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Biomedical subjects

C Goddard

Publications and source records attributed to C Goddard.

At least 37 records · Page 2Linked to original sources
PubMed1996

Decreased muscle cell proliferation in chicks with a deletion in the GH receptor gene.

The increase in muscle weight in neonatal animals is a consequence of increased protein accretion and DNA content. GH increases protein accretion but direct effects of GH on myogenic cell proliferation have not been demonstrated. Sex-linked dwarfism in the chick is caused by mutation or deletion in the GH receptor gene and has provided a useful model to study the physiological consequences of GH insensitivity. This study determined the consequences of GH receptor gene mutation on muscle cell proliferation in vivo. Northern and Southern blotting and PCR analysis revealed restriction fragment length polymorphism patterns and a 1.7 kb deletion of the intracellular domain of the GH receptor gene in commercial dwarf broiler chicks, similar to the Connecticut strain in which there is a dysfunctional GH receptor. Cell proliferation was measured in muscle sections from normal and dwarf chicks after incorporation of 5-bromo-2'-deoxyuridine (BrdU; 25 mg/kg) in vivo at 2, 5 and 13 days of age. Incorporation of BrdU into nuclei was measured in frozen sections, counterstained with propidium iodide to estimate the total number of nuclei by quantitative image analysis, and the labelling index was calculated. Paraffin-embedded sections of breast muscle were stained using an anti-human IGF-I polyclonal antibody. Expression of IGF-I mRNA in muscle from each genotype at 5 days of age was measured by RNAse protection assay. The labelling index was similar in 2-day-old chicks from both genotypes (normal, 20.14 +/- 2.39%; dwarf, 19.79 +/- 5.83%). By day 5 the labelling index had decreased but was significantly higher (P < 0.02) in normal (12.53 +/- 3.36%) compared with the dwarf (6.25 +/- 1.39%). By 13 days of age, there was a further decrease in labelling index but no difference between the groups (normal, 4.92 +/- 1.28%; dwarf, 4.96 +/- 1.51%). IGF-I mRNA was expressed and IGF-I peptide was identified in muscle sections but there was no difference between genotypes. The results show that cell division in breast muscle in vivo is high in neonatal chicks but it declines with increasing age. The absence of a functional GH receptor in the dwarf is associated with a greater decline in DNA synthesis and suggests that GH may directly affect a proportion of cells, since there was no difference in IGF-I mRNA or peptide.

Animals↗
PubMed1996

Transforming growth factor beta in bovine milk: concentration, stability and molecular mass forms.

Transforming growth factor beta (TGF-beta) is one of the predominant growth factors present in milk. The concentration, molecular mass forms and stability of TGF-beta in bovine milk were investigated using a standard bioassay measuring the growth inhibition of a milk lung epithelial cell line. Most of the TGF-beta bioactivity in milk was found to be in a latent form, which was also retained in the whey fraction. After acid activation, the total TGF-beta concentration was 4.3 +/- 0.8 ng and 3.7 +/- 0.7 ng TGF-beta per ml of milk and cheese whey respectively. Cation-exchange chromatography at pH 6.5 was used to concentrate latent whey-derived TGF-beta, which could be activated by transient exposure to extremes of pH, urea or heat. Heparin did not significantly activate milk-derived TGF-beta. Neutral gel filtration of the cationic whey fraction revealed a major peak of latent TGF-beta with a molecular mass of 80 kDa and a smaller peak at 600 kDa. Transient acidification of the cationic whey fraction prior to neutral gel filtration, or gel filtration under acidic conditions, released low molecular mass TGF-beta from both high molecular mass peaks. Whey-derived TGF-beta was purified using a five-step chromatographic procedure. An N-terminal sequence was obtained for TGF-beta 2, which accounted for over 85% of the TGF-beta bioactivity in whey. All TGF-beta activity in whey could be neutralised by a monoclonal antibody directed against TGF-beta 1, -beta 2 and -beta 3. The results suggest that the majority of TGF-beta in bovine milk is present in a small latent complex.

Animals↗
PubMed1996

New opportunities from the isolation and utilization of whey proteins.

Management of dairy whey has often involved implementation of the most economical disposal methods, including discharge into waterways and onto fields or simple processing into low value commodity powders. These methods have been, and continue to be, restricted by environmental regulations and the cyclical variations in price associated with commodity products. In any modern regimen for whey management, the focus must therefore be on maximizing the value of available whey solids through greater and more varied utilization of the whey components. The whey protein constituents offer tremendous opportunities. Although whey represents a rich source of proteins with diverse food properties for nutritional, biological, and functional applications, commercial exploitation of these proteins has not been widespread because of a restricted applications base, a lack of viable industrial technologies for protein fractionation, and inconsistency in product quality. These shortcomings are being addressed through the development of novel and commercially relevant whey processing technologies, the preparation of new whey protein fractions, and the exploitation of the properties of these fractions in food and in nontraditional applications. Examples include the following developments: 1) whey proteins as physiologically functional food ingredients, 2) alpha-lactalbumin and beta-lactoglobulin as nutritional and specialized physically functional food ingredients, and 3) minor protein components as specialized food ingredients and an important biotechnological reagents. Specific examples include the isolation and utilization of lactoferrin and the replacement of fetal bovine serum in tissue cell culture applications with a growth factor extract isolated from whey.

Animals↗
PubMed1996

Divergent selection for high or low growth rate modifies the response of muscle cells to serum or insulin-like growth factor-I in vitro.

Genetic differences in growth potential could result from changes in the levels of growth stimulatory factors or in the response of target tissues. The latter possibility was tested in adult myoblasts prepared from chickens selected for high (HG) or low growth rate (LG). Stimulation of [3H]-thymidine incorporation into DNA by serum was of higher amplitude in HG than LG muscle cells irrespective of whether the cell preparations were enriched in myoblasts or fibroblasts. HG myoblasts were also more responsive to insulin-like growth factor-I (IGF-I) in terms of [3H]-thymidine incorporation. IGF analogues with a reduced affinity for IGF binding proteins gave similar results suggesting that activity of binding proteins could not explain the difference between cells from the HG and LG lines. This difference was restricted to the proliferative stage because in myotubes, basal or IGF-I stimulated glucose and amino acid transports, tyrosine incorporation and protein degradation were not different.

Aging↗
PubMed1995

Porcine osteochondrosis: deficiencies in transforming growth factor-beta and insulin-like growth factor-I.

Osteochondrosis and dyschondroplasia are common multifocal disturbances of endochondral ossification in many species of domestic animals, and are characterized by the retention of avascular cartilage. These cartilage disorders are characterized by a failure of chondrocyte differentiation, matrix mineralisation and its replacement by bone. Rabbit polyclonal antibodies to transforming growth factor-beta (TGF-beta) and to insulin-like growth factor-I (IGF-I) were used to detect the two growth factors in normal and osteochondrotic porcine epiphyses. In the normal pig epiphyses IGF-I and TGF-beta were present in the chondrocytes of the epiphyseal hyaline cartilage and IGF-I was readily localised to the hypertrophic chondrocytes in the growth cartilage adjacent to the epiphyseal ossification centre. Both growth factors were found to be deficient in chondrocytes at sites of osteochondrosis. Both these growth factors are thought to be involved in the cascade of events associated with chondrocyte function during endochondral ossification. Deficiencies in TGF-beta and IGF-I demonstrated in porcine osteochondrosis and previously shown in avian dyschondroplasia suggest further similarities in the pathogenesis of these conditions.

Animals↗
PubMed1995

Increased expression and activity of ornithine decarboxylase in chicks genetically selected for rapid growth rate.

Polyamines are essential requirements for cell proliferation and their role in stimulation of RNA, DNA, and protein synthesis is clearly established. Ornithine decarboxylase is a key enzyme in the biosynthesis of the polyamines and its activity is regulated in response to factors that stimulate cell proliferation. Steady-state ODC mRNA levels and enzyme activity were measured in muscle of chicks genetically selected for increased growth rate or for egg production. In muscle, muscle satellite cells and myotubes, two ODC mRNA transcripts are present of molecular size 2.05 and 1.75 kb. Northern blotting analysis suggest that these transcripts are produced as a result of using different polyadenylation sites. Between day 1 and day 6 after hatching, a period of rapid muscle growth in these animals, a peak in muscle ODC mRNA levels is followed by a peak in enzyme activity in both lines. Significantly higher ODC mRNA levels and enzyme activity are associated with selection for rapid growth in the broiler line. The results are consistent with other data showing that ODC is a major factor in cell growth and provide further evidence that it is a candidate 'trait-gene' for growth.

Animals↗
PubMed1995

Regulation of chick muscle satellite cells by fibroblast growth factors: interaction with insulin-like growth factor-I and heparin.

This study describes the effect of acidic and basic fibroblast growth factor (FGF) on DNA synthesis in chick satellite cells in vitro and interactions with insulin-like growth factor-I (IGF-I) and exogenous heparin. Basic bFGF stimulated incorporation of [3H]thymidine into DNA with a half-maximum concentration (ED50) of 3.23 +/- 0.33 pmol/l, more than 500-fold more potent than acidic FGF (ED50 = 2.13 +/- 0.5 nmol/l). Both bFGF and IGF-I allowed the cells to traverse the cell cycle with an approximate length of the G1 phase of 12 h. When cells were incubated with bFGF and IGF-I together their effects on DNA synthesis were additive rather than synergistic throughout the full concentration range. Incubation of satellite cells with low concentrations of heparin (ng/ml) to mimic the effect of endogenous heparan sulphate proteoglycan caused a small increase in DNA synthesis, whereas higher concentrations (microgram/ml) inhibited DNA synthesis in a dose-related manner. A low concentration of heparin increased DNA synthesis at the highest concentration of bFGF, but high doses of heparin inhibited the response to bFGF throughout the dose-response curve but without altering the ED50. RNAse protection assay showed the expression of bFGF mRNA in proliferating cells which appeared to decrease on differentiation. The results suggest that aspects of neonatal muscle development are regulated by interactions between autocrine/paracrine growth factors such as IGF-I and bFGF, perhaps IGF-I derived from the circulation, and components of the extracellular matrix. Concentrations of the matrix components may change throughout the neonatal period and into adulthood and have an important effect on the regulatory role played by the growth factors.

Animals↗
PubMed1994

Identification of tricyclic analogs related to ellagic acid as potent/selective tyrosine protein kinase inhibitors.

The plant-derived natural product ellagic acid (1) has recently been identified as a potent, though nonselective, inhibitor of the tyrosine-specific protein kinase pp60src. This report details efforts directed toward the identification of tricyclic structures related to ellagic acid, with enhanced specificity for inhibition of pp60src over other protein kinases. Phenanthridinone and carbazole core structures were selected for investigation, since N-functionalization allows for the synthesis of numerous analogs which can be utilized to probe enzyme-inhibitor interactions. These ring systems were prepared via a general sequence of biaryl bond formation followed by cyclization to form the desired tricyclic ring systems. N-Alkylation, -acylation, or -sulfonylation and deprotection with boron tribromide afford the target tetraphenolic phenanthridinones 5 and carbazoles 9. Several analogs from both of these series have potencies comparable to that of 1 and exhibit substantially enhanced selectivities for inhibition of pp60src relative to protein kinase A (PKA), a serine/threonine protein kinase. Carbazole-based analogs 9j,m,p are submicromolar inhibitors of pp60src, with potency for the target tyrosine kinase comparable to that of ellagic acid (1), however with 2 orders of magnitude greater selectivity versus that for PKA. As seen for ellagic acid, members of the phenanthridinone-based series (e.g., 5a) exhibited inhibition of pp60src in a manner which is partial mixed noncompetitive with respect to ATP, while analogs in the carbazole series (e.g., 9a) inhibit pp60src in an ATP competitive manner.

Amino Acid Sequence↗
PubMed1994

Insulin-like growth factor-I in the ovary of the laying hen: gene expression and biological actions on granulosa and thecal cells.

Concentrations of insulin-like growth factor-I (IGF-I) were measured in granulosa and thecal tissue dissected from the three largest follicles in the ovaries of laying hens. The higher concentration was found in extracts of granulosa (0.82 +/- 0.01 pmol/g wet wt) and theca (0.36 +/- 0.02), both of which were greater than that in liver extracts (0.25 +/- 0.01). RNA was extracted from these tissues, and by using reverse transcription and the polymerase chain reaction with primers specific for chicken IGF-I, both granulosa and thecal tissue were shown to express chicken IGF-I mRNA. Granulosa and thecal cell cultures were established and used to measure IGF binding sites and the response to exogenous IGF peptides in terms of DNA synthesis. Both cell types bound [125I]IGF-I, which was displaced by IGF-I, IGF-II, and insulin in descending order of potency, characteristic of a type-I IGF receptor. Treatment of granulosa and thecal cell cultures with IGF-I resulted in a dose-dependent increase in [3H]thymidine incorporation into DNA by both cell types. LH, but not FSH, stimulated DNA synthesis in cultured granulosa cells but not in cultured thecal cells. This effect was enhanced in granulosa cells by the addition of IGF-I to the culture medium. These data are consistent with an autocrine or paracrine role for IGF-I within the developing ovarian follicle of the domestic hen.

Animals↗
PubMed1994

Rapidly growing broiler (meat-type) chickens: their origin and use for comparative studies of the regulation of growth.

1. Rapidly growing (meat-type) chickens have been intensively selected for over 50 years and grow up to four times faster than "layer" strains selected for reproductive traits. 2. Comparison between these lines are increasingly being used to study mechanisms underlying lean tissue growth. 3. Selection for increased growth has resulted in some undesirable consequences such as poor reproductive performance, excessive fatness, increased skeletal abnormalities and ascites. 4. The biochemistry, physiology and molecular biology of these changes are reviewed.

Animals↗
PubMed1994

Plasma concentrations of growth hormone and insulin-like growth factor-I in chickens developing tibial dyschondroplasia.

The concentrations of plasma growth hormone (GH) and insulin-like growth factor-I (IGF-I) were measured in broilers during the initial development of tibial dyschondroplasia. Chicks were fed a standard starter ration, or a diet imbalanced in calcium and phosphorus to increase the incidence of dyschondroplasia. At 14 days of age four blood samples were collected and assayed for GH and IGF-I. The chicks were killed at three weeks of age and sections of bone were assessed histologically for evidence of dyschondroplasia. All the chicks displayed a pulsatile pattern of GH secretion. Eight of the group fed the imbalanced diet developed dyschondroplasia which was accompanied by a significant increase in the mean and peak GH concentrations compared with the control group but no increase in basal concentrations. The chicks fed the imbalanced diet which did not develop dyschondroplasia were not different from the control birds. There were no differences in IGF-I concentrations between the groups.

Animals↗
PubMed1994

Plasma growth hormone and insulin-like growth factor-I (IGF-I) concentrations at the onset of lay in ad libitum and restricted broiler breeder fowl.

1. Plasma concentrations of growth hormone (GH) and insulin-like growth factor I(IGF-I) were determined in individually caged broiler breeder females from 17 to 30 weeks of age. The birds were fed ad libitum or restricted during rearing in a randomised block design. Restricted birds were fed ad libitum after laying an egg and egg production was recorded to 54 weeks. 2. GH concentrations were higher in restricted compared with ad libitum-fed birds at 17 to 21 weeks of age and were similar thereafter. Plasma IGF-I concentrations were higher in restricted compared with ad libitum-fed birds throughout the sampling period of the experiment. 3. The age at first egg was delayed by about two weeks in restricted birds. At 54 weeks of age they had laid 114 compared with 67 eggs in birds fed ad libitum throughout and had produced less than half the number of eggs with defective shells. 4. It was suggested that differences in egg production between ad libitum and restricted broiler breeder females could be related to changes in the pattern of GH and/or IGF-I secretion.

Aging↗
PubMed1993

Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin-like growth factor-I.

The effects of insulin and insulin-like growth factor-I (IGF-I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [3H]-tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [3H]-tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF-I. Subsequent [3H]-aminoisobutyric acid (AIB) uptake was then measured in amino acid-free medium. IGF-I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 +/- 0.03 nmol/l and 34.8 +/- 3.1 nmol/l for IGF-I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 mumol/l) diminished IGF-I-stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF-I-stimulated AIB uptake. This study demonstrates an anabolic effect for IGF-I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation-independent mannose 6-phosphate receptor does not bind IGF-II in chicken cells.

Amino Acids↗
PubMed1993

Regulation of adipocyte precursor DNA synthesis by acidic and basic fibroblast growth factors: interaction with heparin and other growth factors.

The development of adipose tissue is dependent on the growth and differentiation of fibroblast-like adipocyte precursor cells. Culture of adipocyte precursor cells in vitro has provided an ideal system for identifying potential regulators of proliferation and differentiation. We have demonstrated that both acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) stimulate chicken adipocyte precursor DNA synthesis in a dose-dependent manner up to a concentration of 100 micrograms aFGF/l and 1 microgram bFGF/l. The effect of bFGF was biphasic, so that in incubations with 25 micrograms bFGF/l, DNA synthesis was not significantly different from controls. In the presence of heparin, stimulation of DNA synthesis at 25 micrograms bFGF/l was 1.6-fold greater than at a concentration of 1 microgram bFGF/l. Addition of heparin to incubations containing aFGF reduced the concentration required for maximum stimulation of DNA synthesis to 1 microgram/l. Cells incubated with aFGF (1-100 micrograms/l) in combination with insulin-like growth factor-I (IGF-I), platelet-derived growth factor, transforming growth factor-alpha or transforming growth factor-beta 1 (TGF-beta 1) exhibited a marked synergistic increase in DNA synthesis. This was also the case when 1 microgram bFGF/l was used, but at a concentration of 25 micrograms bFGF/l synergy was only seen with IGF-I and TGF-beta 1. These results suggest that both basic and acidic FGF are potentially important regulators of adipocyte hyperplasia and that their effect is modulated by constituents of the extracellular matrix and the presence of other growth factors.

Adipose Tissue↗
PubMed1993

Insulin-like growth factor-I-stimulated glucose transport in myotubes derived from chicken muscle satellite cells.

The effects of insulin and insulin-like growth factor-I (IGF-I) on glucose transport were compared in myotubes derived from chicken breast muscle satellite cells in vitro. Myotubes were incubated (for 0.5 or 4 h) with or without glucose in the presence or absence of insulin or IGF-I. Glucose uptake was subsequently measured by the incorporation of 2-[1,2-3H(N)] deoxy-D-glucose ([3H]2DG) in glucose-free medium (10 min at 20 degrees C). Glucose uptake was almost completely abolished by the addition of cytochalasin B or phloretin. It was increased by a decrease in glucose concentration in the incubation medium. Insulin (5 mg/l) stimulated [3H]2DG uptake to a maximum of 43 +/- 10% above basal after 30-min incubation and 101 +/- 15% after 4-h incubation. IGF-I and insulin at equimolar concentrations (25 micrograms/l and 20 micrograms/l respectively) were almost equipotent after 0.5 h but after 4-h incubation IGF-I was 17-fold more potent, suggesting that this 'late' effect was mediated through the IGF-I receptor. Incubation with cycloheximide suggested that the effect of IGF-I involved increased protein synthesis. The results suggest that chicken myotubes express a glucose transporter which is regulated by IGF-I and glucose concentration. However, they do not appear to express a typical insulin-responsive transport system.

Animals↗
PubMed1992

Effects of transforming growth factor-alpha on chicken adipocyte precursor cells in vitro.

The hyperplastic capacity of adipose tissue resides in a group of fibroblast-like adipocyte precursor cells. There is evidence to suggest that their proliferation and differentiation is regulated by insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) but there is less information about other growth factors which may also participate in adipocyte precursor cell hyperplasia. Transforming growth factor-alpha (TGF-alpha) is a 50 amino acid polypeptide which has been shown to stimulate proliferation in both neoplastic and normal cell types acting through the epidermal growth factor (EGF) receptor. We have studied the regulation of DNA synthesis and the activity of lipoprotein lipase by TGF-alpha in chicken adipocyte precursor cells in vitro. Both TGF-alpha and EGF stimulated incorporation of [3H]thymidine into DNA in a dose-dependent manner. TGF-alpha was approximately 180-fold more potent than EGF. Addition of TGF-alpha in combination with IGF-I, TGF-beta 1 or platelet-derived growth factor produced a synergistic increase in DNA synthesis. Short-term incubation with TGF-alpha reduced lipoprotein lipase activity by 23%. These results show that TGF-alpha is a potent mitogen in these adipocyte precursor cells and can inhibit their differentiation in vitro and may participate in the regulation of adipose tissue development in vivo.

Adipose Tissue↗