Immunochemical detection and characterisation of osteocalcin from moa bone

Osteocalcin (the 6,000 dalton Mr gamma-carboxyglutamate-containing protein of bone) has been detected in acid extracts of bones of the extinct class of New Zealand ratite birds, the moas, using a radioimmunoassay for sheep osteocalcin. The immunoreactive osteocalcin of the extracts of two of these bones (the fibulae from two specimens of Pachyornis elephantopus found in South Island swamps) has been fractionated by gel filtration chromatography and reversed-phase high performance liquid chromatography, and behaves in a manner characteristic of osteocalcin from modern bones. Carbon-14 dating of bones and gizzard contents found in association with these specimens indicates approximate ages of 3,600 and 7,400 years respectively.     

Immunochemical characterisation of megakaryocyte potentiator activity from mouse bone marrow

Megakaryocyte potentiator derived from mouse bone marrow has been shown to be immunologically similar to Interleukin-6 (IL-6). In this study the activity has been characterised by biochemical and immunochemical techniques. The activity is described as a O-linked glycosylated molecule with an apparent MW of 15 Kd and pl of the range pH 5.9–6.35. The data show that mouse bone marrow potentiator activity is a variant of IL-6 and with the potential to enhance megakaryocyte growth. © 1995 Wiley-Liss, Inc.     

Distribution and characterisation of immunoreactive somatostatin in human gastrointestinal tract

Immunoreactive somatostatin (IRS) was measured in acid extracts of human gastrointestinal tissue. The highest levels were found in the duodenum, pancreas, jejunum and stomach with lower levels in the ileum and colon. In the antrum, pylorus, duodenum and pancreas the main peak of IRS (1.6K IRS) coeluted with synthetic somatostatin-14 on both gel filtration chromatography and HPLC. In the body of stomach, jejunum, ileum and colon, a large peak coeluting with synthetic somatostatin-28 (3.5K IRS) on both chromatographic systems was also identified, while minor peaks of IRS assigned molecular weights of 6000 (6K) and greater than 15 000 (15K) were seen in some extracts. The total IRS content and pattern of molecular forms were similar in tissues obtained from adults at surgery or rapid post mortem, and in tissue taken from human fetuses after prostaglandin termination of pregnancy. When tissues were divided into mucosal and muscle layers, greater than 90% of the IRS was in the mucosa with less than 10% in the muscle layer. In the muscle layer the IRS was almost entirely the 1.6K form in all tissues. Immunohistochemical studies showed the IRS in the mucosa to be localised in endocrine-type cells, while in the muscle layer the IRS is present in nerve fibres and neurones of the myenteric plexus. It is suggested that (1) different mechanisms may control the biosynthesis of somatostatin-14 and somatostatin-28 in mucosal cells in different parts of the gut, (2) different biosynthetic controls may operate in endocrine-like and neuronal cells in the same region of the gut.     

Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Genetic characterisation of uninucleated cyst-producing Entamoeba spp. from ruminants

Six ssrRNA gene sequences were obtained by PCR amplification of DNA from uninucleated Entamoeba cysts isolated from fresh faeces of sheep, cows, a roe deer and a reindeer. Phylogenetic analysis using sequences of non-, uni-, quadri- and octonucleate cyst-producing Entamoeba spp. for comparison showed that all six isolates formed a separate clade nested within the clade of quadrinucleate cyst producers. The data indicate that Entamoeba bovis can be isolated from ruminant hosts other than cattle, and we suggest that organisms clustering with the sheep and cattle isolates analysed in the present study be named E. bovis.     

Immunochemical characterisation of the myofibrillar proteinase from cultured rat myocytes as chymase

The proteinase extracted from the myofibrillar fraction of (a) primary rat myocytes and (b) the L-8 myogenic cell line, both maintained in culture, was identified by immunochemical analysis as chymase, the chymotrypsin-like serine proteinase of rat mast cells. Chymase would therefore appear to be an intrinsic protein in the rat myocyte also.     

Immunochemical characterisation of tachykinin immunoreactivity in the nervous system of the garden snail,Helix aspersa

1. Circumoesophageal ganglia and foot muscle of the garden snail. Helix aspersa, were subjected to immunocytochemistry using antisera to the tachykinins, substance P (SP), neurokinin A (NKA), kassinin (KAS) and eledoisin (ELE).2. Immunoreactivity in neuronal somata and fibres was detected only with the SP antiserum.3. SP and NKA radioimmunoassays were performed on extracts of Circumoesophageal ganglia. In common with immunocytochemistry, immunoreactivity was only detected with the SP antiserum.4. Gel permeation chromatography of extracts resolved a single peak of immunoreactivity eluting slightly later than synthetic mammalian SP. Reverse-phase HPLC of immunoreactive fractions resolved two immunoreactive peptides representing oxidised and reduced forms of a single peptide.5. These data suggest that the nervous system of H. aspersa contains a single tachykinin with C-tenninal structural characteristics similar to mammalian SP.     

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.     

Distribution and characterisation of somatostatin receptor mRNA and binding sites in the brain and periphery.

The distribution and nature of (somatostatin) SRIF receptors and receptor mRNAs was studied in the brain and periphery of various laboratory animals using in situ hybridisation, autoradiography and radioligand binding. The messenger RNA (mRNA) expression of SRIF receptors msst1, msst2, msst3, msst4 and msst5 was studied in the adult mouse brain by in situ hybridisation histochemistry using specific oligonucleotide probes and compared to that of adult rats. As observed in rat brain, sst3 receptor mRNA is prominently expressed across the mouse brain, although equivalent binding has not yet been identified in situ. Sst1 and sst2 receptor mRNA expression, was prominent and again comparable to that observed in rat brain, whereas sst4 and especially sst5 receptor mRNA show comparatively low levels, although the former appears to be widely distributed while the latter could only be identified in a few nuclei. Altogether, the data are compatible with current knowledge, i.e. sst1 and sst2 receptor mRNA is prominent (both receptors have been functionally identified in the brain and for sst2 in the periphery), sst3 mRNA is highly expressed but in the absence of any functional correlate remains elusive. The expression of sst4 mRNA is comparatively low (especially when compared to what is seen in the lung, where high densities of sst4 receptors are present) and it remains to be seen whether sst5 receptor mRNA, which is confined to a few nuclei, will play a role in the brain, keeping in mind that high levels are found in the pituitary. Radioligand binding studies were performed in CCL39 cells expressing the five human recombinant receptors and compared to binding in membranes of rat cerebral cortex with [125I]Tyr11-SRIF14 which in the presence of 120 mM labels primarily sst1 receptor as suggested by the better correlation hsst1 and similar rank order of potency. The profile of [125I]Tyr3-octreotide labelled sites in rat cortex correlates better with recombinant sst2 than sst3 or sst5 binding profiles. Finally, [125I]LTT-SRIF28-labelled sites in rat lung express a sst4 receptor profile in agreement with previous findings. SRIF receptor autoradiography was performed in the brain and peripheral tissue of rat and/or guinea-pig using a number of ligands known to label recombinant SRIF receptors: [125I]LTT-SRIF28, [125I]CGP 23996, [125I]Tyr10-CST, or [125I]Tyr3-octreotide. Although, [125I]Tyr10-CST has been shown to label all five recombinant SRIF receptors, it is apparent that this radioligand is not useful for autoradiographic studies. By contrast, the other three ligands show good signal to noise ratios in rat or guinea-pig brain, rat lung, rat pancreas, or guinea-pig ileum. In most tissues, [125I]Tyr3-octreotide represents a prominent part of the binding (when compared to [125I]LTT-SRIF28 and [125I]CGP 23996), suggesting that sst2 receptors are strongly expressed in most tissues; it is only in rat lung that [125I]LTT-SRIF28 and [125I]CGP 23996 show marked binding, whereas [125I]Tyr3-octreotide does apparently label no sites, in agreement with the sole presence of sst4 receptors in this tissue.     

Immunochemical characterisation and epitope mapping of a novel fimbrial protein (Pg-II fimbria) of Porphyromonas gingivalis

Abstract Mouse monoclonal antibody (mAb) Pgf-II specific for a 72-kDa major cell-surface protein (72K-CSP) derived from Porphyromonas gingivalis OMZ 409 was prepared. Immunoblotting analysis revealed that mAb Pgf-II reacted with 72K-CSP but not with 41-kDa fimbrial subunit protein (41K-fimbrilin) derived from P. gingivalis 381. Electron microscopic observation revealed that P. gingivalis OMZ 409 possessed peritrichous, thin fimbriae on their surface. Immunogold electron microscopy also demonstrated that mAb Pgf-II bound to the 72K-CSP examined with the gold particles arranged along the fibril array originating from the cell surface of the bacteria. These findings suggested that P. gingivalis 72K-CSP was identifiable as another fimbriae (termed Pg-II fimbriae) different from the fimbriae (termed Pg-I fimbriae) composed of a 41K-fimbrilin. Using multipin peptide synthesis technology, 102 sequential overlapping peptides covering the entire 514 amino-acid stretch of Pg-II fimbriae were synthesised. Seven immunodominant regions within Pg-II fimbrial protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected.     

Poly-unsaturated fat in young ruminants

Lambs trained to suck daily liquid milk emulsified with various amounts of sunflower oil retained their suckling reflex up to 16 weeks of age, when they were slaughtered. Lambs receiving 12.5–25.0 ml sunflower oil daily between 13 and 16 weeks of age inclusive grew at slightly slower rates than lambs receiving no oil whereas a daily intake of 50 ml oil significantly suppressed growth.The addition of sunflower oil significantly increased the linoleic acid content of perinephric, subcutaneous and muscle fat (11.6–30.1%) compared with control samples (3.2–8.1%). The increase was highest in perinephric fat, and in all fats was accompanied by decreased palmitic, palmitoleic, stearic, oleic and linolenic acids. Oil feeding increased cholesterol levels in plasma but not in the carcass. No significant amounts of phytosterols were found in any of the groups.Taste panel evaluation of cooked meat showed that the increase in poly-unsaturated acids did not adversely affect its acceptability.     

Population regulation in trichostrongylids of ruminants

Since regulation of population size requires the existence of one or more density-dependent processes affecting parasite numbers, the literature was examined for evidence of density-dependence in establishment, reproduction and mortality of trichostrongylids of ruminants. Because of differences between the two environments required for completion of the trichostrongylid life-cycle, this evidence was sought in processes occurring within the host, rather than in dung-pats or on pasture. Evidence was found of at least one density-dependent population process in several economically important species of the genera Haemonchus, Ostertagia and Trichostrongylus, although further observations on populations derived from continuous infection at several rates of larval intake are required to substantiate much of this evidence. It is suggested that populations of trichostrongylids of ruminants are regulated at the level of the suprapopulation by density-dependent constraints on egg production of constituent infrepopulations; rates of larval intake may play a central role in coordinating regulation within each ecosystem.     

Acute phase proteins in ruminants

The physiological response to infections and injuries involves local inflammation and the initiation of events leading to a systemic response, also called acute phase reaction (APR). This multiplicity of changes is distant from the site of injury, and includes fever, leukocytosis and quantitative and qualitative modification of a group of non-structurally related proteins present in blood and other biological fluids, collectively named Acute Phase Proteins (APP). Proteomic investigations of serum or plasma following natural or experimental infection frequently reveal substantial alterations in the APP, several of which are high abundance proteins in these fluids. The present review will focus on the results of recent research on ruminant APP. Highlight points will include: - The structure and the functions of the main APPs in ruminants, as well as the regulatory mechanisms that trigger their systemic and local expression in both physiological and pathological conditions.- The clinical aspects of APPs in ruminants, including the current and future application to veterinary diagnosis and animal production.- The APP in small and wildlife ruminants.- Alteration in APP detected by proteomic investigations.     

Anthelmintics for ruminants

There is now a wide range of potent broad-spectrum anthelmintics for cattle and small ruminants, including the benzimidazoles, the avermectins, tetrahydropyrimidines and imidothiazoles which are highly efficacious against parasite gastroenteritis and bronchitis. Fascioliasis can be controlled with the salicy-lanilide and related phenolic compounds, the benzimidazoles, albendazole and triclabendazole the latter being the first flukicide with excellent activity against all stages of liver fluke infestation.The major developments for anthelmintic therapy have been and will be in the use of intraruminal boluses with either sustained release or pulse release of anthelmintic and other methods for group medication. The implications of such strategies for helminth resistance and individual immunity require further evaluation.