Mannheimia haemolytica is the major cause of severe pneumonia in bovine respiratory disease (BRD). Early M. haemolytica bacterins were either ineffective or even enhanced disease in vaccinated cattle, which led to studies of the bacterium's virulence factors and potential immunogens to determine ways to improve vaccines. Studies have focused on the capsule, lipopolysaccharide, various adhesins, extracellular enzymes, outer membrane proteins, and leukotoxin (LKT) resulting in a strong database for understanding immune responses to the bacterium and production of more efficacious vaccines. The importance of immunity to LKT and to surface antigens in stimulating immunity led to studies of individual native or recombinant antigens, bacterial extracts, live-attenuated or mutant organisms, culture supernatants, combined bacterin-toxoids, outer membrane vesicles, and bacterial ghosts. Efficacy of several of these potential vaccines can be shown following experimental M. haemolytica challenge; however, efficacy in field trials is harder to determine due to the complexity of factors and etiologic agents involved in naturally occurring BRD. Studies of potential vaccines have led current commercial vaccines, which are composed primarily of culture supernatant, bacterin-toxoid, or live mutant bacteria. Several of those can be augmented experimentally by addition of recombinant LKT or outer membrane proteins.

The aim of this study was to evaluate transcriptome changes in the muscle tissue of Bos taurus indicus cull cows subjected to recovery weight gain under grazing conditions. In all, 38 Nellore cull cows were divided randomly into two different management groups: (1) Maintenance (MA) and (2) Recovery gain (RG) from weight loss by moderate growth under high forage availability. After slaughter, RNA analysis was performed on the Longissimus thoracis muscle. Semaphorin 4A, solute carrier family 11 member 1, and Ficolin-2 were expressed in the RG, which may indicate an inflammatory response during tissue regrowth. Signaling factors, such as Myostatin, related to fibroblast activation, negative control of satellite cell proliferation in adults and muscle protein synthesis were less abundant in the RG group. The only gene related to anabolic processes that were more abundant in the MA group was related to fat deposition. The genes that were differentially expressed in the experiment showed muscle repair-related changes during RG based on the greater expression of genes involved in inflammatory responses and the lower expression of negative regulators of muscle cell proliferation and hypertrophy.

Proteolytic enzymes present in extracts of third (L3) and fourth (L4) stage larvae and adults of the cattle nematode Ostertagia ostertagi were defined on the basis of pH optima and proteinase inhibitor sensitivity in spectrophotometric assays using azocasein and elastin-orcein as protein substrates. Evidence that different classes of proteinases are expressed in a stage specific manner was provided by the contrasting pH optima and inhibitor sensitivities shown by the enzymes in the different parasite stages. Stage specificity was confirmed by gelatin-substrate analysis. In addition, proteolytic activity was sought in the excretory/secretory products (ES) of the L4 following simple in vitro culture. Contrasting pH and inhibitor sensitivities as well as gelatin-substrate analysis showed that different proteinases were present in somatic L4 extracts and L4 ES products. The secreted proteinases may be useful targets for serodiagnosis or vaccination.

Protease expression among TCI and TCII field isolates was analysed. Gelatin-containing gels revealed hydrolysis bands with molecular masses ranging from 45 to 66 kDa. The general protease expression profile showed that TCII isolates presented higher heterogeneity compared to TCI. By utilizing protease inhibitors, we showed that all active proteases at acid pH are cysteine-proteases and all proteases active at alkaline pH are metalloproteases. However, the expression of cruzipain, the T. cruzi major cysteine-protease, did not reproduce a heterogeneous TCII cysteine zymogram profile. Dendogram analyses based on presence/absence matrices of proteases and cruzipain bands showed a TCI separation from the TCII group with 50–60% similarity. We suggest that the observed cysteine protease diversification contributes to differential host infection between TCI and II genotypes.

This paper summarises the progress towards vaccine development against the major blood-feeding nematodes of man and livestock, the hookworms and Haemonchus contortus, respectively. The impact of the diseases and the drivers for vaccine development are summarized as well as the anticipated impact of the host immune response on vaccine design. The performance requirements are discussed and progress towards these objectives using defined larval and adult antigens, many of these being shared between species. Specific examples include the Ancylostoma secreted proteins and homologues in Haemonchus as well as proteases used for digestion of the blood meal. This discussion shows that many of the major vaccine candidates are shared between these blood-feeding species, not only those from the blood-feeding stages but also those expressed by infective L3s in the early stages of infection. Challenges for the future include: exploiting the expanding genome information for antigen discovery, use of different recombinant protein expression systems, formulation with new adjuvants, and novel methods of field testing vaccine efficacy.

Proteolytic activities of 5 strains of Leishmania (Viannia) braziliensis isolated from Brazilian and Colombian patients, presenting distinct clinical manifestations, were characterized and compared using whole-promastigote extracts and extracellular secretions. Zymographic assays concerning whole-cell extracts and supernatants resulted in the detection of high molecular weight bands, ranging from 50 to 125 kDa. Proteolytic activities from both whole-cell extracts and supernatants were optimal in a pH range 5·5 to 9·0 for all analysed strains. Such protease activities were inhibited when 10 mM 1,10-phenanthroline was assayed, strongly suggesting that the enzymes responsible for hydrolysis of the substrate belong to the metalloproteases class. Distinct profiles of metalloproteases were observed among the studied L. (V.) braziliensis strains. Differences among the microorganisms might be related to the geographical origin of the strains and/or to the clinical presentation.

Sarcoptes scabiei is an important parasitic mite in both man and animals. Little is known about the molecular interactions between this pathogen and its host. This is in part explained by the paucity of mite-derived material, including antigens. To extend the knowledge of the molecular repertoire in S. scabiei, we have performed a gene survey by an expressed sequence tag (EST) analysis. A total of 1020 ESTs were generated from an S. scabiei cDNA library. The average sequence read was 510 bp after editing and the overall sequencing success was 89%. Clustering of the sequences resulted in 76 clusters, comprising 36% of the ESTs. Sequence similarity searches showed that almost half of the S. scabiei ESTs could be assigned a putative identity. Many of these transcripts shared similarity with genes involved in basic metabolism and cellular organization. In the data set we also identified several proteases and other types of potential allergens implicated in various disease mechanisms. A relatively large fraction of the ESTs coded for different proteins carrying protease inhibitor-like domains. The clones with no similarity to previously identified genes constituted 11% of our transcripts. The EST data generated in this study will be a valuable resource in further studies of the biology of S. scabiei and in the identification of genes that can serve as potential targets in the control of the parasite.

Proteases and protease inhibitors were detected in the seminal plasma, blood plasma, skin mucus and spermatozoa. Their molecular weights were estimated using SDS-PAGE under non-reducing conditions. The results demonstrate that the two main bands of anti-proteinase activity (APA) detected earlier in common carp seminal plasma with molecular weight of approximately 47 and 58 kDa are also present in other fluids. The intensity of staining was highest in blood and seminal plasma. The intensity in skin mucus was visible, but in a sperm extract it was faint. An additional fast migrating band (30 kDa) was observed only in seminal plasma. Highest APA was found in blood and seminal plasma followed by skin mucus. The activity in sperm extracts was low. The two serine-like proteases with molecular weight of 79 and 189 kDa in seminal and blood plasma have also been found. In skin mucus, protease of 79 kDa was also present. Metalloproteinases with molecular weights of 61 and 69 kDa were found in seminal and blood plasma but metalloproteinases of 44 and 38 kDa were observed only in seminal plasma. Although metalloproteinases, of molecular weight ranging between 61 and 75 kDa, were also visible in a sperm extract, the experimental approach used in this study did not allow unequivocal identification of unique proteases of spermatozoa. Blood plasma contains a serine protease and protease inhibitor not present in seminal plasma. Skin mucus also showed the profile of three unique proteases (two EDTA stimulated and one metalloproteinase). These results indicate that the analysis of proteases and their inhibitors makes it possible to distinguish contamination of milt with either blood or skin mucus. The physiological role of the detected protease-inhibitory system in fish seminal plasma is still unknown. It is possible that the protease inhibitor and proteases, unique for seminal plasma, are involved in a specific function of milt or testis (e.g. the control of spermatogenesis).

Introduction. Le babaco (Carica pentagona Heilb.), plante équatoriale de mon- tagne, est originaire d'Équateur. Ses fruits sont particulièrement appréciés pour leur arôme caractéristique. La plante. Le babaco est un arbuste semi-ligneux de la famille des Caricacées. Cet hybride naturel se cultive en Équateur entre (1500 et 2500) m. Les rendements de production sont compris entre (60 et 80) fruits par plante et par an. Le fruit. Le fruit du babaco est une baie asperme jaune à maturité. Son arôme caractéristique se rapproche à la fois de l'ananas, du citron et de la papaye. Quand le fruit est vert, il exsude un latex qui con- tient une activité protéolytique semblable à celle de la papaye. Transformation. Le fruit est consommé essentiellement à l'état frais. La fabrication de pulpe, conserves et de fruits déshy- dratés est également possible. L'extraction et la purification des enzymes protéolytiques du latex des fruits verts pourrait être une nouvelle voie de valorisation industrielle du babaco. Tendances économiques. Grâce à des rendements de production élevés, ce fruit a éveillé un grand intérêt commercial. L'Équateur et la Nouvelle-Zélande sont les deux seuls pays où le babaco est cultivé à grande échelle. Conclusion. Le babaco présente d'intéressantes perspec- tives économiques, grâce notamment à ses propriétés sensorielles et à ses potentialités comme source d'enzymes protéolytiques.

It is fairly easy to control the enzymic hydrolysis of proteins in alkaline conditions by measuring the base consumption required to keep the pH constant in the reactor. Unfortunately, however, base consumption is not related in any simple way to the degree of hydrolysis reached at any given moment and to establish this relationship it is essential to find out the mean pK of the α-amino groups released during the hydrolytic process. We have shown here that the correct mean pK value varies according to the pH of the working conditions and that the relationship between these values may depend upon the kind of protein and protease used. We have put forward a method for determining this relationship experimentally by using a given protein–protease system, consisting of an alkaline titration of the raw protein and when partially hydrolysed. We have tested the results predicted by our theoretical model by applying it to the hydrolysis of whey proteins with a bacterial protease from Bacillus licheniformis at 50 °C, pH 8·0. This model can easily be applied to any hydrolytic process involving the appearance of functional groups that are partially protonizable under the working conditions in question in order to follow the kinetics of the reaction via the consumption of the neutralizing agent required to keep pH constant.

The presence of protease inhibitors in different ingredients used in fish feeds may negatively affect their digestive utilisation. The effect of inhibitors present in several protein sources on the activity of digestive proteases of sea bream was assessed using ‘in vitro’ assays. Inhibition produced by extracts of plant proteins ranged from 25 to 50% of total activity, whereas that obtained using animal protein sources ranged from 1 to 20 %. The exception was ovoalbumin, which showed the highest measured value (62 %). A plot of the inhibition values obtained by changing the relative concentrations enzyme/inhibitor resulted in different curves for different protein sources. The use of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) zymograms allowed visualisation of the aforementioned differences in inhibition. The importance of measuring protease inhibition in the preliminary evaluation of fish feed ingredients is discussed.

Glossina morsitans morsitans were maintained on rabbits immunized with crude tsetse midgut proteases. Newly emerged flies fed on these rabbits showed a depressed trypsin/proteinase VI activity, 6.5% of these flies failed to empty their crops as readily as controls, and 7.8% cases stayed as long as 11 days with undigested bloodmeal. Of these flies 4% exhibited premature larviposition.