High-intensity exercise induces structural, compositional and metabolic changes in cuboidal bones--findings from an equine athlete model.

Fatigue fracture of cuboidal bones occurs in the human foot as well as the equine carpus. The racehorse provides a naturally-occurring model to study the effects of high-intensity exercise on the morphology and metabolism of cuboidal bones. We studied both the mineral and the collagenous matrix of the third (C(3)) and radial (C(r)) carpal bones of raced and non-raced Thoroughbred (TB) horses. We hypothesised that racehorses would show increases in the mineral component of these bones and post-translational modifications of the collagenous matrix alongside changes in markers of collagen remodelling and bone formation. C(3) and C(r) carpal bones were retrieved from raced TB horses (n=14) and non-raced TB horses (n=11). Standardised proximal-distal sections were taken from each bone and these were sliced transversely to study the proximal-distal differences in bone metabolism from the subchondral plate through to trabecular bone. Histomorphometry and bone mineral density measurements were performed in parallel with biochemical analyses including total collagen, collagen synthesis and cross-links, matrix metalloproteinases-2 and 9 and their inhibitors, calcium and phosphate, and bone alkaline phosphatase. The results of this study show that, while there is a net increase in bone formation in the racehorses, there is additionally an increase in bone collagen synthesis and remodelling, particularly within the trabecular regions of the bone. The increase in bone density would lead to greater stiffness, particularly in the cortical bone, and failure of this 'stiffer' cortical bone may result from its lack of support from the rapidly remodelling and structurally weakened underlying trabecular bone.

The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria of Magnaporthe grisea.

Histochemical and ultrastructural studies were carried out on a wild-type strain (Guy11) and a melanin-deficient mutant (buf1) of the rice-blast pathogen, Magnaporthe grisea (= Pyricularia oryzae), in order to investigate the destination of lipid storage reserves during appressorium development. Lipid droplets were abundant in conidia and were mobilised upon germination, accumulating in the appressorial hook which developed at the tip of each germ tube. Following the formation of a septum at the base of the nascent appressorium, one or a few closely appressed central vacuoles became established and were observed to enlarge in the course of appressorium maturation. On unyielding artificial surfaces such as glass or plastic, appressoria matured to completion within 36-48 h, by which time the enlarged vacuole filled most of the inside volume of the appressorium. Light and transmission electron microscopical observations revealed that the lipid droplets entered the vacuole by autophagocytosis and were degraded therein. Histochemical approaches confirmed the vacuole as the key lytic element in maturing appressoria. Endocytosis of a vital dye, Neutral Red, progressed via endosomes which migrated into the vacuole and lysed there, releasing their dye content into the vacuolar lumen. Furthermore, activity of the lysosomal marker enzyme, acid phosphomonoesterase, was strongly localised in the vacuole at all stages of appressorium maturation. It is therefore envisaged that vacuoles are involved in the degradation of lipid storage reserves which may act as sources of energy and/or osmotically active metabolites such as glycerol, which generate the very high turgor pressure known to be crucial for penetration of hard surfaces. On softer surfaces such as onion epidermis, appressoria of M. grisea were able to penetrate before degradation of lipid droplets had been completed.

Effects of estrogen and progesterone on tibia histomorphometry in growing rats.

The present study was performed to evaluate possible interactions between estrogen and progesterone on peak cancellous bone mass. Ovariectomized (OVX) growing rats were treated with 17beta-estradiol (4.8 microg/day), progesterone (4.8 mg/day), a combination of the two sex steroids, or with vehicle for 14 days beginning 7 days after OVX. The tibiae were removed for histomorphometric analysis of the proximal metaphysis. OVX and growth each resulted in net resorption of cancellous bone at a sampling site adjusted for longitudinal bone growth. Estradiol and progesterone treatment each antagonized bone loss by inhibiting the decrease in trabecular number. Estradiol increased but progesterone had no effect on trabecular thickness. Progesterone did not influence either osteoclast number or the resorption of the pretreatment fluorochrome label. Estradiol reduced osteoclast number and inhibited label resorption, the latter change being accentuated by combination treatment. Estradiol reduced and progesterone enhanced the mineral apposition and bone formation rates. The results indicate that estradiol and progesterone have independent activities on cancellous bone turnover during growth. Whereas estradiol reduced bone turnover, progesterone had a stimulatory effect on bone formation. These findings suggest that progesterone has a role in establishing and maintaining peak cancellous bone volume during growth.

Histochemical and ultrastructural characterization of vacuoles and spherosomes as components of the lytic system in hyphae of the fungus Botrytis cinerea.

An integrated approach to acid phosphatase (EC 3.1.3.2) histochemistry by the azo-dye and lead-capture ('Gomori') methods in phosphate-starved hyphae of the fungus Botrytis cinerea revealed strikingly different patterns of localization of activity staining. Reaction product formed with the azo-dye method was found in numerous small organelles (<0.5 microm diameter), which also accumulated the lipophilic dye Nile Red and mislocalized the formazan indicating mitochondrial succinate dehydrogenase activity. Such small organelles were stained only weakly and sporadically with the lead-capture method; instead, lead phosphate deposits were produced mainly in large vacuoles (up to 2.5 microm diam.), similar to those accumulating the vital dye Neutral Red. Additionally, acid phosphatase activity was detected in apical secretory vesicles with the lead-capture method but not with the azo-dye method. Ultrastructural studies by transmission electron microscopy confirmed the presence of large vacuoles which showed evidence of autophagic activity, and of small moderately osmiophilic organelles. The latter are considered to be spherosomes rather than lysosomes because of their weak reaction with the lead-capture method and their high lipid content. It is suggested that their apparently strong reaction with the azo-dye method is caused partly by false localization due to the lipophilic nature of the reaction product.

Glucocorticoids regulate the expression of the human osteoblastic endothelin A receptor gene.

The endothelial cell-derived peptide endothelin 1 (ET1) stimulates cell proliferation and differentiated functions of human osteoblastic cells (HOC), and HOC constitutively express the endothelin A receptor (ETRA). Therefore, ET1 may play an important role in the regulation of bone cell metabolism. As glucocorticoids (GC) exert a profound influence on bone metabolism and increase the effects of ET1 on bone cell metabolism in vitro, the effects of GC on ETRA expression in HOC were investigated. Dexamethasone (DEX) increased ETRA mRNA levels in a dose- and time-dependent fashion. The effects of dexamethasone, prednisolone, and deflazacort on the increase of ETRA mRNA levels correlate positively with their binding affinity to the GC receptor. Scatchard analysis of ET1 binding data to HOC revealed that DEX increased the binding capacity for ET1 from 25,300 to 62,800 binding sites per osteoblastic cell, leading to an enhanced mitogenic effect of ET1 on HOC after preincubation with DEX. Transiently transfected primary HOC with a reporter gene construct, containing the 5'-flanking region of the ETRA gene fused to luciferase gene, showed a promoter-dependent expression of the reporter gene and the induction of reporter gene expression by DEX treatment. Total RNA extracts of femoral head biopsies with osteonecrotic lesions from GC-treated patients showed threefold higher ETRA mRNA levels compared with extracts of bone biopsies from patients with traumatically induced osteonecrosis and coxarthrosis. Furthermore, GC treatment increased plasma ET1 levels by 50% compared with pretreatment values. These findings suggest that GC induced upregulation of ETRA, and ET1 plasma levels enhance ET1's anabolic action on bone cell metabolism. Increased ET1 concentrations may also impair bone perfusion by vasoconstriction in a metabolically activated skeletal region.

Complementation of the mpg1 mutant phenotype in Magnaporthe grisea reveals functional relationships between fungal hydrophobins.

The functional relationship between fungal hydrophobins was studied by complementation analysis of an mpg1(-) gene disruption mutant in Magnaporthe grisea. MPG1 encodes a hydrophobin required for full pathogenicity of the fungus, efficient elaboration of its infection structures and conidial rodlet protein production. Seven heterologous hydrophobin genes were selected which play distinct roles in conidiogenesis, fruit body development, aerial hyphae formation and infection structure elaboration in diverse fungal species. Each hydrophobin was introduced into an mpg1(-) mutant by transformation. Only one hydrophobin gene, SC1 from Schizophyllum commune, was able partially to complement mpg1(-) mutant phenotypes when regulated by its own promoter. In contrast, six of the transformants expressing hydrophobin genes controlled by the MPG1 promoter (SC1 and SC4 from S.commune, rodA and dewA from Aspergillus nidulans, EAS from Neurospora crassa and ssgA from Metarhizium anisopliae) could partially complement each of the diverse functions of MPG1. Complementation was always associated with partial restoration of a rodlet protein layer, characteristic of the particular hydrophobin being expressed, and with hydrophobin surface assembly during infection structure formation. This provides the first genetic evidence that diverse hydrophobin-encoding genes encode functionally related proteins and suggests that, although very diverse in amino acid sequence, the hydrophobins constitute a closely related group of morphogenetic proteins.

Gonadal and adrenal androgens are potent regulators of human bone cell metabolism in vitro.

Androgens stimulate bone formation and play an important role in the maintenance of bone mass. Clinical observations suggest that both gonadal and adrenal androgens contribute to the positive impact of androgenic steroids on bone metabolism. We investigated the mechanism of action of the adrenal androgen dehydroepiandrosterone (DHEA) and its sulfated compound dehydroepiandrosterone sulfate (DHEAS) on human osteoblastic cells (HOCs) in vitro. The DHEA- and DHEAS-induced effects were analyzed in parallel with the actions elicited by the gonadal androgen dihydrotestosterone (DHT). There was no qualitative difference between the effects of gonadal and adrenal androgens on HOC metabolism in vitro. Both were stimulatory as regards cell proliferation and differentiated functions, but the gonadal androgen DHT was significantly more potent than DHEA. The actions of DHT and DHEA on HOC proliferation and alkaline phosphatase (ALP) production could be prevented by the androgen receptor antagonist hydroxyflutamide and inhibitory transforming growth factor beta antibodies (TGF-beta ab), respectively, but were not affected by the presence of the 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 5-alpha-reductase (5-AR) inhibitor 17 beta-N,N-diethylcarbamoyl-4-methyl- 4aza-5 alpha-androstan-3-one (4-MA). This indicates that DHT and DHEA (1) exert their mitogenic effects by androgen receptor-mediated mechanisms, (2) stimulate ALP production by increased TGF-beta expression, (3) that the action of DHT is not affected by the presence of 4-MA, and that (4) DHEA does not need to be metabolized by 3 beta HSD or 5-AR first to exert its effects on HOCs in vitro.

Short-term effects of high dose estrogen on tibiae of growing male rats.

The short-term effects of estrogen at a single high dose (4 mg/kg body weight/day for 14 days) were determined on tibiae in the normal (noncastrate) growing male rat. In cortical periosteal bone, at a middiaphyseal site devoid of resorbing activity, estrogen suppressed periosteal bone formation and apposition rates, resulting in a smaller cross-sectional area. In middiaphyseal endocortical bone, estrogen had no effect on apposition and formation rates and, because medullary area was unchanged, probably had no effect on endocortical bone resorption. In the proximal tibial metaphysis, estrogen greatly suppressed longitudinal growth rate. In a site within the metaphysis adjusted for the effects of growth, cancellous mineral apposition was greatly reduced by the hormone. Estrogen-treated rats retained more of a fluorochrome label deposited in cancellous bone at the beginning of the study than vehicle-treated animals, indicating a reduced net bone loss. As a result of the lowered resorption induced by estrogen, cancellous bone mass (area and perimeter) were both significantly higher in estrogen-treated rats. No evidence was found for an anabolic action of the hormone in the male rat; indeed, estrogen reduced indices of bone formation.

MPG1 Encodes a Fungal Hydrophobin Involved in Surface Interactions during Infection-Related Development of Magnaporthe grisea.

The rice blast fungus expresses a pathogenicity gene, MPG1, during appressorium formation, disease symptom development, and conidiation. The MPG1 gene sequence predicts a small protein belonging to a family of fungal proteins designated hydrophobins. Using random ascospore analysis and genetic complementation, we showed that MPG1 is necessary for infection-related development of Magnaporthe grisea on rice leaves and for full pathogenicity toward susceptible rice cultivars. The protein product of MPG1 appears to interact with hydrophobic surfaces, where it may act as a developmental sensor for appressorium formation. Ultrastructural studies revealed that MPG1 directs formation of a rodlet layer on conidia composed of interwoven ~5-nm rodlets, which contributes to their surface hydrophobicity. Using combined genetic and biochemical approaches, we identified a 15-kD secreted protein with characteristics that establish it as a class I hydrophobin. The protein is able to form detergent-insoluble high molecular mass complexes, is soluble in trifluoroacetic acid, and exhibits mobility shifts after treatment with performic acid. The production of this protein is directed by MPG1.

Lack of an effect of sodium zeolite A on rat tibia histomorphometry.

Cell culture studies suggest that the aluminum silicate polymer sodium Zeolite A (SZA) increases bone cell proliferation and extracellular matrix production. This study in rats investigated the short-term (2 weeks) and long-term (18 weeks) in vivo effects of SZA on growth rate (weight gain) and tibia histomorphometry. In separate short-term experiments, female (experiment 1) or male (experiment 2) Sprague-Dawley rats grown and maintained on normal calcium diets were gavaged daily during a 2 week treatment period with 30 mg/kg, 100 mg/kg, or 500 mg of SZA/kg of rat body weight. In the long-term study (experiment 3) ovariectomized (OVX) rats were fed a low calcium diet containing 0, 1.80, and 9.00 g of SZA/kg of diet (0, 125, and 617 mg/kg of body weight, respectively). Short- and long-term treatment of growing rats with SZA resulted in a dose-dependent increase in bone aluminum. In the first experiment, growing intact female rats showed no significant SZA dose-dependent response in growth rate (weight gain) or histomorphometry of cortical bone in the tibial diaphysis or cancellous bone in the secondary spongiosa of the tibial metaphysis. In the second experiment, growing male rats, with right hind limbs immobilized by unilateral sciatic neurotomy, showed no SZA dose-dependent response in growth rate. The longitudinal growth of cancellous bone in the tibia of the denervated limb and the intact contralateral limb were not influenced by sciatic neurectomy and/or by treatment with SZA. Histomorphometry demonstrated that cortical bone mass and formation was reduced in the sciatic-sectioned limb when compared with the contralateral intact limb of vehicle-treated rats, as evidenced by significant reductions in static measurements of cortical bone area (-8.5%) and cross-sectional area (-4.8%) and in calculations of the periosteal formation rate (-33.8%) and mineral apposition rate (-31.6%), and the endocortical formation rate (-35.5%) and mineral apposition rate (-37.9%). The cancellous bone mass of denervated limbs of vehicle-treated rats was also deficient, as evidenced by decreased cancellous bone area (-39.1%) and perimeter (-31.9%). The bone mineral apposition rate was decreased (-26.7%) indicating reduced osteoblast activity. Treatment with SZA did not influence these indices in the tibiae of either sciatic-sectioned limbs or contralateral intact limbs. In the long-term experiment, OVX resulted in a dramatic 88% decrease in cancellous bone volume which was prevented by treatment with 17 beta-estradiol and not influenced by treatment with Zeolite A. The increases in osteoblast and osteoclast number following OVX were not influenced by SZA. The results indicate that SZA treatment has no anabolic effect on cortical and cancellous bone formation and mass in normal growing female rats and that this compound does not protect against osteopenia due to reduced load bearing in the growing male rat or gonadal hormone deficiency in adult female rats.

Spaceflight results in depressed cancellous bone formation in rat humeri.

Two experiments investigating the effects of short-term spaceflight on cancellous bone turnover were carried out: Physiological Systems Experiment-1 (PSE-1) (a 4-d orbital spaceflight) and PSE-2 (a 10-d flight). Cancellous bone area was not significantly altered by spaceflight in the humerus during either flight. The calculated bone formation rate was unchanged during the 4-d flight but decreased during the 10-d flight. The decrease in calculated bone formation during the longer flight was due to a combination of a statistically significant decrease in mineral apposition rate and a nonsignificant decrease in double label perimeter. The dynamic measurements suggest that spaceflight results in decreases in osteoblast number and activity. Further, the decreases in osteoblast perimeter and osteoid perimeter indicate that a 2-d reloading period was insufficient to restore bone formation to normal. Resorption of the preflight fluorochrome label was not influenced by spaceflight, suggesting that there was no net change in bone resorption. This conclusion is supported by the unremarkable effects of spaceflight on osteoclast number and osteoclast perimeter. Our findings indicate that short-term spaceflight results in depressed osteoblast number and/or activity. Continued decreases in bone formation with normal bone resorption could ultimately lead to cancellous osteopenia and reduced bone strength.

Reduced chondroclast differentiation results in increased cancellous bone volume in estrogen-treated growing rats.

These studies were designed to investigate the role of altered growth processes in mediating estrogen-induced changes in cancellous bone volume in growing female rats. Ovariectomy resulted in an increase in the longitudinal growth rate of the tibia throughout the growth period and estrogen treatment of ovariectomized (OVX) rats resulted in a dose-dependent decrease in longitudinal growth. Estrogen treatment also resulted in decreases in growth plate thickness, chondroclast number in the zone of vascular invasion, and osteoclast number in the secondary spongiosa. There were simultaneous increases in mineralized cartilage in the zone of vascular invasion; total mineralized tissue, mineralized cartilage, and bone in the primary spongiosa; and bone in the secondary spongiosa. Ovariectomy increased and estrogen treatment of OVX rats decreased [3H]thymidine-labeled nuclei in chondroclasts after 24 h, but after 7 days, the labeling indices were similar in the OVX and intact groups and only slightly decreased in the estrogen-treated rats. We interpret these results as evidence that estrogen impairs chondroclast differentiation. We propose that this impairment leads to decreased chondroclast number and reduced resorption of mineralized cartilage in the zone of vascular invasion, which, in turn, results in an increased cancellous bone volume.

Estrogen does not increase bone formation in growing rats.

The purpose of these experiments was to test the hypothesis that estrogen has an anabolic effect on cancellous bone formation in long bones of growing rats. In the present studies, estrogen decreased measurements related to cancellous bone formation, including osteoblast number, fluorochrome-labeled bone perimeter, and incorporation of [3H]proline into osteoid. Steady state mRNA levels for bone matrix proteins and mineral apposition rate were unchanged or reduced. The [3H]thymidine labeling index for osteoblasts was very low and was reduced by estrogen treatment. Finally, estrogen decreased the rate of removal of tetracycline previously incorporated into cancellous bone as well as the growth-dependent decrease in cancellous bone area, indicating that hormone treatment reduced net bone resorption. In summary, no evidence was secured to support the hypothesis that estrogen has an anabolic action on bone formation; the results obtained in these studies consistently demonstrated a pronounced inhibitory action of the hormone on bone turnover.

Mechanism of action of estrogen on cancellous bone balance in tibiae of ovariectomized growing rats: inhibition of indices of formation and resorption.

Ovariectomy results in cancellous osteopenia in rat long bones, a condition that is prevented by treatment with estrogens. The purpose of these studies was to clarify the effects of estrogen on cancellous bone turnover using dynamic bone histomorphometry. Treatment of ovariectomized rats with diethylstilbestrol (DES) reduced the mineral apposition rate, double-label perimeter, osteoblast number, and osteoclast number, suggesting that the hormone had inhibitory effects on bone formation as well as bone resorption. However, we could not estimate the bone formation rate because of rapid resorption of tetracycline-labeled bone in the ovariectomized rat. The magnitude of loss was documented by a time course study: 58% of the tetracycline initially incorporated into the secondary spongiosa of the tibial metaphysis was resorbed after 11 days and 89% was resorbed after 22 days. Similarly, cancellous bone area was decreased by 67% after 11 days and by 88% after 22 days. Administration of either DES or tamoxifen (TAM) dramatically reduced resorption of tetracycline as well as the decrease in cancellous bone area. These results demonstrate that (1) estrogen prevents osteopenia in ovariectomized (OVX) rats, in part by inhibiting bone turnover, (2) TAM is an estrogen agonist on bone resorption, and (3) resorption of tetracycline-labeled bone leads to serious underestimation of the bone formation rate in OVX rats.

Disuse osteopenia is accompanied by downregulation of gene expression for bone proteins in growing rats.

Unilateral sciatic neurectomy (USN) resulted in cortical osteopenia in tibiae from the sciatic nerve-sectioned limb of growing rats. The bone deficit resulted from decreased periosteal addition; there were no changes in the indexes of bone resorption. The periosteal bone formation rate was reduced in the nerve-sectioned limb within 7 days of sciatic neurectomy, and this decrease persisted for at least 56 days. Steady-state mRNA levels for bone proteins were determined in periosteum isolated from tibiae and femurs 7 and 14 days after sciatic nerve section. Nerve section resulted in decreased levels of mRNA for osteocalcin, alkaline phosphatase, and possibly the prepro-alpha (I)-subunit of type I collagen (collagen). The effects were more pronounced in tibiae than femurs, corresponding to the greater degree of immobility induced by USN in the former bone. The results demonstrate that decreased bone formation precedes establishment of disuse cortical osteopenia in growing rats with no evidence for a change in bone resorption. Furthermore, the decreased bone formation is associated with, and may be due to, reduced mRNA levels for matrix proteins and other important bone proteins.

Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers.

A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.

Incorporation of sodium fluoride into cortical bone does not impair the mechanical properties of the appendicular skeleton in rats.

Clinical studies on the use of sodium fluoride (NaF) in osteoporotic patients have demonstrated increased spinal bone mass without a reduction in vertebral fracture incidence, and a trend towards reduced appendicular bone mass with an increase in peripheral fracture incidence. As previous reports have suggested that NaF becomes incorporated into bone's crystal structure, possibly affecting bone strength, we sought to examine the relationship among bone fluoride content, bone mass, and skeletal fragility. Twenty-one-day-old female Sprague-Dawley rats were treated with four different doses of NaF. The tibiae were subjected to histomorphometric and biochemical analyses, and the femora were tested in torsion for the properties of strength, stiffness, energy storage capacity, and angular deformation. The results showed that over 50% of the skeleton in these rats was turned over in the presence of NaF. The four different doses resulted in a linear increase in bone F concentration and suggested excellent absorption and incorporation of this drug. No changes in histomorphometric indices of bone formation or turnover were found. Despite the large fraction of bone formed during NaF treatment, and the linear increase in bone fluoride content in relation to dose, there were no changes observed in any of the mechanical properties. These results suggest that, even extensive incorporation of fluoride into bone, in the absence of an effect on bone mass or remodeling, does not significantly alter its capacity to withstand mechanical loads.