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C Secchi

Publications and source records attributed to C Secchi.

At least 19 recordsLinked to original sources
PubMed2006

Heterophile antibody interference in a solid phase sandwich immunoassay for detection of equine growth hormone in plasma.

Heterophile antibodies (HAs) present in serum recognize animal immunoglobulins and are one of the most unpredictable causes of false results in immunoassays. However, no study has yet reported their interference on the diagnostic reliability of immunochemical analyses on horse plasma. Recently, we developed a sandwich ELISA for detection of equine growth hormone (eGH) in plasma. In a pilot study to measure basal eGH levels (blood samples were drawn from 13 horses every 10 min for 1h), we noted one horse with abnormally high eGH (>100 ng/mL). We demonstrate here that this plasma eGH level was falsely elevated due to interference from HAs. The interfering antibodies were polyspecific immunoglobulins, with fairly broad species-specificity, which affected the eGH immunoassay by bridging the mouse IgG capture antibody and the rabbit IgG conjugate. This produced artificial sandwiches which led to overestimation of the eGH plasma concentration. Spiking horse plasma with pure mouse and rabbit immunoglobulins or whole plasma of several species significantly reduced but did not totally eliminate the HAs interference. Immunoglobulins and whole plasma differed in their ability to block the interference, suggesting that HAs may recognize other proteins beside immunoglobulins in animal sera. To investigate whether HAs have any implications in equine clinical practice, we decided to seek information on the incidence of HAs interference in normal animals. We collected single plasma samples from another 114 horses and we found that 5 of these had plasma HAs. Therefore, in total 6 out of the 127 horses examined (4.7%) had plasma HAs generating falsely elevated eGH measures. In conclusion, this study provides the first evidence of HAs in horse plasma interfering with an immunoassay and indicates that veterinary surgeons and diagnostic laboratory staff should be aware of this potential for interference in tests on horse plasma using monoclonal or polyclonal antibody reagents.

Animals↗
PubMed2005

Effects of disease progression and L-dopa therapy on the control of reaching-grasping in Parkinson's disease.

The present study aimed to determine whether the bradykinesia of Parkinson's disease (PD) patients during the execution of reaching-grasping movements (i) is related to an impaired implementation of movement parameters and (ii) selectively involves the control of reach and/or grasp movements. We compared the kinematics of reaching to grasp of differently sized objects placed at different positions, among PD patients in the early stage of disease (ESPD), in the advanced stage of disease (ASPD) without L-dopa medication (off-state), and in healthy controls. In addition, we analysed the effects of L-dopa replacement therapy by comparing the kinematics of the patients in the advanced stage of disease after L-dopa administration with those of the other groups. Bradykinesia increased with disease progression, but only in the initial phases of the reach and grasp components. However at both stages of the disease, the kinematics of reaching and grasping responded to extrinsic and intrinsic object properties just as in controls. L-dopa administration improved the performance of PD patients, though this was more evident for the reach than for the grasp. We suggest that the basal ganglia (BG) are involved in implementing kinematic parameters, but neither (or only marginally) in the initial movement parameterization itself, nor in the on-line control of movement. Specifically, the BG dysfunction in PD induces a slowed implementation of movement parameters. The lack of effect of L-dopa administration on grasp kinematics may be because the motor control of distal effectors is less represented in the motor circuitry formed by the supplementary motor area (SMA), thalamus and BG.

Aged↗
PubMed2001

Amino acid modifications in canine, equine and porcine pituitary growth hormones, identified by peptide-mass mapping.

Modified amino acid residues in porcine, canine and equine growth hormones purified from pituitary glands were characterised by tryptic mapping and high-performance liquid chromatography with on-line coupled electrospray ionisation mass spectrometry (HPLC-ESI-MS) detection. Hormones from all three species showed the same changes. Conversion of Asp128 to iso-Asp128 was a component of native hormones, while deamidation of Asn12 and Asn98 to Asp and iso-Asp, oxidation of Met4, and cyclisation to the pyroglutamyl derivative of Gln139, probably occurred in vitro, during isolation, storage or hydrolysis. Porcine and canine hormones had indistinguishable protein fingerprints, confirming the assumption, based on their cDNA sequences, that their mature primary structures are identical.

Amino Acids↗
PubMed1999

Binding and signalling properties of a growth hormone enhancing monoclonal antibody.

We have used a sequential, qualitative biosensor based assay to demonstrate that OA15, a monoclonal antibody which enhances in vivo the activity of bovine growth hormone (bGH) does not disrupt the interaction between bGH and its cognate receptor (as represented by recombinant bovine GH binding protein -rbGHBP). We have confirmed this using a classical cell-based radio-receptor assay with the GH-responsive mouse pre-adipocyte cell line 3T3-F442A. The fact that OA 15 binding to bGH still allows hormone to interact with its receptor, allows us to test the hypothesis that there is any amplification of signalling events following hormone-MAb treatment of 3T3-F442A cells. We have used as a reporter of GH activity the rapid stimulation of JAK-2 tyrosine phosphorylation which is a critical first step in GH signalling events. We demonstrate that binding of rbGH by OA15 attenuates hormone stimulation of JAK-2 tyrosine phosphorylation. We conclude that although OA15 does not disrupt GH-GH receptor (GHR) interactions it does interfere with subsequent GH activity at the molecular and cellular level. We further speculate therefore that the biological enhancing activity of this antibody is most likely due to an in vivo effect as presentation of antibody-hormone complexes to a GH-target cell inhibits hormone activity.

3T3 Cells↗
PubMed1999

Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems.

We describe the properties of three monoclonal antibodies (MAbs) to ovine GH, two of which have previously been shown to enhance, in vivo, the biological activity of bovine and ovine growth hormone. We have examined the effects of these MAbs on GH activity in two appropriate GH-responsive cell culture systems, investigating both acute signalling effects (Janus-activated kinase (Jak)-2 tyrosine phosphorylation -5 min) and longer-term (MTT-formazan production -24 h) effects of hormone-antibody complexes. In the 3T3-F442A pre-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the two enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, OA14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, stably transfected with the full-length ovine GH receptor (oGHR), we demonstrate that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cause a decrease in this stimulatory activity of rbGH over a hormone concentration range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhancing MAbs are most probably related to the time-courses over which these two assays are performed, and also to the relative affinities between antibody, hormone and receptor. In addition, the in vitro inhibitory activity of the enhancing MAb OA11 in both short- and long-term bioassay lends further support to an exclusively in vivo model for MAb-mediated enhancement of GH action.

Adipocytes↗
PubMed1998

Plasma levels of GH and PRL and concentrations in the fluids of bovine ovarian cysts and follicles.

Prolactin and GH have been detected within the ovary, and it has become increasingly evident that they have a role as intrafollicular regulatory factors. The aim of the present work was to gain an insight into the elements influencing intraovarian GH and PRL in bovine species and to see whether cystic degeneration was accompanied by abnormal bovine GH (bGH) and PRL (bPRL) plasma patterns. We followed the relationships between plasma and ovarian fluid bGH and bPRL concentrations over an entire year in Friesian cows whose ovaries showed distinct types of structures. To assess the presence of bGH and bPRL within ovarian cells, we assayed selected ovarian structures by immunohistochemistry. The results demonstrated that: 1) plasma and ovarian fluid hormonal concentrations were independent, and their ratio was independent of the ovarian structure classes, subclasses and period of the year; 2) in the majority of the cows the concentration of bGH in ovarian fluid was no more than 80% of the level in plasma, whereas in about half the animals bPRL concentrations were higher in the ovary than in peripheral plasma; 3) mean bPRL concentrations in ovarian fluids were significantly higher in summer than in winter; 4) immunoreactive bGH and bPRL were present within granulosa and luteal cells. Thus, it is suggested that in the cow bGH and bPRL levels in the ovary might be regulated in some way independently of the pituitary.

Animals↗
PubMed1997

Structure and function of bovine growth hormone. Bovine growth hormone as an experimental model for studies of protein-protein interactions.

Growth hormone (GH) is a polipeptide that controls the differentiation, growth and metabolism of many cell types, and is secreted from the hypophysis of all vertebrate species tested so far. Despite the overlapping evolutionary, structural, immunological and biological properties, it is well-known that GHs from distinct mammalian species have significant species-specific characteristics. The main purpose of this review is to highlight bovine GH (bGH) structural features related to its species-specific properties. Novel interest in bGH is also aroused by the advent of biotechnological methods for production of recombinant proteins. In fact recombinant bGH will have a great importance in veterinary medicine research and as a 'high tech' drug that needs to be monitored in zootechnical productions.

Amino Acid Sequence↗
PubMed1996

Growth hormone stimulates the secretion of insulin-like growth factor binding protein-2 (IGFBP-2) by monolayer cultures of sheep costal growth plate chondrocytes.

Using monolayer cultures of costal chondrocytes established from four week old Clun Forest lambs, we have demonstrated that, under serum free conditions the cells release three IGFBPs (32, 29 and 21 kDa) into the medium. The most abundant of these--the 32 kDa BP--was shown to be IGFBP-2 by Western blotting. Furthermore we demonstrate that the levels of IGFBP-2 in conditioned medium are acutely increased (6, 12 and 24 h time points) following treatment of cells with bovine GH (1-100 ng/ml). In a parallel set of experiments, using ovine fibroblasts (derived from dermis) we show that IGFBPs of Mr 32, 29 and 21 kDa are also secreted by this cell type. However the relative abundance of these BPs differed from that seen in the chondrocyte cultures, with the 21 kDa species now the most abundant. In addition, prolonged exposure of autoradiographs indicated that fibroblasts secreted a higher Mr IGFBP (most probably ovine BP-3) that was not detected in any of our chondrocyte cultures. Most significant however was the demonstration that bGH did not dramatically affect the levels of IGFBPs in fibroblast cell cultures. We conclude that GH stimulates BP-2 production from chondrocytes and this is a cell-type specific effect in as much as it is not replicated in cultures of dermal fibroblasts.

Animals↗
PubMed1996

Growth hormone but not prolactin concentrations in the fluid of bovine ovarian cysts are related to the cystic stage of luteinization.

Regulation of follicular growth and ovulation as well as steroid production by the ovary depends principally on gonadotropins. However nonsteroid systemic hormones and autocrine and paracrine factors contribute to the regulation of ovarian function. The objectives of the present work were 1) to asses the presence of growth hormone (GH) and prolactin (PRL) in fluid drawn from normal bovine ovarian follicles, cysts or cystic corpora lutea; 2) to relate the stage of luteinization of the cyst with the GH and PRL concentrations in fluids; and 3) to asses the feasibility of providing a defined nonsteroid hormone marker to distinguish between normal and pathological ovarian structures. Cysts were classified according to histological and morphological appearance as follicular or luteal. Concentrations of GH, PRL, estrogens (E2), progesterone (P4) and testosterone (T) were measured in follicular and cystic fluids. On the basis of the E2 to P4 ratio, ovarian formation classes were further divided into two subclasses (E2 dominant and P4 dominant). The results provide evidence of 1) the presence of immunoreactive GH and PRL in all the follicular and cystic fluids assayed, 2) an increasing concentration of GH correlated to the stage of luteinization of the cyst and a direct correlation between GH and P4 concentrations, 3) a significant variability of intraovarian fluid PRL concentration not related to the histological class of the cyst nor to the concentrations of steroid hormones examined, and 4) the possibility of distinguishing 6 different ovarian formation classes by merely measuring GH, P4, E2 and T concentrations in fluids. These data contribute to a better understanding of the endocrine milieu of bovine ovarian cystic degeneration.

Journal Article↗
PubMed1996

Monoclonal antibodies against recombinant human growth hormone as probes to study immune function.

Monoclonal antibodies were raised against human recombinant growth hormone (rhGH) and those that did not cross-react with other human recombinant proteins like prolactin (PRL), interleukin 2 (IL-2), insulin, or bovine pituitary growth hormone were selected. The selected hybridoma supernatants were studied for their ability to influence T lymphocyte proliferation when induced either by a mitogen, such as phytohemagglutinin (PHA), or by alloantigen. All supernatants inhibited proliferation. Three MAbs were then purified by several passages on antimouse IgG (or IgM)-agarose columns, and characterized. These MAbs recognized three different epitopes, as revealed by competition study, although their inhibitory effect on PHA-induced T cell proliferation was quite similar. The data demonstrate that the MAbs were not cytolytic, that they did not interfere with the PHA binding to T cell membranes, and, as revealed by FACS analysis, did not bind to the membrane. Finally, these MAbs immunoprecipitated a 44-kDa molecule from PHA-activated T cell-concentrated supernatants. These data indicate that the MAbs recognized a soluble factor that plays a central role in T cell proliferation and that is probably the immune growth hormone.

Animals↗
PubMed1995

Matrix-assisted laser desorption mass spectrometry for the detection of recombinant bovine growth hormone in sustained-release form.

We employed matrix-assisted laser desorption mass spectrometry (LD-MS) to detect recombinant bovine growth hormone (r-bGH) in sustained-release preparations. After preliminary extraction in phosphate buffer, LD-MS provided a precise determination of the molecular mass (M(r)) of the r-bGH contained in 38 sustained-release preparations. The hormone was characterised using enzyme immunoassay, immunoblotting and amino acid sequencing. Rapid detection is essential for analysing large numbers of samples, and for monitoring the use of r-bGH in zootechnical productions and its administration as a "high-tech" drug for therapeutic purposes.

Amino Acid Sequence↗
PubMed1995

Inverse control of growth hormone and prolactin secretion in clonidine-stimulated dairy cattle.

Clonidine is a specific alpha-2-adrenoreceptor agonist that stimulates growth hormone (GH) release in animals and humans. This drug was used to study the GH and prolactin (PRL) secretory response in dairy cows and heifers. An i.v. infusion of 10 micrograms/kg body weight induced GH release to a peak concentration after 30-60 min, while 2 micrograms/kg had no effect on GH secretory patterns. Plasma PRL decreased significantly (P < 0.01) starting 15-60 min after both doses of clonidine, this effect lasting up to 6 h. Clonidine significantly lowered plasma insulin (P < 0.01) and raised plasma glucose (P < 0.01). The changes in plasma GH, PRL, insulin and glucose differed significantly between doses, the 10 micrograms/kg dose being more effective (P < 0.01). The results of our investigation in dairy cattle provide evidence of (i) an increase in GH release after 10 micrograms/kg clonidine; (ii) a concomitant decrease in PRL secretion, hence GH and PRL secretion in cattle appear inversely controlled; (iii) a significant difference between the effects of the 2 and 10 micrograms/kg doses and (iv) no relationship between the changes in plasma GH and PRL after clonidine and plasma hormone levels before treatment.

Animals↗
PubMed1994

Circadian and circannual plasma secretory patterns of growth hormone and prolactin in Fresian heifers: hormonal profiles and signal analysis.

The circadian and circannual secretory patterns of growth hormone (GH) and prolactin (PRL) in Fresian heifers were analysed to identify the significant pulses, the shape and the periodic components of the pulsatile signal that may be relevant to intercellular communication. The possible influence of temperature and photoperiod over these hormonal secretions has also been investigated. In the bovine GH (bGH) secretory patterns the basal levels and number, magnitude and amplitude of significant secretory peaks seem to be independent and not affected by seasonal changes. All the bGH circadian patterns show two periodic components with distinct frequencies. The bovine PRL (bPRL) circadian secretion shows basal levels, number and magnitude of the secretory peaks greater in summer than in winter; the periodic components are only one in winter, but two in summer. There is a significant increase of plasma bPRL in the May-July period and a correlation of the circannual hormone profile with both temperature and daylength.

Animals↗
PubMed1994

Monoclonal antibodies can reveal immunoreactivity differences between pituitary and recombinant bovine growth hormone.

Monoclonal antibodies (MAbs) to pituitary bovine growth hormone (bGH) were used to assay the immunoreactivity of a recombinant form of bGH. The recombinant hormone used differed from the pituitary principally in the presence of a short amino acid sequence starting with methionine added to the N-terminal end of the molecule. Monoclonal antibody 1D2 recognized the recombinant hormone with greater affinity than the pituitary hormone, whereas MAb 5G1 bound the recombinant molecule with a lower strength than the pituitary. The other MAbs showed different behavior depending on the type of immunoassay used. Results indicate that the recombinant bGH molecule has been altered in its immunological structure, and suggest a possible interaction of the added N-terminal fragment with the three-dimensional structure of the hormone.

Amino Acid Sequence↗
PubMed1993

An immuno-dominant sequential epitope recognized by mouse anti-recombinant bovine growth hormone antisera.

The sequential epitopes on bovine growth hormone (bGH) recognised by five polyclonal mouse antisera have been identified by scanning with multiple pin-bound peptides. Four main epitopes were identified at residues 29-40, 101-110, 139-152 and 181-191. Of these, only epitope 139-152 is recognised by all five mouse antisera indicating that, for the mouse, this may represent an immuno-dominant region of the bGH molecule.

Amino Acid Sequence↗
PubMed1992

Chemical modification of functional arginyl residues in beef kidney D-aspartate oxidase.

Chemical modification of beef kidney D-aspartate oxidase by phenylglyoxal is a biphasic process involving the transient formation of an enzymatic species with a decreased activity versus dicarboxylic substrates, an increased activity versus D-proline and a new activity versus other monocarboxylic D-amino acids which is absent in the native protein. Prolonged incubation with the modifier causes complete inactivation of the enzyme. The presence of the competitive inhibitor L-tartrate in the incubation mixture prevents enzyme inactivation. Kinetic and structural data suggest that complete loss of activity is paralleled by modification of eight arginine residues, of which two are critical for the specificity and the activity of the enzyme. We propose that the two essential arginine residues are located in the substrate binding site of D-aspartate oxidase.

Amino Acid Oxidoreductases↗
PubMed1991

Purification to homogeneity of bovine prolactin by high-performance ion-exchange chromatography.

Homogeneous bovine prolactin (bPRL) has been obtained using a procedure based on high-performance anion-exchange chromatography. The procedure enables up to 6 mg of 99.4% pure bPRL to be obtained per hour, with a recovery of 32.4%. The purity of the protein was checked by N-terminal sequencing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The highly purified bPRL obtained with this method is suitable for complete structural and immunochemical studies.

Animals↗