates and surgical castrates, which was significant only for the low-energy diet (P<0.01). Energy conversion ratio (ECR; expressed as MJ NEf/kg gain) was better for boars than for surgical castrates (P<0.05). For immunocastrates, ECR was in between that of boars and surgical castrates, and was better than for surgical castrates on the low-energy diet (P<0.05). Meat percentage was higher for boars than for surgical castrates and immunocastrates (P<0.05). On the high-energy diet, meat percentage was lower in immunocastrates than in surgical castrates (P<0.05) but on the low-energy diet it was similar. The results of the present study demonstrate that there was a tendency towards a better growth performance for immunocastrates as compared to surgical castrates, especially on the low-energy diet.

ABSTRACT Erns is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. Erns is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine cysteines present in the mature dimer was established by analysis of the proteolytically cleaved protein. Fragments were obtained after digestion with multiple proteolytic enzymes and subsequently analyzed by liquid chromatography-electrospray ionization mass spectrometry. The analysis demonstrates which cysteine is involved in dimerization and reveals an extremely rare vicinal disulfide bridge of unknown function. With the assistance of the disulfide arrangement, a three-dimensional model was built by homology modeling based on the alignment with members of the Rh/T2/S RNase family. Compared to these other RNase family members, Erns shows an N-terminal truncation, a large insertion of a cystine-rich region, and a C-terminal extension responsible for membrane translocation. The homology to mammalian RNase 6 supports a possible role of Erns in B-cell depletion.

In this study, the performance of male pigs immunized against GnRH was determined in relation to the onset of their biological response to the immunization. Pigs were immunized at 9 and 17 wk of age and were housed in a pen together with both a surgically castrated and an intact boar littermate. Feed intake was restricted to 2.8 to 3.2 times maintenance requirement for energy. Animals were weighed weekly and slaughtered at 108 kg BW. Depending on the time of onset of the response after immunization in terms of biological effects, immunized pigs were retrospectively grouped into two categories. One category consisted of the immunized pigs, which had undetectable or low levels of LH and testosterone at the time of booster immunization-known as "early" responding immunocastrates (E-IM, n = 8), whereas the "late" responding immunocastrates (L-IM, n = 7) had substantial LH and testosterone levels at that time. This dichotomy of the response to immunization also was reflected in testis weight, with 17 g and 40 g for E-IM and L-IM pigs, respectively. At slaughter, testis size and weight were reduced (P < 0.001) in the immunocastrated pigs as compared to the intact boars. Androstenone concentrations in backfat of all immunocastrated pigs were undetectable. Growth performance (i.e., ADG and feed efficiency [FE, g gain/kg feed]), was better in boars and L-IM pigs than in surgical castrates and E-IM pigs (P < 0.05). Average daily gain and FE did not differ between E-IM pigs and the surgical castrates, but intact boars performed better than L-IM (P < 0.02). There were no significant differences in carcass quality (backfat thickness and meat percentage) between boars and surgical castrates at slaughter. However, for both characteristics L-IM pigs and intact boars performed better (P < 0.03) than E-IM pigs. Thus, growth performance in L-IM is better than in either E-IM or surgical castrates.

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×Large White) male pigs were randomly assigned to one of the five treatments as follows: (I) 0 μg (control, n=8); (II) 10 μg (n=8); (III) 62.5 μg (n=8); (IV) 125 μg (n=8); (V) 250 μg (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 μg dose group (53.6 and 50.5% binding, respectively) were significantly higher than in the 10 or 250 μg 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 μg 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 μg peptide is not suitable. We conclude that, within the dose-range studied, the 62.5 μg dose is optimal for future GnRH immunization studies or future practical use in immunocastration of Chinese male pigs.

Surgical castration of young female pigs is common practice in Chinese pig farming today. The purpose of the present study is to investigate anti-GnRH immunization as a practical alternative to surgical castration for female pigs. Thirty-six Chinese female crossbred pigs (Chinese Yanan×Yorkshire) were selected from 12 litters, three pigs from each litter, at the age of 10–13 weeks. One pig from each litter was immunized with 62.5 μg d-Lys6-GnRH-tandem-dimer peptide conjugated to ovalbumin in Specol adjuvant at Week 0 (0 week post-vaccination, wpv), and a booster vaccination was given 8 weeks later (8 wpv). Its intact and castrate littermates (surgically castrated at the time of weaning, i.e. at 6 weeks of age) were administered the vehicle and served as controls. Antibody titers, serum LH and inhibin A were determined at the day of first vaccination, every 4 weeks thereafter and at the day of slaughter (18 wpv). At slaughter, ovaries were inspected for the presence of follicles and corpora lutea, and ovarian and uterine weights were recorded. Ten of twelve immunized pigs responded well to the immunization (immunocastrated animals), while the remaining two pigs responded poorly (nonresponders). Antibody titres in immunocastrated animals steadily increased after immunization, became maximal at 12 wpv and remained high until slaughter. Serum LH levels were reduced (P<0.05) in immunocastrated pigs as compared to intact controls and surgical castrates. Serum inhibin A levels decreased after vaccination, and equaled surgical castrate levels from 8 wpv until the end of the experiment. Ovarian and uterine weights (1.3±0.2 and 43.9±11.4 g, respectively; mean±S.E.M.) were significantly lower (P<0.05) in immunocastrates than in intact controls (9.4±1.1 and 390.9±67.2 g, respectively). Antibody titers were significantly lower (P<0.05) in nonresponders than in immunocastrated pigs from 12 wpv to slaughter. Ovarian and uterine weights were similar in nonresponders and in intact controls. Macroscopically, no follicular structures were found in ovaries of immunocastrated pigs, while large follicles or corpora lutea were observed in the ovaries of both nonresponders and intact controls. Although not significant, immunocastrates had a numerically higher average daily gain than surgical castrates and intact controls (0.74±0.04 versus 0.66±0.04 versus 0.66±0.03 kg per day, respectively; mean±S.E.M., P=0.09). Results obtained in the present study demonstrate that anti-GnRH immunization can be an attractive alternative to surgical castration for Chinese crossbred female pigs. Our results also question the beneficial effect of surgical castration on growth as compared to intact controls.

Rs-AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs-AFPs have been isolated (Rs-AFP1-4). The structure of Rs-AFP1 consists of three beta-strands and an alpha-helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure-function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs-AFP2 is concentrated mainly in the beta2-beta3 loop. In this study, we synthesized linear 19-mer peptides, spanning the entire beta2-beta3 loop, that were found to be almost as potent as Rs-AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19-mer loop peptides, cysteines can be replaced by alpha-aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19-mer peptides, forced to adopt a hairpin structure by the introduction of one or two non-native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19-mer peptides, like Rs-AFP2 itself, cause increased Ca2+ influx in pregerminated fungal hyphae.

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.

Monoclonal antibody 3C9 was the starting material in the definition of the epitope that led to the synthesis of the first efficient peptide vaccine against a viral disease (canine parvovirus) in the natural host (dog). In this report, we have analysed the specificity of the antibody at the single amino acid level and the contribution of each residue to the binding, using multiple length analysis. Moreover, a replacement analysis allowed determining those critical residues for the binding. Finally, in an attempt to optimise the production cost of the vaccine, we have determined that the minimal dose required for induction of protective antibodies can be as low as 0.5 μg of peptide. Also, KLH can be replaced as a carrier for a much cheaper alternative such as ovalbumine. All these findings implicate a substantial reduction in the cost of the vaccinal dose.