Ryno J. Naudé

The function of rhamnose-binding lectin in innate immunity by restricted binding to Gb3

L-rhamnose-binding lectins (RBLs) have been isolated from various kinds of fish and invertebrates and interact with various kinds of bacteria, suggesting RBLs are involved in various inflammatory reactions. We investigated the effect of RBLs from chum salmon (Oncorhynchus keta), named CSL1, 2 and 3, on the peritoneal macrophage cell line from rainbow trout (Oncorhynchus mykiss) (RTM5) and an established fibroblastic-like cell line derived from gonadal tissue of rainbow trout (RTG-2). CSLs were bound to the surface of RTM5 and RTG-2 cells and induced proinflammatory cytokines, including IL-1beta1, IL-1beta2, TNF-alpha1, TNF-alpha2 and IL-8 in both cells by recognizing globotriaosylceramide (Gb3). In addition, CSLs had an opsonic effect on RTM5 cells and this effect was significantly inhibited by L-rhamnose, indicating that CSLs enhanced their phagocytosis by binding to Gb3 on cell surfaces. This is the first finding that Gb3 plays a role in innate immunity by cooperating with natural ligands, RBLs.

The roles of the proteasome, and cathepsins B, L, H and D, in ostrich meat tenderisation

As very little research has been conducted on ostrich meat tenderisation, this study aims at investigating the roles of the proteasome and cathepsins B, L, H, and D in the tenderisation process. The enzyme activities in meat from eight ostriches during a 12-day ageing period and the corresponding physical characteristics (e.g. pH, shear force) and myofibril patterns were determined. After 12 days, substantial high remaining activities were found, especially of the proteasome, thus implicating their possible roles in the tenderisation process. The mean shear force values, however, showed no improvement in tenderness, but the myofibril patterns showed the appearance of a M(r) 32 K component. Myofibril degradation studies of the proteasome, analysed electrophoretically, also revealed a possible role of the proteasome, but under activating conditions. This study provides further insights into the tenderisation process, particularly of ostrich meat, which may ultimately be used for the advantageous manipulation of the process.

An investigation into the effect of selenium supplementation on microcystin hepatotoxicity.

Toxin-producing cyanobacteria pose a worldwide health threat to humans and animals due to their increasing presence in both drinking and recreational waters. Little work has, however, been done on a preventative therapy for anyone at risk of exposure to cyanobacterial toxins. The potential benefits of dietary supplementation of selenium, an antioxidant, to protect against the mouse liver injury induced by the toxin, microcystin-LR, has been investigated. BALB/c mice were pretreated for two weeks with sodium selenite (1.5 microg/mouse/day) before an intraperitoneal injection of microcystin-LR. Selenium-supplementation was found to provide some protection to the action of the toxin. In addition selenium pretreatment reduced the liver damage caused by lethal and sub-lethal toxin doses as reflected in liver pathology, decreased serum ALT and lipid peroxidation levels as well as prevention of glycogen loss compared to non-selenium supplemented toxin treated mice. The increased level of liver glutathione peroxidase activity following selenium-supplementation may indicate the possible route of selenium protection in the mice.

Characterization of ostrich (Struthio camelus) β‐microseminoprotein (MSP): Ideication of homologous sequences in EST databases and analysis of their evolution during speciation

β‐Microseminoprotein, alternatively called prostatic secretory protein of 94 amino acids, is a hydrophilic, unglycosylated, small protein rich in conserved half‐cystine residues. Originally found in human seminal plasma and prostatic fluids, its presence was later shown in numerous secretions and its homologs were described in many vertebrate species. These studies showed that this protein had rapidly evolved, but they failed to unambiguously idey its biological role. Here, we show that a protein isolated from ostrich pituitary gland is closely related to a similar one isolated from chicken serum and that the two are structurally related to the mammalian β‐microseminoprotein. The complete 90–amino acid sequence of the ostrich molecule was established through a combination of automated Edman degradation and matrix‐assisted laser desorption ionization–time of flight (MALDI‐TOF) mass spectrometric procedures, including postsource decay (PSD) and ladder sequencing analyses. This study documents for the first time that β‐microseminoprotein is present in aves. It is also the first report of a C‐terminal amidated form for a member of this protein family and the first in which the disulfide linkages are established. Database searches using the herein‐described amino acid sequence allowed ideication of related proteins in numerous species such as cow, African clawed frog, zebrafish, and Japanese flounder. These small proteins show a strikingly high rate of amino acid substitutions, especially across phyla boundaries. Noticeably, no β‐microseminoprotein–related gene could be found in the recently completed fruit fly genome, indicating that if such a gene exists in arthropods, it must have extensively diverged from the vertebrate ones.

Adrenocorticotropin 53. The amino acid sequence of the hormone from the ostrich pituitary gland

Abstract The amino acid sequence of corticotropin from the ostrich pituitary gland has been determined. It consists of 39 amino acids with the following sequence: H-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Arg-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro-Asn-Gly-Val-Gln-Glu-Glu-Thr-Ser-Glu-Gly-Phe-Pro-Leu- Glu-Phe-OH. This is the first report on the primary structure of corticotropins from avian species.

Comparative blood coagulation studies in the ostrich.

Blood coagulation of the ostrich was compared to that of mammalian (man and sheep), avian (chicken) and reptilian (puff adder) systems. The international normalised ratio (INR), partial thromboplastin time (PTT), thrombin time and fibrin degradation were determined, as well as the various coagulation factors in venous ostrich plasma, using human physiological substrates. Thromboplastin was isolated from fresh brain tissue with the exception of the reptile for which lung tissue was used. The levels of markers of the coagulation [antithrombin III (AT), factor X (FX) and prothrombin], the fibrinolytic (alpha2-antiplasmin) and the kallikrein system were determined using chromogenic substrates. Elevated values for INR, PTT and thrombin time were obtained as compared to known human standards. It was found that factors VII, IX, X, XI and XII were absent from ostrich plasma. A study of the homologous and heterologous thromboplastin activities indicated that ostrich plasma exhibited a lower thromboplastic activity when compared to human standards, but was comparable to avian and reptilian values. Ostrich plasma revealed 42.2% FX, 72.9% AT, 35.3% prothrombin, 115.6% alpha2-antiplasmin and 19.8% plasma kallikrein, relative to human plasma. All the results suggest that the ostrich coagulatory system has not evolved to include all the complex myriad of reactions found in the human system.

Ostrich (Struthio camelus) carboxypeptidase B : purification, kinetic properties and characterization of the pancreatic enzyme

Carboxypeptidase B has been isolated from numerous mammalian and invertebrate species. In contrast, very little is known about carboxypeptidases of avian origin. To provide information for a comparative study, we have undertaken an investigation of the kinetic and physical properties of ostrich carboxypeptidase B. Carboxypeptidase B from the pancreas of the ostrich was purified by water extraction of acetone powder and aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. The effects of pH and temperature on CPB activity were examined. K(i)-values for numerous inhibitors (PCI, ABSA, hipp-D-lys, epsilon-aminocaproic acid, D-arg and 3-phenylproprionic acid) and kinetic parameters (K(m), k(cat) and k(cat)/K(m)) for several substrates (hipp-arg, hipp-lys, FAAA, FAAL and hipp-AA) were determined. N-terminal sequencing and amino acid analysis were also performed. Purified ostrich carboxypeptidase B was assessed to be homogeneous by SDS-PAGE with a M(r) value of approx. 35,000. For ostrich carboxypeptidase B the K(m) values for the different substrates were of the same order as those reported for other species, whereas the k(cat) values were 8- to 21-fold lower than the reported values. FAAA and hipp-AA were the preferred substrates. PCI was the most effective inhibitor, with a K(i) in the nM region, and no inhibition was shown with 3-phenylpropionic acid. The N-terminal sequence showed a high degree of homology when aligned with CPB from other species. Amino acid analysis showed significantly lower levels of Asx and Cyh and higher levels of Trp and Leu when compared with other species. Ostrich carboxypeptidase B would appear to show many physical, chemical and kinetic properties similar to those of other known carboxypeptidases.

Ostrich (Struthio camelus) carboxypeptidase A: Purification, kinetic properties and characterization of the pancreatic enzyme

1. Carboxypeptidase A beta and carboxypeptidase A tau-type from the pancreas of the ostrich were purified by water extraction of acetone powder, aminobenzylsuccinic acid affinity and hydroxylapatite chromatography. 2. The final preparations were homogeneous when subjected to SDS-PAGE and PAGE. The M(r) values obtained from SDS-PAGE for CPA beta and CPA tau-type were 34,600 and 34,400, respectively. 3. The effects of inhibitors (1,10 phenanthroline and indole-3-acetic acid), pH and temperature on CPA activity were examined. Ki-values for CPI, PPA, D-phe, D-trp and aminobenzylsuccinic acid were determined. 4. Km, kcat and kcat/Km values were determined for hipp-phe, cbz-gly-phe, cbz-(gly)2-phe, cbz-gly-leu, cbz-(gly)2-leu and cbz-(gly)2-val. 5. N-terminal sequencing and amino acid analysis were performed for CPA beta and CPA tau-type.

The ostrich pituitary contains a major peptide homologous to mammalian chromogranin A(1-76)

A major peptide related to the NH2-terminal fragment (position 1 to 76) of mammalian chromogranin A was isolated from ostrich adenohypophyses following acid-acetone extraction. The complete amino acid sequence of the homogenous peptide was deduced following automatic Edman degradation of the native peptide as well as of CNBr-, tryptic- and Lysobacter-derived peptides. The 76 amino acid sequence is strikingly homologous to bovine (80.3% sequence identity), porcine (79.0%), human (79.0%) and rat (72.4%) corresponding sequences, but much less so to human chromogranin B (22.4%). As this peptide is followed in bovine, porcine and human structure by a pair of basic residues (Lys-Lys), it could conceivably be produced during maturation in secretory granules. Finally, its structure appears to contain two potential amphipathic helices joined by the single disulfide bridge present in all chromogranin A and B molecules.

The isolation and partial characterization of trypsinogen, pancreatic secretory trypsin inhibitor and multiple forms of chymotrypsinogen and trypsin from the pancreas of the ostrich (Struthio camelus)

1. PSTI, two chymotrypsinogens and two trypsins were purified to homogeneity by acid extraction, salt fractionation, SP-Sephadex C-50 chromatography and RP-HPLC. 2. A third chymotrypsinogen, a trypsinogen and another trypsin were purified using an alkaline extraction procedure, followed by Trasylol- and Benzamidine-Sepharose affinity chromatography and hydroxylapatite chromatography. 3. The enzymes differed in amino acid composition as well as in specific activities towards synthetic amidase and esterase substrates. 4. N-terminal amino acid sequences were determined for one chymotrypsinogen and one trypsin.