The effect of inhibition of heat-shock proteins on thiram-induced tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is a skeletal abnormality that can cause economic losses and animal welfare concerns. Thiram-induced TD is characterized by enlarged, unvascularized growth plates, low levels of the vascular endothelial growth factor receptor Flk-1, abnormal chondrocyte differentiation, and lameness. Recently we reported the involvement of heat-shock protein 90 (Hsp90) in chondrocyte differentiation and growth-plate vascularization. Inhibition of Hsp90 activity in thiram-induced TD resulted in increased Flk-1 levels, re-instated normal growth-plate angiogenesis and morphology, and abrogated lameness. In the present study, we evaluated the efficacy of various concentrations of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90 activity, in preventing growth-plate histopathology and lameness in TD-affected chicks. Low doses of 17-DMAG (2 injections, each of 100 or 300 µg) did not prevent TD development even though Flk-1 levels were restored, which suggests that Flk-1 is not the only rate-limiting factor in growth-plate angiogenesis. High doses of 17-DMAG (2 injections, each of 600 or 900 µg) prevented BW loss, decreased the TD score, reduced lesion width, restored proper chondrocyte differentiation, increased blood vessel invasion, and eliminated lameness. To assess the specificity of Hsp90, we evaluated the efficacy of the flavonoid quercetin, an inhibitor of Hsp70 synthesis, in preventing TD development; it decreased Hsp70 levels but not those of Hsp90 in the control growth plates and prevented upregulation of Hsp70 in the TD-affected growth plates. Dietary quercetin (at 100 or 500 ppm) did not prevent the hypoxia that is characteristic of the TD-affected growth plate or development of thiram-induced TD and lameness. The present results demonstrate the specificity and the major role of Hsp90 in chondrocyte differentiation and growth-plate vascularization. In contrast to the anti-angiogenic effect of 17-DMAG observed in mammals, inhibition of Hsp90 activity in the unvascularized TD-affected growth plates resulted in activation of the angiogenic switch and restored normal growth-plate morphology.

Effect of an 8.0% arginine and calcium carbonate desensitizing toothpaste on shear dentin bond strength.

OBJECTIVE: The objective of this study was to determine the effect of an 8.0% arginine and calcium carbonate desensitizing toothpaste (Colgate Sensitive Pro-Relief) on shear bond strength of composites to bovine incisor dentin. METHODS: Bovine incisors were sectioned and prepared into 27 dentin specimens. The experimental group had 13 specimens treated for 10 sessions of two-minute brushing with an 8.0% arginine and calcium carbonate desensitizing toothpaste, followed by a 30-second agitated water wash. The control group had 14 specimens treated with flour of pumice only. Each specimen was dried, etched with 35% phosphoric acid for 15 seconds, and washed clean. A bonding agent was applied and polymerized. A 2.38 mm diameter column of Filtek Supreme A2 was bonded to the surface and polymerized as per manufacturer's instructions. Specimens were stored in water for at least 48 hours, subjected to a shear force at a crosshead speed of 0.5 mm/minute on an Instron mechanical testing device, and force at failure was recorded. A one-sided t-test was used to evaluate significant differences among the groups as measured by mean shear strength. RESULTS: Mean shear force was 19.6 +/- 9.4 (SD) for the experimental group and 15.4 +/- 6.0 for the control group with p = 0.0291. CONCLUSION: No significant differences were found for bond strength to dentin treated with an 8.0% arginine and calcium carbonate desensitizing toothpaste or pumice. Dentists can still achieve optimal dentin bonding results if a patient is using Colgate Sensitive Pro-Relief to manage dentin hypersensitivity.

Keloid histopathology after intralesional cryosurgery treatment.

BACKGROUND: Keloid presents a great healthcare challenge. The patients suffer from aesthetic disfiguration and occasionally from pruritus, pain and discomfort. Although various treatments are recommended, a single, highly effective treatment represents a great clinical need. OBJECTIVE: The cellular events and histopathology that follow intralesional cryosurgery were evaluated including cell proliferation, the number of cells expressing fibroblast markers, collagen synthesis and organization and mast cell infiltration. METHODS: Biopsies were collected before and after intralesional cryoneedle procedure. Collagen structure was evaluated with confocal microscopy. Mast cells, blood vessels and cell proliferation were evaluated using immunohistochemistry. RESULTS: Keloids contain abnormally thick collagen bundles, organized in swirls comprising closely bound fibrils. After intralesional cryosurgery, the collagen bundles lost their swirl structure, the thickness of the collagen layer decreased, and the bundles became more compact with less space between the fibres. A clear distinct transition zone separated the treated from the unaffected area. The frozen tissue was devoid of proliferating cells and mast cells whereas the number of blood vessels remained unaltered. Most of the fibroblasts expressed all tested myofibroblast markers although some exclusively expressed one and not the other. Few nuclei were observed in the affected area after treatment and very few of them expressed any fibroblast markers. CONCLUSIONS: Intralesional cryosurgery resulted in major changes in collagen structure and organization. The treatment reduced the number of proliferating cells, of myofibroblasts and of mast cells. These results may explain the reduction in no-response rate and the amelioration of the clinical symptoms after intralesional cryosurgery treatment.

Accumulation of ammonia and other potentially noxious gases on live export shipments from Australia to the Middle East.

Noxious gases on ships are irritant pollutants that have potential impacts on the comfort and health of both livestock and humans. Identification of environmental influences on the pollutants will assist live exporters to control them. Ammonia, hydrogen sulphide and carbon dioxide, as well as wet and dry bulb temperature, dew point, air speed and depth of faeces that the sheep stood in, were measured on two ship voyages in which sheep were transported from Australia to the Middle East. Daily measurements were made at 20 measurement locations over 12 days. At four sites, the mean ammonia concentration for the voyage was above the recommended maximum limit for the live export industry (25 ppm). The mean ammonia concentrations at the remaining 16 sites were below 18 ppm and considered safe. High ammonia concentrations were localised and occurred particularly on closed decks, as well as at the front of the vessel and near the engine block on open decks. Ammonia concentration on the open decks was correlated with cumulative wind during the voyage, air speed, dew point, wet bulb temperature and faecal pad depth, and on the closed decks with dew point, and wet and dry bulb temperature. Hydrogen sulphide (<1.8 ppm) and carbon dioxide (<1900 ppm) concentrations were low and did not pose a risk to animal or human welfare or health. The results suggest that high ammonia concentrations occur in those parts of the ship where there is insufficient ventilation and/or high temperatures and humidity.

Osteopontin and calbindin gene expression in the eggshell gland as related to eggshell abnormalities.

Eggshell quality is a major concern to the poultry industry: eggs with poor-quality shells hatch poorly and are rejected in the processing plant. The eggshell gland (ESG) proteins and the matrix proteins, which participate in crystallization, fulfill important functions during formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We selected layers that consistently produced eggshells with specific abnormalities, and continued to do so after molting, and evaluated the expression of 2 genes-osteopontin (OPN) and calbindin-as related to particular eggshell abnormalities. These genes are synthesized by the ESG and appear to participate in the calcification process. When the ESG produces normal eggshells, OPN was expressed uniformly by all of the epithelial cells facing the lumen, and calbindin was expressed by the glandular epithelium. In contrast, in the layers producing pimpled eggs, OPN was expressed only in sections of the pseudostratified epithelium, separated by areas of cells devoid of OPN gene expression, whereas calbindin was expressed at much greater levels throughout the glandular epithelium. Almost no OPN gene expression was observed in the ESG of layers producing corrugated shells, but their pattern of calbindin expression was similar to but somewhat greater than that in ESG that produced normal eggshells. In cases in which eggs had cracks at the sharp or blunt poles, OPN was expressed only at the side opposite to the cracks, whereas calbindin was expressed at both sides equally independent of the cracks. The results suggest that synthesis of the proteins associated with the formation of eggshells with the various abnormalities is controlled by different mechanisms. This may imply that more than 1 strategy will be required to improve eggshell quality.

Damage maps for layered ceramics under simulated mastication.

Ceramic restorations, whether monolithic (single layer) or porcelain-veneered, often chip and fracture from repeated occlusal loading. Occlusion involves the opposing tooth sliding along the cuspal incline surface with an applied biting force (off-axis loading). We hypothesized that off-axis contact-load-slide-liftoff fatigue, as compared with normal axial fatigue loading, produces different fracture modes and fatigue lifespans of layered ceramics. Monolithic glass plates were epoxy-bonded to polycarbonate substrates as a transparent model for an all-ceramic crown on dentin. Off-axis and axial (control) cyclic loading was applied through a hard sphere in water, with a mouth-motion machine. The off-axis loading is more deleterious for contact-induced occlusal surface fracture, but less harmful for flexure-induced cementation surface fracture of brittle layers than the axial loading. This is because of the tangential load component associated with the off-axis loading. Clinical relevance is discussed.

Hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha), and heat-shock proteins in tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is one of the most prevalent skeletal abnormalities in avian species; it causes economic losses and is an animal welfare problem. It has been hypothesized that the absence of vasculature in the lesion of the TD growth plates at the ends of the long bones is involved in the etiology of the disease. We evaluated the hypoxia status of normal and thiram-induced TD growth plates by immunostaining the protein adducts after pimonidazole hydrochloride administration. In addition, we evaluated the expression of hypoxia-inducible factor-1alpha (HIF-1alpha), the major regulator of the hypoxic response that is essential for chondrogenesis, and that of heat-shock proteins (Hsp) downstream from HIF-1alpha. We demonstrated that, in contrast to the normal growth plates, those afflicted by TD were hypoxic. A major increase in hypoxia was observed in the proliferative, hypertrophic, and calcified zones. In the normal growth plate, HIF-1alpha was expressed in chondrocytes of the articular cartilage and of the maturation zone, whereas in cases of TD, HIF-1alpha was also expressed in chondrocytes below the lesion. The expression level of HIF-1alpha was related to the severity of the disease, but was independent of its cause; the same pattern of expression was observed in growth plates of chicks selected for a high incidence of TD. No differentiation-dependent expression of HIF-1alpha was observed in response to hypoxia, as demonstrated by the use of primary cultures of growth plate chondrocytes. In the normal growth plates, Hsp90 and Hsp70 were localized to the maturation zone. More cells expressed both Hsp in the TD lesion. In conclusion, we demonstrated that the TD growth plate, in contrast to the normal one, is hypoxic, probably because of the lack of vascularization. Hypoxia leads to an increase in the transcription factor HIF-1alpha, causing increases in the levels of Hsp90 and Hsp70.

Fracture of porcelain-veneered structures in fatigue.

Porcelain-veneered crowns are widely used in modern dentistry, and their fracture remains problematic, especially in all-ceramic systems. We hypothesized that substructure properties have a significant effect on the longevity of porcelain-veneered crowns. Flat porcelain/metal or porcelain/ceramic structures were cemented to dentin-like composite, and a mouth-motion cyclic load of 200 N was delivered by means of a tungsten carbide spherical indenter (r = 3.18 mm), emulating occlusal loading on crowns supported by dentin. Findings indicated that porcelain on a low-hardness gold-infiltrated alloy was vulnerable to both occlusal surface contact damage and porcelain lower surface radial fracture, while porcelain on a higher-hardness palladium-silver alloy fractured chiefly from occlusal surface damage. The advantage of a high-modulus metal substructure was less pronounced. Fracture in the porcelain/zirconia system was limited to surface damage in the veneer layer, similar to that in the porcelain/palladium-silver system. Bulk fracture, observed in veneered alumina layers, was not found for zirconia.

Effect of temperature during the incubation period on tibial growth plate chondrocyte differentiation and the incidence of tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is one of the most prevalent skeletal abnormalities in avian species, causing enormous economic losses and major animal welfare problems. Irregular cell differentiation of the chondrocytes that populate the growth plate has been hypothesized to be involved in the etiology of the disease. We evaluated the effect of incubation temperature at various stages of embryo development and bone formation on growth plate chondrocyte differentiation and the incidence of TD. Eggs were incubated either at a control temperature of 37.8 degrees C, or at 36.9 or 39 degrees C, each for 6 h/ d, during early (0 to 8 d) or late (10 to 18 d) embryo development. At 14 d of incubation and at hatch, tibias were collected and weighed, and their ash and calcium contents were determined. Growth plate chondrocyte differentiation was evaluated by alkaline phosphatase activity and collagen type II and osteopontin gene expression. In addition, the level of the heat-shock protein 90 (Hsp90) was evaluated by immunohistochemistry. The rest of the chicks were raised to 49 d and the incidence of TD was recorded. The incidence of TD increased only when the temperature was altered at the early stages of embryo development, and it was correlated with an increase in tibia ash but not with tibia weight or calcium content. Moreover, increased TD incidence was correlated with delayed chondrocyte differentiation. Early changes in incubation temperature caused an increase in the level of Hsp90 in articular and differentiated chondrocytes of the hypertrophic zone and in the numbers of distinct undifferentiated chondrocytes arranged in columns in the proliferative zone of the growth plate. In summary, the early stages of embryo development and bone formation are of utmost importantance for appropriate growth plate chondrocyte differentiation, and any temperature deviation will increase the subsequent incidence of TD. The increase in TD incidence is probably the result of delayed Hsp90-driven chondrocyte differentiation, supporting the hypothesis that TD is the result of abnormal chondrocyte differentiation.

Inhibition of bladder carcinoma angiogenesis, stromal support, and tumor growth by halofuginone.

We have previously demonstrated that halofuginone, a widely used alkaloid coccidiostat, is a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 gene expression. Halofuginone also suppresses extracellular matrix deposition and cell proliferation. We investigated the effect of halofuginone on transplantable and chemically induced mouse bladder carcinoma. In both systems, oral administration of halofuginone resulted in a profound anticancerous effect, even when the treatment was initiated at advanced stages of tumor development. Although halofuginone failed to prevent proliferative preneoplastic alterations in the bladder epithelium, it inhibited further progression of the chemically induced tumor into a malignant invasive stage. Histological examination and in situ analysis of the tumor tissue revealed a marked decrease in blood vessel density and in both collagen alpha1(I) and H19 gene expression. H19 is regarded as an early marker of bladder carcinoma. The antiangiogenic effect of halofuginone was also demonstrated by inhibition of microvessel formation in vitro. We attribute the profound antitumoral effect of halofuginone to its combined inhibition of the tumor stromal support, vascularization, invasiveness, and cell proliferation.

Inhibition of aldosterone secretion by atrial natriuretic peptide in chicken adrenocortical cells.

Dispersed chicken adrenocortical cells were preincubated with atrial natriuretic peptide (rANP), sodium nitroprusside (SNP) or 8-bromo cyclic GMP, followed by incubations with ACTH, chicken PTH, cholera toxin or various steroid intermediates of aldosterone production. Cyclic AMP production and aldosterone secretion were evaluated, in order to determine the sites of ANP inhibition in the sequence of events leading to aldosterone secretion. Dose-dependent inhibitory effects on ACTH-stimulated aldosterone secretion by rANP and SNP were observed. Both agents appeared to stimulate cGMP production by the particulate fraction of the avian adrenocortical cells. Aldosterone production, stimulated by cyclic AMP agonists such as ACTH, chicken PTH and cholera toxin, was significantly inhibited by ANP. On the other hand, ANP did not interfere with production or degradation of cAMP. Each of the aldosterone intermediates--pregnenolone, progesterone, 11-deoxycorticosterone and corticosterone--promoted aldosterone production when included in the incubation media. Atrial natriuretic peptide and SNP inhibited aldosterone secretion when enhanced by the intermediates, by about 40-60%, but the ACTH-stimulated secretion was inhibited by over 90%. The results suggest two sites of inhibition by ANP in the pathway of aldosterone synthesis and secretion: synthesis of cholesterol or pregnenolone, and conversion of corticosterone to aldosterone. The inhibition by 8-bromo cGMP of aldosterone secretion and the similar sites of inhibition for ANP and SNP suggest that cyclic GMP mediates the inhibition in both cases.

Effect of hydrocortisone and glucagon on glycogen metabolism in the fetal rat liver.

1. Hydrocortisone increases in vivo incorporation of [14C] glucose into fetal liver glycogen in the last days of gestation, whereas in glucagon-treated fetuses, a slight decrease in the incorporation rate was found. 2. Hydrocortisone increases total synthetase activity as that of synthetase a but was without effect on fetal liver glycogen phosphorylase. 3. Glucagon causes a slight increase in phosphorylase a activity on days 19-21, and was without effect on the activities of synthetase a and total synthetase. 4. Dibutyryl cyclic AMP had no effect on the key enzymes of glycogen metabolism 1 h after injection in utero, whereas after 6 h an increase in phosphorylase a activity was found without any change in synthetase a activity.

Halofuginone: an inhibitor of collagen type I synthesis.

The effect of halofuginone--a plant alkaloid used as a coccidiostat in birds--on collagen metabolism was studied in various avian and mammalian cell cultures. In avian skin fibroblasts halofuginone attenuated the incorporation of [3H]proline into collagenase-digestible proteins (CDP) at concentrations as low as 10(-11) M, without affecting production of [3H]collagenase-nondigestible proteins (NCDP), cell proliferation or collagen degradation. Halofuginone depressed specifically the expression of alpha 1 gene of collagen type I but not that of collagen type II. This was demonstrated in skin fibroblasts and growth-plate chondrocytes using probes containing inserts sequences corresponding to the alpha 1(I) and alpha 1(II) mRNAs. A slight inhibition of the expression of alpha 2(I) was observed in avian skin fibroblasts but not in growth-plate chondrocytes. The inhibition of gene expression of both polypeptides of collagen type I in skin fibroblasts resulted in a decrease in synthesis, as demonstrated by immunoprecipitation with specific type I collagen antiserum. In primary cultures of mouse skin fibroblasts, avian epiphyseal growth plate chondrocytes and a rat embryo cell line--all of which produce and secrete collagen type I--halofuginone inhibited the incorporation of [3H]proline into CDP, the Rat-1 line being the most sensitive to the drug. These results suggest that halofuginone affects specifically type I collagen synthesis by repressing gene-expression. The need for extremely low concentrations of halofuginone to inhibit collagen type I synthesis, regardless of the tissue or animal species, contributes to the potential usefulness of the substance in studying collagen metabolism.

Weight loading young chicks inhibits bone elongation and promotes growth plate ossification and vascularization.

The mechanical stimuli resulting from weight loading play an important role in mature bone remodeling. However, the effect of weight loading on the developmental process in young bones is less well understood. In this work, chicks were loaded with bags weighing 10% of their body weight during their rapid growth phase. The increased load reduced the length and diameter of the long bones. The average width of the bag-loaded group's growth plates was 75 +/- 4% that of the controls, and the plates showed increased mineralization. Northern blot analysis, in situ hybridization, and longitudinal cell counting of mechanically loaded growth plates showed narrowed expression zones of collagen types II and X compared with controls, with no differences between the relative proportions of those areas. An increase in osteopontin (OPN) expression with loading was most pronounced at the bone-cartilage interface. This extended expression overlapped with tartarate-resistant acid phosphatase staining and with the front of the mineralized matrix in the chondro-osseous junction. Moreover, weight loading enhanced the penetration of blood vessels into the growth plates and enhanced the gene expression of the matrix metalloproteinases MMP9 and MMP13 in those growth plates. On the basis of these results, we speculate that the mechanical strain on the chondrocytes in the growth plate causes overexpression of OPN, MMP9, and MMP13. The MMPs enable penetration of the blood vessels, which carry osteoclasts and osteoblasts. OPN recruits the osteoclasts to the cartilage-bone border, thus accelerating cartilage resorption in this zone and subsequent ossification which, in turn, contributes to the observed phenotype of narrower growth plate and shorter bones.

Inhibition of matrix metalloproteinase-2 expression and bladder carcinoma metastasis by halofuginone.

Matrix metalloproteinase-2 (MMP-2) plays a critical role in tumor cell invasion and metastasis. Inhibitors of this enzyme effectively suppress tumor metastasis in experimental animals and are currently being tested in clinical trials. MMP-2 transcriptional regulation is a part of a delicate balance between the expression of various extracellular matrix (ECM) constituents and ECM degrading enzymes. Halofuginone, a low-molecular-weight quinazolinone alkaloid, is a potent inhibitor of collagen type alpha1 (I) gene expression and ECM deposition. We now report that expression of the MMP-2 gene by murine (MBT2-t50) and human (5637) bladder carcinoma cells is highly susceptible to inhibition by halofuginone. Fifty percent inhibition was obtained in the presence of as little as 50 ng/ml halofuginone. This inhibition is due to an effect of halofuginone on the activity of the MMP-2 promoter, as indicated by a pronounced suppression of chloramphenicol acetyltransferase activity driven by the MMP-2 promoter in transfected MBT2 cells. There was no effect on chloramphenicol acetyltransferase activity driven by SV40 promoter in these cells. Halofuginone-treated cells failed to invade through reconstituted basement-membrane (Matrigel) coated filters, in accordance with the inhibition of MMP-2 gene expression. A marked reduction (80-90%) in the lung colonization of MBT2 bladder carcinoma cells was obtained after the i.v. inoculation of halofuginone-treated cells as compared with the high metastatic activity exhibited by control untreated cells. Under the same conditions, there was almost no effect of halofuginone on the rate of MBT2 cell proliferation. These results indicate that the potent antimetastatic activity of halofuginone is due primarily to a transcriptional suppression of the MMP-2 gene, which results in a decreased enzymatic activity, matrix degradation, and tumor cell extravasation. This is the first description, to our knowledge, of a drug that inhibits experimental metastasis through the inhibition of MMP-2 at the transcriptional level. Combined with its known inhibitory effect on collagen synthesis and ECM deposition, halofuginone is expected to exert a profound anticancerous effect by inhibiting both the primary tumor stromal support and metastatic spread.

Modulation of responsiveness of the adenylate cyclase system in avian chondroprogenitor cells by pertussis toxin, PTH, and PGE2.

Chondroprogenitor cells, derived from avian tibia epiphyseal growth plate, were cultured in vitro. Incubation of these cells with pertussis toxin augmented their cAMP response to parathyroid hormone (PTH), attenuated the response to forskolin, but did not modify the response to PGE2. Pertussis toxin modulation of the cAMP response was accompanied by ADP ribosylation of two proteins with molecular weights of 39 and 40 kD. Using specific antibodies, the 39 kD protein was identified as the inhibitory guanine nucleotide binding protein (Gi) of the adenylate cyclase system. The other ADP-ribosylated protein has not been identified. Preincubation of the chondroprogenitor cells with PTH or PGE2 resulted in time-dependent heterologous desensitization of the cAMP response to a second challenge of either hormone. The cells did not recover from the densitization for at least 18 h after removal of the hormones. PTH and PGE2 treatment did not affect the cAMP response to forskolin and cholera toxin. The PTH-dependent cAMP production was also not altered by forskolin treatment. PTH homologous desensitization was not affected by pertussis toxin treatment, but the heterologous desensitization due to PGE2 was significantly attenuated. These results suggest that exposure of chondroprogenitor cells to PTH and PGE2 results in heterologous desensitization of the cAMP response. The desensitization is not due to changes in the adenylate cyclase activity. The pertussis toxin-sensitive G proteins are involved in the PTH heterologous rather than homologous desensitization of the cAMP response.

Expression of chicken 75-kDa gelatinase B-like enzyme in perivascular chondrocytes suggests its role in vascularization of the growth plate.

UNLABELLED: A newly cloned avian 75-kDa gelatinase B-like enzyme is expressed by the cells surrounding the blood vessels of the growth plate and upregulated by angiogenic substances in cultured chondrocytes. Despite its low homology to mammalian gelatinase-B, the avian 75-kDa seems to function similarly in the context of endochondral bone formation. INTRODUCTION: Gelatinase B/metalloproteinase (MMP)-9, a zinc-dependent protease of the MMP family, is a key regulator in the final step of endochondral ossification. Recently an avian 75-kDa gelatinase B-like enzyme that shows low sequence similarity to the mammalian enzyme (59% on the protein level) was cloned and characterized. However, its expression pattern in the chicken growth plate and its role in bone formation have not, so far, been examined. RESULTS: Based on the published sequence, we cloned a 700-bp fragment from cDNA of the chicken growth plate and studied its expression pattern in primary chondrocytes. Because the basal expression level of gelatinase B was almost undetectable, we induced its expression by different culturing conditions, the most dramatic induction achieved by treatment with retinoic acid, which is known as an inducer of vascular invasion in the epiphyseal plates. The gelatinolitic activity, checked by zymography, detected bands corresponding to the gelatinase A and B as well as a new high-molecular weight band of approximately 200 kDa. We further studied the expression pattern of gelatinase B by in situ hybridization. The gelatinase B was expressed by the cells surrounding the blood vessels penetrating the growth plate and by chondrocytes located in the front of these vascular invasions in the borders between the bone and the cartilage, resembling the expression of mouse gelatinase B in the growth plate. The induction of rickets by a vitamin D-deficient diet reduced the expression levels of gelatinase B in the growth plate of 12-day-old chickens but did not affect the expression of gelatinase A mRNA. CONCLUSION: The chicken growth plate has a distinctly different structure from the mammalian one: it is much wider, it contains more cells in each zone, and the blood vessels penetrate deeper into the hypertrophic zone. Nevertheless, the upregulation of the avian 75-kDa gelatinase B-like enzyme by vitamins A and D, coupled with its perivascular expression pattern in the growth plate, implies a similar role for the mammalian and avian genes in bone formation.

Nucleotide sequence of cloned cDNAs encoding chicken preproparathyroid hormone.

In order to characterize an avian parathyroid hormone gene, a lambda gt10 cDNA library constructed from chicken parathyroid gland mRNA was screened with a human preproparathyroid hormone (preproPTH) cDNA probe. Nucleotide sequence analysis of three independent clones confirmed that they encoded chicken preproPTH. This analysis, complemented by primer extension and Northern blot analysis of mRNA, demonstrated a 5'-untranslated region for chicken preproPTH of 127 nucleotides, a coding region of 357 nucleotides, and a 3'-untranslated region of approximately 2500 nucleotides. The coding sequence predicts a mature chicken PTH of 88 amino acids in contrast to the 84 amino acids of the mammalian hormones. Comparison of the avian and the mammalian hormones shows striking homology in the region of amino acids 1-32. The middle and carboxyl-terminal portions of chicken PTH, however, differ considerably from the mammalian hormones and include deletions of sequences conserved in mammalian PTH and insertions of novel peptide sequences. Comparison of the avian and mammalian structures suggests potential alterations of the mammalian sequences that may lead to altered bioactivity and/or hormone metabolism.

Inhibition of neovascularization and tumor growth, and facilitation of wound repair, by halofuginone, an inhibitor of collagen type I synthesis.

Halofuginone, an inhibitor of collagen alpha1(I) gene expression was used for the treatment of subcutaneously implanted C6 glioma tumors. Halofuginone had no effect on the growth of C6 glioma spheroids in vitro, and these spheroids showed no collagen alpha1(I) expression and no collagen synthesis. However, a significant attenuation of tumor growth was observed in vivo, for spheroids implanted in CD-1 nude mice which were treated by oral or intraperitoneal (4 microg every 48 hours) administration of halofuginone. In these mice, treatment was associated with a dose-dependent reduction in collagen alpha1(I) expression and dose- and time-dependent inhibition of angiogenesis, as measured by MRI. Moreover, halofuginone treatment was associated with improved re-epithelialization of the chronic wounds that are associated with this experimental model. Oral administration of halofuginone was effective also in intervention in tumor growth, and here, too, the treatment was associated with reduced angiogenic activity and vessel regression. These results demonstrate the important role of collagen type I in tumor angiogenesis and tumor growth and implicate its role in chronic wounds. Inhibition of the expression of collagen type I provides an attractive new target for cancer therapy.

Inhibition of collagen synthesis and changes in skin morphology in murine graft-versus-host disease and tight skin mice: effect of halofuginone.

The effect of halofuginone, a plant alkaloid known to inhibit collagen type I synthesis, on skin collagen content and skin morphology was evaluated in two in vivo models of scleroderma: the murine chronic graft-versus-host disease (cGvHD) and the tight skin mouse. Skin collagen was assessed by hydroxyproline levels in skin biopsies and by immunohistochemistry using anti-collagen type I antibodies. Daily intraperitoneal injections of halofuginone (1 microgram/mouse) for 52 d starting 3 d before spleen cell transplantation, abrogated the increase in skin collagen and prevented the thickening of the dermis and the loss of the subdermal fat, all of which are characteristic of the cGvHD mice. Halofuginone had a minimal effect on collagen content of the control mice. The halofuginone-dependent decrease in skin collagen content was concentration-dependent and was not accompanied by changes in body weight in either the cGvHD or the control mice. Injections of halofuginone (1 microgram/mouse) for 45 d caused a decrease in the collagen content and dermis width in tight skin mice, but did not affect the dermis width of control mice. Collagen content determination from skin biopsies confirmed the immunohistochemical results in the same mice. The low concentration of halofuginone needed to prevent collagen deposition in fibrotic skin without affecting body weight suggests that halofuginone may serve as a novel and promising anti-fibrotic therapy.