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Biomedical subjects

K Hannon

Publications and source records attributed to K Hannon.

At least 19 recordsLinked to original sources
PubMed2006

Ankyrin repeat and SOCS box protein 15 regulates protein synthesis in skeletal muscle.

Ankyrin repeat and SOCS box protein 15 (ASB15) is an Asb family member expressed predominantly in skeletal muscle. We have previously reported that ASB15 mRNA abundance decreases after administration of beta-adrenergic receptor agonists. Because beta-adrenergic receptor agonists are known to stimulate muscle hypertrophy, the objective of this study was to determine whether ASB15 regulates cellular processes that contribute to muscle growth. Stable myoblast C2C12 cells expressing full-length ASB15 (ASB15-FL) and ASB15 lacking the ankyrin repeat (ASB15-Ank) or SOCS box (ASB15-SOCS) motifs were evaluated for changes in proliferation, differentiation, protein synthesis, and protein degradation. Expression of ASB15-FL caused a delay in differentiation, followed by an increase in protein synthesis of approximately 34% (P<0.05). A consistent effect of ASB15 overexpression was observed in vivo, where ectopic expression of ASB15 increased skeletal muscle fiber area (P<0.0001) after 9 days. Expression of ASB15-SOCS altered differentiation of myoblasts, resulting in detachment of cells from culture plates. Expression of ASB15-Ank increased protein degradation by 84 h of differentiation (P<0.05), and in vivo ectopic expression of an ASB15 construct lacking both the ankyrin repeat and SOCS box motifs decreased skeletal muscle fiber area (P<0.0001). Together, these results suggest ASB15 participates in the regulation of protein turnover and muscle cell development by stimulating protein synthesis and regulating differentiation of muscle cells. This is the first study to demonstrate a role for an Asb family member in skeletal muscle growth.

Adrenergic beta-Agonists↗
PubMed2000

Loss of FGF receptor 1 signaling reduces skeletal muscle mass and disrupts myofiber organization in the developing limb.

The identities of extracellular growth factors that regulate skeletal muscle development in vivo are largely unknown. We asked if FGFs, which act as repressors of myogenesis in culture, play a similar role in vivo by ectopically expressing in the developing limb a truncated FGF receptor 1 (dnFGFR1) that acts as a dominant negative mutant. Hind limbs and the adjacent somites of Hamburger and Hamilton (HH) stage 17 chickens were infected with a replication-competent RCAS virus encoding dnFGFR1. By ED5, the virus had spread extensively within the limb and the adjacent somites with little rostral or caudal expansion of the infection along the axial midline. Viral infection and mutant receptor expression were coincident as revealed by the distribution of a viral coat protein and an HA epitope tag present on the carboxy terminus of dnFGFR1. Within 48 h following injection of dnFGFR1, we could detect no obvious changes in skeletal muscle precursor cell migration into the hind limb as compared to control limbs infected with an empty RCAN virus. However, by 3 days following infection of RCAS-dnFGFR1 virus, the level of skeletal muscle-specific myosin heavy chain was decreased and the expression pattern altered, suggesting disruption of skeletal muscle development. Two striking muscular phenotypes were observed in dnFGFR1-expressing limbs, including an average loss of 30% in skeletal muscle wet weight and a 50% decrease in myofiber density. At all ages examined the loss of skeletal muscle mass was accompanied by a loss of myoblasts and an unexpected concomitant loss of fibroblasts. Consistent with these observations, explants of infected cells revealed a reduction in the number of myonuclei in myotubes. Although the myofiber density per unit area was decreased over 50% compared to controls there were no detectable effects on myofiber diameter. The loss in myofiber density was, however, accompanied by an increase in the space surrounding individual myofibers and a generalized loss of myofiber integrity. It is noteworthy that long-bone development was unaffected by RCAS-dnFGFR1 infection, suggesting that FGFR2 and FGFR3 signaling was not disrupted. Our data provide conclusive evidence that FGFR1 signaling is necessary to maintain myoblast number and plays a role in myofiber organization.

Animals↗
PubMed2000

FGF5 stimulates expansion of connective tissue fibroblasts and inhibits skeletal muscle development in the limb.

FGF5 is expressed in the mesenchyme and skeletal muscle of developing and adult mouse limbs. However, the function of FGF5 during development of the limb and limb musculature is unknown. To elucidate the inherent participation of FGF5 during limb organogenesis, a retroviral delivery system (RCAS) was used to overexpress human FGF5 throughout developing hind limb of chicken embryos. Misexpression of the soluble growth factor severely inhibited the formation of mature myocytes. Limbs infected with RCAS-FGF5 contained smaller presumptive muscle masses as evidenced by a decrease in MyoD and myosin heavy chain expressing cells. In contrast, ectopic expression of FGF5 significantly stimulated proliferation and expansion of the tenascin-expressing, connective-tissue fibroblast lineage throughout the developing limb. Histological analysis demonstrated that the increase in tenascin immunostaining surrounding the femur, ileum, and pubis in the FGF5 infected limbs corresponded to the fibroblasts forming the stacked-cell perichondrium. Furthermore, pulse labeling experiments with the thymidine analog, BrdU, revealed that the increased size of the perichondrium was attributable to enhanced cell proliferation. These results support a model whereby FGF5 acts as a mitogen to stimulate the proliferation of mesenchymal fibroblasts that contribute to the formation of connective tissues such as the perichondrium, and inhibits the development of differentiated skeletal muscle. These results also contend that FGF5 is a candidate mediator of the exclusive spatial patterning of the hind limb connective tissue and skeletal muscle.

Animals↗
PubMed2000

Isolation of a spontaneously fusing BC3H1 muscle cell line: fusion alters the response to serum stimulation.

Differentiation of skeletal muscle cells involves two distinct events: exit from the cell cycle and expression of muscle-specific contractile genes and formation of multinucleated myocytes. Although many studies have shown that growth factors regulate the initial step of differentiation, little is known about regulation of fusion. BC3H1 cells are a skeletal muscle cell line characterized by a nonfusing phenotype and an ability to dedifferentiate. When subjected to serum or growth factors, differentiated BC3H1 cells lose muscle-specific gene expression and re-enter the cell cycle. In this study, we describe a spontaneously fusing clone of BC3H1 cells. We demonstrate that this fusion capability is not due to altered muscle regulatory factor or adhesion molecule expression. Furthermore, we show that fusion inhibits dedifferentiation. Multinucleated BC3H1 cells do not lose myosin expression, nor do they re-enter the cell cycle. Fused BC3HI cells react to serum stimulation with a hypertrophic response. Our results suggest that the state of differentiation, mono- or multi-nucleated, is essential to how myocytes react to growth stimulation and may provide a mechanism for how differentiation, fusion, and hypertrophy are regulated in vivo.

Blood Proteins↗
PubMed1999

Expression of fibroblast growth factor family during postnatal skeletal muscle hypertrophy.

The potential role of the fibroblast growth factor (FGF) family during stretch-induced postnatal skeletal muscle hypertrophy was analyzed by using an avian wing-weighting model. After 2 or 11 days of weighted stretch, anterior latissimus dorsi (ALD) muscles were, on average, 34 (P < 0.01) and 85% (P < 0.01) larger, respectively, than unweighted ALD control muscles. By using quantitative RT-PCR, FGF-1 mRNA expression was found to be significantly decreased in ALD muscles stretched for 2 or 11 days. In contrast, FGF-4 and FGF-10 mRNA expression was significantly increased 2 days after initiation of stretch. FGF-2, FGF-10, fibroblast growth factor receptor 1, and FREK mRNA expression was significantly increased at 11 days poststretch. Increases in FGF-2 and FGF-4 protein could be detected throughout the myofiber periphery after 11 days of stretch. On a cellular level, FGF-2 and FGF-4 proteins were differentially localized. This differential expression pattern and protein localization of the FGF family in response to stretch-induced hypertrophy suggest distinct roles for individual FGFs during the postnatal hypertrophy process.

Animals↗
PubMed1996

Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms.

Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in either proliferating or differentiated skeletal muscle cells. FGF-I and FGF-2 proteins were present in proliferating skeletal muscle cells, but undetectable after terminal differentiation. We show that transfection of expression constructs encoding FGF-1 or FGF-2 mimics the effects of exogenously applied FGFs, inhibiting skeletal muscle cell differentiation and stimulating DNA synthesis. These effects require activation of an FGF tyrosine kinase receptor as they are blocked by transfection of a dominant negative mutant FGF receptor. Transient transfection of cells with FGF-1 or FGF-2 expression constructs exerted a global effect on myoblast DNA synthesis, as greater than 50% of the nontransfected cells responded by initiating DNA synthesis. The global effect of cultures transfected with FGF-2 expression vectors was blocked by an anti-FGF-2 monoclonal antibody, suggesting that FGF-2 was exported from the transfected cells. Despite the fact that both FGF-l and FGF-2 lack secretory signal sequences, when expressed intracellularly, they regulate skeletal muscle development. Thus, production of FGF-1 and FGF-2 by skeletal muscle cells may act as a paracrine and autocrine regulator of skeletal muscle development in vivo.

Actins↗
PubMed1994

Role of FGFs in skeletal muscle and limb development.

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridge-derived factor that participates in limb outgrowth and patterning.

Animals↗
PubMed1994

Genesis of olfactory receptor neurons in vitro: regulation of progenitor cell divisions by fibroblast growth factors.

Olfactory receptor neurons are produced continuously in mammalian olfactory epithelium in vivo, but in explant cultures neurogenesis ceases abruptly. We show that in vitro neurogenesis is prolonged by fibroblast growth factors (FGFs), which act in two ways. FGFs increase the likelihood that immediate neuronal precursors (INPs) divide twice, rather than once, before generating neurons; this action requires exposure of INPs to FGFs by early G1. FGFs also cause a distinct subpopulation of explants to generate large numbers of neurons continually for at least several days. The data suggest that FGFs delay differentiation of a committed neuronal transit amplifying cell (the INP) and support proliferation or survival of a rare cell, possibly a stem cell, that acts as a progenitor to INPs.

Animals↗
PubMed1994

Are fibroblast growth factors regulators of myogenesis in vivo?

Recent advances in understanding of skeletal muscle differentiation implicate fibroblast growth factors (FGFs) as regulators of myogenesis; however, the identity and actions of factors that repress myogenesis in vivo remain to be established. This review will focus on the fibroblast growth factor family and the evidence for its role in regulating myogenesis in culture and in vivo.

Animals↗
PubMed1993

Synthesis of PCR-derived, single-stranded DNA probes suitable for in situ hybridization.

We report the novel synthesis of polymerase chain reaction (PCR)-derived single-stranded DNA (ssDNA) probes and their subsequent application in in situ hybridizations. Serial transverse sections of an 11.5-day postcoitum mouse embryo were hybridized to a 33P-ssDNA, 33P-RNA, or 35S-RNA probe corresponding to the same 181-bp sequence in the myogenin cDNA. Signal obtained using 33P-ssDNA was more intense than that using 33P-RNA probe, while signal/noise ratios obtained with both 33P-probes were far superior to those obtained with 35S-probe. Digoxigenin-labeled chicken growth hormone (GH) ssDNA gave slightly more intense signal than did digoxigenin-labeled chicken GH RNA when hybridized to chicken pituitary sections. 32P-ssDNA probes were found to be suitable for Northern blot hybridization. Advantages of using ssDNA probes for in situ hybridization include: (1) The ssDNA technique is rapid and simple. There was no need to clone a DNA template into a special RNA vector or order special T7-containing PCR primers. ssDNA probes can be synthesized in less than 1 day using any primers which currently exist in a laboratory (optimal probe length for in situ hybridization is between 50 and 200 bp). (2) In three separate in situ experiments, ssDNA probes yielded more intense signal than RNA probes. (3) ssDNA probes are potentially more stable than RNA probes. (4) Since the RNAse rinse is eliminated, posthybridization rinses are shortened when hybridizing with ssDNA probes. The ssDNA probes produced by this protocol can be labeled with a variety of different isotopes (both radioactive and nonradioactive), and are excellent probes for use in in situ hybridizations.

Animals↗
PubMed1993

Single-stranded RNA probes generated from PCR-derived DNA templates.

The following report outlines the use of the polymerase chain reaction (PCR) in combination with in vitro transcription to generate single-stranded radiolabelled RNA probes useful for nuclease protection and in situ hybridization experiments. Specific DNA fragments with bacteriophage promoter (T3 and/or T7) sequences at the 5' or 3' end are generated by repeated rounds of amplification. Following purification, these PCR-generated DNA products are used as templates for in vitro transcription with the correct DNA-dependent RNA polymerase. The resultant radiolabelled, single-stranded RNA (ssRNA) can be used for in situ hybridization, Southern or Northern blot analysis, and ribonuclease protection experiments. Sub-cloning or hydrolysis of large fragments is not required. Probes can be made from virtually any sequence using a variety of template sources.

Animals↗
PubMed1993

Expression of bovine myf5 induces ectopic skeletal muscle formation in transgenic mice.

myf5 is one of a family of four myogenic determination genes that control skeletal muscle differentiation. To study the role of myf5 in vivo, we generated transgenic mice harboring the bovine homolog, bmyf, under control of the murine sarcoma virus promoter. Ectopic expression of the full-length bmyf transgene was detected in brain and heart tissue samples of F1 progeny from transgenic founder mice. Ectopic bmyf expression activated endogenous skeletal myogenic determination genes in the hearts and brains of transgenic animals. Incomplete skeletal myogenesis in most hearts gave rise to cardiomegaly and focal areas of cardiomyopathy. In brains in which ectopic expression led to a more complete myogenesis, focal areas of multinucleated, striated myotubes containing actin, desmin, and myosin were observed. These unexpected results show that myf5 can initiate myogenic differentiation in vivo, supporting the hypothesis that myf5 is responsible for determination of cells to the myogenic lineage in normal embryogenesis.

Animals↗
PubMed1992

Temporal and quantitative analysis of myogenic regulatory and growth factor gene expression in the developing mouse embryo.

Using a reverse transcription/polymerase chain reaction method, the temporal pattern of expression of the myogenic regulatory genes (myf5, myogenin, MRF4, myo D) was quantitated in developing mouse muscle (whole embryo: 6.5 to 12.5 days postcoitum (dpc); front limb buds: 9.5 to 12.5 dpc; hind limb buds: 11.5 to 14.5 dpc) and related to expression of TGF-beta 1, b-FGF, IGF-I, and IGF-II. Myf5 was the first myogenic regulatory factor to appear in both the whole embryo and front limb bud, with expression evident 7.5 and 9.5 dpc, respectively. A transient peak of MRF4 expression occurred 10.5 dpc in both the whole embryo and the front limb bud. Myogenin and myo D expression in the whole embryo was detected 8.5 and 9.5 dpc, respectively. In the front limb bud myogenin and myo D expression was not detected until 10.5 dpc. In the hind limb bud myf5, myogenin, and MRF4 expression was detected 11.5 dpc. Myo D expression was not detected until 12.5 dpc. With respect to growth factor expression, in the front limb bud TGF-beta 1, IGF-I, and IGF-II were evident 9.5 dpc, while bFGF was not detected until 10.5 dpc. In the hind limb bud TGF-beta 1, bFGF, IGF-I, and IGF-II expression was detected 11.5 dpc. These results show that in both the whole embryo and limb buds, all four myogenic regulatory factors are involved in the initiation of the myogenic program. We also show myf5 expression in the 9.5-dpc front limb bud, suggesting its expression in the somite-derived migrating muscle precursor cells. Correlations between growth factor-mediated myoblast proliferation and myogenic differentiation are discussed.

Animals↗
PubMed1992

An Irish out-patient based in-vitro fertilisation service.

The first year's activity of a fully out-patient based, self financing, in vitro fertilisation service is reported. 98 couples had superovulation regimes tailored to need. 138 cycles were started. 109 retrievals were attempted using the vaginal ultrasound probe under Fentanyl and Midazolam sedation. In 106, oocytes were successfully retrieved (average seven per patient). In 80, embryos were transferred (average three). 13 patients achieved clinical pregnancies giving an overall pregnancy rate of 9.4% per cycle, 12% per oocyte retrieval, 16.5% per embryo transfer and 13% per patient who embarked on the programme. One pregnancy was ectopic, two aborted, and 10 are ongoing; this includes seven singleton foetuses and three sets of twins. All pregnant patients were referred back to their unit of origin for antenatal care and delivery.

Adult↗
PubMed1991

Relationship of thyroid status to growth hormone and insulin-like growth factor-I (IGF-I) in plasma and IGF-I mRNA in liver and skeletal muscle of cattle.

Steers were made hyperthyroid or hypothyroid to study the effects of physiological alterations in thyroid hormone status on plasma growth hormone (GH) profiles, plasma insulin-like growth factor-I (IGF-I) concentrations, and relative abundance of IGF-I mRNA in skeletal muscle and liver. Eighteen yearling crossbred steers (360 to 420 kg) were randomly allotted to hyperthyroid (subcutaneous injection 0.6 microgram/kg BW L-thyroxine for 10 d), hypothyroid (oral thiouracil; 0.25% diet plus 12.5 g capsule/d for 17 d), or control (subcutaneous injection 0.9% NaCl) treatment groups. Blood samples were taken for measurement of GH, IGF-I, thyroxine (T4) and triiodothyronine (T3) by RIA. Samples of liver and skeletal muscle were taken by biopsy for measurement of IGF-I mRNA by solution hybridization. Steers receiving thiouracil had 57 and 53% (P less than .05) lower T4 and T3, respectively, than control steers (84.1 and 1.7 ng/ml). The hyperthyroid steers had 228 and 65% greater (P less than .05) T4 and T3 than control steers. Neither increased nor decreased thyroid status had any significant effects on plasma GH profiles, liver IGF-I mRNA, or plasma concentration of IGF-I. There was no effect of thyroid hormone alteration on skeletal muscle IGF-I mRNA concentrations. The results of this study suggest that short-term changes in thyroid status of cattle had no major impact on the GH-IGF-I axis or skeletal muscle IGF-I mRNA.

Animals↗
PubMed1991

Relationship of liver and skeletal muscle IGF-1 mRNA to plasma GH profile, production of IGF-1 by liver, plasma IGF-1 concentrations, and growth rates of cattle.

Growth hormone (GH), insulin-like growth factor-1 (IGF-1), and thyroid hormone (T3 and T4) concentrations in blood plasma of 18 crossbred cattle (six bulls, six steers, and six heifers) were measured over an 8-hr period. One week later at slaughter, IGF-1 production by liver slices and IGF-1 mRNA concentrations in skeletal muscle and liver were measured. Bulls had higher (P less than 0.05) mean plasma GH and GH peak amplitudes (P less than 0.01) than heifers, and values for steers were intermediate between bulls and heifers. Baseline GH concentrations and number of GH peaks were not significantly different for the three groups. Bulls had 1.6-fold (P less than 0.01) and 3.0-fold (P less than 0.01) greater liver IGF-1 mRNA concentrations than steers or heifers, respectively, whereas the steers had 1.8-fold (P less than 0.05) greater IGF-1 mRNA in liver than heifers. Production of IGF-1 by liver slices was greater (P less than 0.05) in bulls than steers or heifers. Bulls had 1.3-fold greater plasma IGF-1 than steers (P less than 0.01), whereas steers had 1.8-fold greater plasma IGF-1 than heifers (P less than 0.01). There were no significant differences in concentrations of skeletal muscle IGF-1 mRNA between the three groups of animals. Liver IGF-1 mRNA, liver IGF-1 production, and plasma IGF-1 were all significantly correlated with gain and mean GH peak amplitude, but not with GH baseline, GH peak frequency, or concentrations of T3 and T4. Concentrations if IGF-1 mRNA in skeletal muscle were not correlated to gain or any parameter of the GH profile. Plasma concentrations of T3 were significantly (P less than 0.05) negatively correlated to plasma GH baseline concentrations. Muscle IGF-1 mRNA concentration was negatively related to plasma T4 and T3. The results of this study suggest that the cascade of events starting with secretion of GH from the pituitary, expression of liver IGF-1 mRNA, and secretion of IGF-1 by the liver are important phenomena for growth of cattle.

Animals↗
PubMed1990

Evaluation of condensed molasses fermentation solubles as a nonprotein nitrogen source for ruminants.

Condensed molasses fermentation solubles (CMS), an effluent from the production of lysine, was evaluated as a nonprotein nitrogen supplement for ruminants by measuring the availability of its nitrogen to rumen microorganisms grown in batch cultures and by comparing CMS to urea as a source of supplemental nitrogen for growing cattle. In vitro dry matter digestion studies showed that, with 1 ml or less of rumen inoculum, microbial digestion was enhanced more (P less than .05) by the addition of CMS than by the addition of urea to 100 mg of cellulose. These stimulatory effects of CMS were absent when either the amount of inoculum (5.0 ml) or cellulose (250 mg) was increased and when wheat straw or alfalfa replaced cellulose as the substrate. Growth rate and feed intake for cattle fed a high-cob/cracked-corn diet containing 2.5 or 5.0% CMS were lower (P less than .05) than for cattle fed the control diet containing urea. Digestibility of dry matter, crude protein, neutral detergent fiber and acid detergent fiber were reduced (P less than .05) by the addition of CMS. Addition of CMS also decreased feed utilization, although the differences were not statistically significant. In conclusion, the nitrogen in CMS was available to rumen microorganisms growth in batch culture; however, CMS was not satisfactory as a substitute for all the urea in a diet for growing cattle containing over 45% of dietary N from the supplemental N source.

Animal Feed↗