Increased sclerostin associated with stress fracture of the third metacarpal bone in the Thoroughbred racehorse.

OBJECTIVES: The exact aetiology and pathogenesis of microdamage-induced long bone fractures remain unknown. These fractures are likely to be the result of inadequate bone remodelling in response to damage. This study aims to identify an association of osteocyte apoptosis, the presence of osteocytic osteolysis, and any alterations in sclerostin expression with a fracture of the third metacarpal (Mc-III) bone of Thoroughbred racehorses. METHODS: A total of 30 Mc-III bones were obtained; ten bones were fractured during racing, ten were from the contralateral limb, and ten were from control horses. Each Mc-III bone was divided into a fracture site, condyle, condylar groove, and sagittal ridge. Microcracks and diffuse microdamage were quantified. Apoptotic osteocytes were measured using TUNEL staining. Cathepsin K, matrix metalloproteinase-13 (MMP-13), HtrA1, and sclerostin expression were analyzed. RESULTS: In the fracture group, microdamage was elevated 38.9% (sd 2.6) compared with controls. There was no difference in the osteocyte number and the percentage of apoptotic cells between contralateral limb and unraced control; however, there were significantly fewer apoptotic cells in fractured samples (p < 0.02). Immunohistochemistry showed that in deep zones of the fractured samples, sclerostin expression was significantly higher (p < 0.03) than the total number of osteocytes. No increase in cathepsin K, MMP-13, or HtrA1 was present. CONCLUSION: There is increased microdamage in Mc-III bones that have fractured during racing. In this study, this is not associated with osteocyte apoptosis or osteocytic osteolysis. The finding of increased sclerostin in the region of the fracture suggests that this protein may be playing a key role in the regulation of bone microdamage during stress adaptation.Cite this article:Bone Joint Res 2018;7:94-102.

The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.

The effects of ethanol on the brain are concentration dependent. Low concentrations (mM) intoxicate, while greater than 100 mM anaesthetize. Of most relevance to human alcohol addiction are mechanisms of intoxication. Previously, Caenorhabditis elegans has been employed in genetic screens to define effectors of intoxication. Here, we inform interpretation of these studies by providing evidence that ethanol rapidly equilibriates across C. elegans cuticle. Importantly, the effect of ethanol on muscle activity rapidly reaches steady-state, and the concentration-dependence of the effect is very similar in intact animals and exposed muscle. Thus the cuticle does not present an absorption barrier for ethanol, and furthermore the internal concentration is likely to approach that applied externally. Thus, modelling intoxication in C. elegans requires exposure to external ethanol less than 100 mM. Furthermore, the permeability of the cuticle to ethanol enables analysis of precisely controlled concentration-dependent effects of acute, chronic, and episodic ethanol exposure on behaviour.

Molecular characterization of the metabotropic glutamate receptor family in Caenorhabditis elegans.

mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that play an important neuromodulatory role in the brain. Glutamatergic transmission itself plays a fundamental role in the simple nervous system of the model organism Caenorhabditis elegans, but little is known about the contribution made by mGluR signalling. The sequenced genome of C. elegans predicts three distinct genes, mgl-1, mgl-2 and mgl-3 (designated Y4C6A.2). We have used in silico and cDNA analyses to investigate the genes encoding mgls. Our results indicate that mgl genes constitute a gene family made up of three distinct subclasses of receptor. Our transcript analysis highlights potential for complex gene regulation with respect to both expression and splicing. Further, we identify that the predicted proteins encoded by mgls harbour structural motifs that are likely to regulate function. Taken together, this molecular characterization provides a platform to further investigate mGluR function in the model organism C. elegans.

Primary hypothyroidism in a child mimicking a pituitary macroadenoma.

We report the case of an 11-year-old girl with primary autoimmune hypothyroidism causing secondary pituitary enlargement. She presented with headaches and a pituitary mass on MRI thought to be due to a pituitary macroadenoma. Resolution of the pituitary mass and symptoms occurred with thyroxine therapy. It is mandatory to rule out primary hypothyroidism as a cause of pituitary enlargement before surgery is considered.

Requirements of multiple domains of SLI-1, a Caenorhabditis elegans homologue of c-Cbl, and an inhibitory tyrosine in LET-23 in regulating vulval differentiation.

SLI-1, a Caenorhabditis elegans homologue of the proto-oncogene product c-Cbl, is a negative regulator of LET-23-mediated vulval differentiation. Lack of SLI-1 activity can compensate for decreased function of the LET-23 epidermal growth factor receptor, the SEM-5 adaptor, but not the LET-60 RAS, suggesting that SLI-1 acts before RAS activation. SLI-1 and c-Cbl comprise an N-terminal region (termed SLI-1:N/Cbl-N, containing a four-helix bundle, an EF hand calcium-binding domain, and a divergent SH2 domain) followed by a RING finger domain and a proline-rich C-terminus. In a transgenic functional assay, the proline-rich C-terminal domain is not essential for sli-1(+) function. A protein lacking the SH2 and RING finger domains has no activity, but a chimeric protein with the SH2 and RING finger domains of SLI-1 replaced by the equivalent domains of c-Cbl has activity. The RING finger domain of c-Cbl has been shown recently to enhance ubiquitination of active RTKs by acting as an E3 ubiquitin-protein ligase. We find that the RING finger domain of SLI-1 is partially dispensable. Further, we identify an inhibitory tyrosine of LET-23 requiring sli-1(+) for its effects: removal of this tyrosine closely mimics the loss of sli-1 but not of another negative regulator, ark-1. Thus, we suggest that this inhibitory tyrosine mediates its effects through SLI-1, which in turn inhibits signaling upstream of LET-60 RAS in a manner not wholly dependent on the ubiquitin-ligase domain.

ARK-1 inhibits EGFR signaling in C. elegans.

A screen for synthetic enhancers of sli-1 identified ark-1 (forAck-related tyrosine kinase), a novel inhibitor of let-23 EGFR signaling in C. elegans. An ark-1 mutation synergizes with mutations in other negative regulators of let-23, resulting in increased RAS signaling. Genetic analysis suggests that ARK-1 acts upstream of RAS and is dependent upon SEM-5. ARK-1 inhibits LET-23-mediated ovulation, a RAS-independent function. ARK-1 physically interacts with SEM-5 in the yeast two-hybrid assay. We find that sem-5 also has a negative function in let-23-mediated ovulation and suggest that this negative function is mediated by the recruitment of inhibitors such as ARK-1.

Caenorhabditis elegans SOS-1 is necessary for multiple RAS-mediated developmental signals.

Vulval induction in Caenorhabditis elegans has helped define an evolutionarily conserved signal transduction pathway from receptor tyrosine kinases (RTKs) through the adaptor protein SEM-5 to RAS. One component present in other organisms, a guanine nucleotide exchange factor for Ras, has been missing in C.ELEGANS: To understand the regulation of this pathway it is crucial to have all positive-acting components in hand. Here we describe the identification, cloning and genetic characterization of C.ELEGANS: SOS-1, a putative guanine nucleotide exchanger for LET-60 RAS. RNA interference experiments suggest that SOS-1 participates in RAS-dependent signaling events downstream of LET-23 EGFR, EGL-15 FGFR and an unknown RTK. We demonstrate that the previously identified let-341 gene encodes SOS-1. Analyzing vulval development in a let-341 null mutant, we find an SOS-1-independent pathway involved in the activation of RAS signaling. This SOS-1-independent signaling is not inhibited by SLI-1/Cbl and is not mediated by PTP-2/SHP, raising the possibility that there could be another RasGEF.

cudA: a Dictyostelium gene with pleiotropic effects on cellular differentiation and slug behaviour.

The Dictyostelium cudA gene encodes a nucleoplasmic protein that is essential for normal culmination. There are no functionally characterised homologues in other organisms but there is a related gene of unknown function in Entamoeba histolytica. The cudA gene is expressed by the prestalk cells that constitute the slug tip (the pstA cells), it is not detectably expressed in the band of prestalk cells that lies behind the tip (the pstO cells) but it is expressed in the prespore cells. This unusual pattern of expression suggests a role on both the stalk and spore pathways of differentiation and cudA- mutant cells are indeed defective in both stalk and spore formation. Furthermore, the slugs formed by cudA- cells continue to migrate under environmental conditions where normal slugs culminate immediately. This aspect of their behaviour can be reversed when the cudA gene is selectively expressed in the pstA cells. This shows that processes occurring in the pstA cells regulate entry into culmination.

Extracellular cAMP depletion triggers stalk gene expression in Dictyostelium: disparities in developmental timing and dose dependency indicate that prespore induction and stalk repression by cAMP are mediated by separate signaling pathways.

During Dictyostelium development, amoebae differentiate into spores and stalk cells. Earlier studies showed that extracellular cAMP is essential for induction of prespore differentiation and that cAMP represses stalk gene expression in vitro. We show that the repressive pathway is operative in vivo, because activation of the stalk-specific promoter region of the ecmB gene is strongly enhanced by overexpression of a phosphodiesterase that depletes extracellular cAMP. To test whether a single cAMP transduction pathway controls the choice between prespore or stalk cell differentiation, we compared the timing and dose dependency of the effects of cAMP on both responses. Cells acquire competence for cAMP repression of ecmB promoter activity 4 hr later than for prespore gene induction. Half-maximal prespore induction requires 30 microM stable cAMP analog Sp-cAMPs, while ecmB induction is half-maximally repressed by 200 nM Sp-cAMPs, which is equivalent to about 3 to 13 nM cAMP. At concentrations exceeding 10 microM, Sp-cAMPs stimulates ecmB expression from the intact promoter, but not from the stalk-specific subregion. These data suggest that distinct signaling pathways operating at different developmental stages control induction of prespore genes on one hand and repression of stalk genes on the other. Both stalk gene repression and prespore gene induction by Sp-cAMPs are antagonized by millimolar adenosine concentrations. However, an adenosine analog that is resistant to extracellular metabolism is active at 10 microM. Since adenosine inhibits cAMP binding to cAMP receptors, it may facilitate stalk gene expression by reducing the perceived cAMP concentration.

Cerebral artery blood flow velocity changes following rapid release of lower body negative pressure.

BACKGROUND: Circulatory changes occur during exposure to Lower Body Negative Pressure (LBNP). These changes may have some similarities to exposure to moderately and slowly increased G-loads in a relaxed subject without anti-G suit. HYPOTHESIS: Changes will also occur in cerebral blood circulation during a rapid release of LBNP. METHODS: Transcranial Doppler ultrasound (TCD) was used to measure middle cerebral artery blood flow velocity (CBFV) in 14 human subjects following rapid release of a ramped lower body negative pressure (LBNP) (0.33 mm Hg.s) to presyncope (mean peak negative pressure of -124 mm Hg). RESULTS: The mean CBFV decreased to an average of 60% (p < 0.05) of the baseline value at peak LBNP. Mean CBFV was still decreased to 65% and 84% of the baseline value (p < 0.05) at the third heart beat and 30 s, respectively, after pressure release. The systolic CBFV decreased similarly to 57% (p < 0.05) of baseline during peak LBNP, and was still 63% (p < 0.05) at the third heart beat after pressure release. Heart rate increased by a mean of 51% (p < 0.001) and systolic heart level blood pressure decreased by 28% (p < 0.001) during peak negative pressure. Both heart rate and blood pressure returned to baseline levels within 30 s after pressure release. CONCLUSIONS: Following a presyncopal LBNP, the CBFV is not fully restored up to 30 s after the release of the negative pressure. This delayed returning of cerebral circulation following orthostatic stress may have some similarities to what occurs after the release of a gradual onset G-load in a relaxed subject without anti-G suit.

Protein kinase A is a positive regulator of spore coat gene transcription in Dictyostelium.

The cotA, cotB, and cotC genes encode the major spore coat proteins of Dictyostelium. All three cot genes are coordinately expressed as aggregation is nearing completion. Induction and maintenance of their expression is dependent upon the presence of extracellular cAMP. We show that expression of a dominant inhibitor of the cAMP dependent protein kinase (PKA) in prespore cells greatly reduces the transcription rates of the cotB and cotC genes. All three cot genes contain, in their upstream regulatory regions, short sequence elements that have a high content of cytosine and adenosine residues. These CA-rich sequences are essential for optimal cot gene transcription. We show that expression of the dominant PKA inhibitor results in a greatly reduced level of the binding activity that recognizes the CA-rich sequences upstream of the cotB gene. Thus PKA acts, either directly or indirectly, to control expression of the cot genes and it may do so by modulating the activity of a DNA binding protein. However, we find that mutant cells where PKA is constitutively active still require exogenous cAMP for optimal cot gene expression in dissociated cells, suggesting that a separate, PKA-independent, signalling pathway is also involved in the regulation of cot gene expression by extracellular cAMP.

A role for cAMP-dependent protein kinase in determining the stability of prespore cell differentiation in Dictyostelium.

When Dictyostelium slugs are disaggregated and shaken in suspension prespore mRNAs disappear from the cells very rapidly but in the presence of extracellular cAMP the loss of prespore mRNA sequences is retarded. In a mutant which lacks a functional regulatory subunit of the cAMP-dependent protein kinase (PKA), and where the catalytic subunit is thereby rendered constitutively active, two prespore mRNA sequences persist for extended times after cellular disaggregation. Thus PKA is a component of the signaling pathway that allows prespore cells rapidly to dedifferentiate when extracellular cAMP signaling is disrupted. Analysis of cells expressing a dominant inhibitor of PKA shows there to be both a PKA-requiring and a non-PKA-requiring intracellular signaling pathway directing prespore-specific gene transcription and suggests that there may also be post-transcriptional regulation by PKA.

Induction of terminal differentiation of Dictyostelium by cAMP-dependent protein kinase and opposing effects of intracellulr and extracellular cAMP on stalk cell differentiation.

Expression of the catalytic (C) subunit of the cAMP-dependent protein kinase (PKA) of Dictyostelium under the control of heterologous, cell-type-specific promoters causes ectopic terminal differentiation. When expressed under the control of a prespore-specific promoter, development is accelerated, to yield highly aberrant fruiting bodies that contain a basal mass of spore cells surrounding a central stalk-like structure. When expressed under the control of a prestalk-specific promoter, development arrests much earlier, at the tight mound stage. Prestalk cells move to the apices of these mounds, apparently normally, but no tip is formed. Most of the prestalk cells remain arrested in their development but there are a few isolated stalk cells scattered within such mounds. We show that extracellular cAMP represses stalk cell-specific gene expression in cells where the kinase is constitutively active, suggesting that inhibition of stalk cell differentiation by cAMP in normal cells (Berks and Kay, 1988) occurs because of an effect of extracellular cAMP on an intracellular signalling pathway independent of PKA. We propose a scheme whereby two separate events, a rise in intracellular cAMP levels and a fall in extracellular cAMP concentration, are required to induce stalk cell differentiation.

Activation of the prespore and spore cell pathway of Dictyostelium differentiation by cAMP-dependent protein kinase and evidence for its upstream regulation by ammonia.

Expression of a dominant inhibitor of the Dictyostelium cAMP-dependent protein kinase in prespore cells blocks their differentiation into spore cells. The resultant structures comprise a normal stalk supporting a bolus of cells that fail to express a sporulation-specific gene and that show greatly reduced levels of expression of several prespore-specific genes. The latter result suggests that in addition to activating spore formation, the cAMP-dependent protein kinase may play a role in initial prespore cell differentiation. Development of the strain expressing the dominant inhibitor is hypersensitive to the inhibitory effects of ammonia, the molecule that is believed to repress entry into culmination during normal development. This result supports a model whereby a decrease in ambient ammonia concentration at culmination acts to elevate intracellular cAMP and hence induce terminal differentiation.

Regulation of Dictyostelium morphogenesis by cAMP-dependent protein kinase.

During formation of the Dictyostelium slug extracellular cAMP signals direct the differentiation of prespore cells and DIF, a chlorinated hexaphenone, induces the differentiation of prestalk cells. At culmination the slug transforms into a fruiting body, composed of a stalk supporting a ball of spores. A dominant inhibitor of cAMP-dependent protein kinase (PKA) expressed under the control of a prestalk-specific promoter blocks the differentiation of prestalk cells into stalk cells. Analysis of a gene specifically expressed in stalk cells suggests that PKA acts to remove a repressor that prevents the premature induction of stalk cell differentiation by DIF during slug migration. PKA is also necessary for the morphogenetic movement of prestalk cells at culmination. Expression of the PKA inhibitor under control of a prespore-specific promoter blocks the accumulation of prespore mRNA sequences and prevents terminal spore cell differentiation. Thus PKA is essential for progression along both pathways of terminal differentiation but with different mechanisms of action. On the stalk cell pathway it acts to regulate the action of DIF while on the spore cell pathway PKA itself seems to act as the inducer of spore cell maturation. Ammonia, the extracellular signal which regulates the entry into culmination, acts by controlling the intracellular concentration of cAMP and thus exerts its effects via PKA. The fact that PKA is necessary for both prespore and spore gene expression leads us to postulate the existence of a signalling mechanism which converts the progressive rise in cAMP concentration during development into discrete, PKA-regulated gene activation events.

Interacting signalling pathways regulating prestalk cell differentiation and movement during the morphogenesis of Dictyostelium.

Analysis of the expression patterns of two genes encoding extracellular matrix proteins shows there to be an unexpectedly complex pattern of prestalk cell differentiation and movement during the morphogenesis of Dictyostelium. The organism employs both cell sorting and positional differentiation to generate a patterned structure but these two mechanisms are used at different times during development. During slug formation prestalk cells arise at scattered positions within the aggregate and then move to its apex to form the tip. In contrast, during culmination, stalk cell differentiation occurs in a positionally localized manner at the entrance to the stalk tube. Two interacting signalling pathways regulate the differentiation of prestalk and stalk cells. Prestalk cell differentiation is induced by DIF, a chlorinated hexaphenone, and a repression mechanism prevents DIF acting to induce premature stalk cell differentiation during slug migration. At culmination intracellular cAMP levels rise, the cAMP dependent protein kinase (PKA) is activated and the block to stalk cell differentiation is lifted. Activation of PKA is also necessary in order that prestalk cells move to the entrance of the stalk tube at culmination. Thus, in Dictyostelium, PKA plays a role both in the regulation of cellular differentiation and in morphogenetic cell movement.

Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase.

We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.