Boning up on glypicans--opportunities for new insights into bone biology.

Bone formation is remarkable for the convergence in the activity of four major signalling pathways, the bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH) and wingless-integrated (WNT) pathways. These pathways cooperate in morphogenetic, proliferative and differentiative processes that underpin the development, growth and repair of skeletal structures. They are regulated by pathway-specific modulators and by another class of molecules, the glypicans. Glypicans are proteoglycans located on the cell surface, where they act as coreceptors to promote or inhibit signalling by ligands of the BMP, FGF, HH and WNT pathways, through protein-protein and protein-carbohydrate interactions. In this review, we discuss glypican structure, expression and function in the context of bone development and growth, with emphasis on the long bone growth plate where five of the six glypicans are expressed in overlapping patterns in the chondrogenic zone. Analyses of gene knockout models and the human conditions of Simpson-Golabi-Behmel syndrome and omodysplasia, which arise from mutations in glypican 3 (GPC3) and GPC6, respectively, highlight both subtle and striking effects of glypicans on bone growth. We draw attention to challenges and areas of opportunity, where the actions of glypicans on BMP, FGF, HH and WNT signalling might be profitably studied to help illuminate the complex interplay of signalling that drives bone growth.

Repression of basal transcription by vitamin D receptor: evidence for interaction of unliganded vitamin D receptor with two receptor interaction domains in RIP13delta1.

Repression of basal transcription of a 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) responsive 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter construct as observed in kidney cells in the absence of ligand and this repression was dependent on a functional vitamin D response element (VDRE). Basal repression was also seen with a construct where a consensus DR-3-type VDRE was fused to the thymidine kinase promoter. Expression of a dominant negative vitamin D receptor (VDR) isoform that strongly bound to the VDRE motif in the CYP24 promoter ablated basal repression. This VDR isoform lacked sequence in the hinge- and ligand-binding domains implicating one or both of these domains in basal repression. It is well known that thyroid hormone and retinoic acid receptors silence basal transcription of target genes in the absence of ligands and this repressor function can be mediated by the nuclear receptor corepressor N-CoR. Two variants of N-CoR have been described, RIP13a and RIP13delta1. N-CoR and the variants contain two receptor interaction domains, ID-I and ID-II, which are identical except region ID-II in RIP13delta1 has an internal deletion. We have used the mammalian two hybrid system to investigate whether VDR, in the absence of ligand 1,25-(OH)2D3, can interact with these domains. The data showed that unliganded VDR does not interact with either ID-I or ID-II from RIP13a and RIP13delta1, but does interact strongly with a composite domain of ID-I and ID-II from RIP13delta1 (but not from RIP13a) and this strong interaction is abrogated in the presence of ligand. This finding implicates RIP13delta1 in VDR-dependent basal repression of the promoter constructs under investigation. However, over-expression of RIP13delta1 in kidney cell lines did not alter basal expression of the CYP24 promoter construct. It is concluded that either the level of endogenous RIP13delta1 in these kidney cells permits maximal repression or that repression occurs by a mechanism that is independent of RIP13delta1. Alternatively, repression may be dependent on RIP13delta1 but requires an additional cofactor that is limiting in these cells.

Calcitonin stimulates expression of the rat 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter in HEK-293 cells expressing calcitonin receptor: identification of signaling pathways.

Regulation of the gene for renal 25-hydroxyvitamin D-24-hydroxylase (CYP24) is important for controlling the level of circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We report here for the first time that the peptide hormone calcitonin significantly stimulates expression of a rat CYP24 promoter-luciferase construct in both transiently and stably transfected kidney HEK-293 cells. A GC box at -114/-101 and a CCAAT box at -62/-51 have been identified that underlie both basal expression of the CYP24 promoter and the calcitonin inductive response. Data from overexpression studies suggested that Sp1 and NF-Y are the proteins that function through the GC and CCAAT boxes respectively. ERK1/2 signaling pathways were not involved in the calcitonin-mediated response, since stimulation of the promoter was unaffected by the pharmacological ERK1/2 inhibitor PD98059 and by a dominant negative mutant of ERK1/2 (ERK1K71R). In contrast, calcitonin induction but not basal expression was dependent on protein kinase A and protein kinase C (PKC) activities with the inhibitors H89 and calphostin C lowering induction by 50-60%. The atypical PKC, PKCzeta contributes to calcitonin induction, but not to basal expression of the CYP24 promoter, since overexpression of a dominant negative clone PKCzetaK281 M lowered induction by 50%. Cotransfection of a dominant negative form of Ras resulted in calcitonin-mediated induction being reduced also by about 50%. A Ras-PKCzeta signaling pathway for calcitonin action is proposed, which acts through the GC box. The findings have been extrapolated to the in vivo situation where we suggest that induction of renal CYP24 by calcitonin could be important under hypercalcemic conditions thus contributing to the lowering of circulating 1,25(OH)2D3 levels.

Modulation of benzene induced toxicity by protein A.

Administration of benzene (i.p. 1.0 mL/kg body weight) for 3 consecutive days produced leucopenia and lymphocytopenia in female albino rats. In addition, the total iron content, lipid peroxidation and superoxide dismutase activity of the liver and bone marrow were significantly (P < 0.001) increased. Low molecular weight (LMW) bleomycin-detectable iron accumulated only in bone marrow. Prior administration of Protein A (PA), a multipotent immunostimulant and interferon inducer (60 micrograms/kg body weight, i.v. twice weekly for 2 weeks), ameliorated most of the adverse effects of benzene. PA restored the changes in hepatic histological architecture, reversed leucopenia and superoxide dismutase activity, lipid peroxidation, total iron content and LMW iron content of bone marrow were normalized. Isozymes of glutathione-S-transferase (alpha, pi, mu) which decreased following benzene exposure increased in PA pretreated benzene exposed rats. This study suggests that pretreatment with PA modulates the toxicity of benzene.

Overview of regulatory cytochrome P450 enzymes of the vitamin D pathway.

Cytochromes P450c1 and P450c24 are regulated hydroxylase enzymes that direct the bioactivation and metabolic degradation of vitamin D. The bioactivation pathway is regulated by cytochrome P450c1 through its synthesis of 1alpha,25(OH)(2)D(3), the hormonally active form of the vitamin. Expression of the P450c1 gene is regulated at the transcription level. Promoter regions within the P450c1 gene have been identified that respond to cAMP and 1alpha,25(OH)(2)D(3) during the respective up- and down-regulation of P450c1 gene expression. The diametric action of 1alpha,25(OH)(2)D(3) to up-regulate P450c24 gene expression is discussed in the context of two vitamin D response elements (VDREs) that are linked functionally to an adjoining Ets-binding site. It is apparent from sequence-derived data that the P450c1 and P450c24 enzymes share only 10-25% sequence identity, yet they display functionally similar domains that are conserved across the family of cytochrome P450 enzymes. Expression of E. coli recombinant P450c1 and P450c24 enzymes, and the substrate-binding parameters for P450c24 are discussed. Finally, the natural point mutations in human P540c1 from patients with pseudovitamin D-deficiency rickets (PDDR) are discussed in the context of the enzyme's structure and function.

Evaluation of LD50 of some polyaminocarboxylic acids used as chelating drugs in metal intoxication.

The LD50 of the following metal-binding chelating drugs, EDTA, diethylenetriaminepentaacetic acid (DTPA), hydroxyethylenediaminetriacetic acid (HEDTA), cyclohexanediaminotetraacetic acid (CDTA) and triethylenetetraminehexaacetic acid (TTHA) was evaluated in terms of mortality in rats after intraperitoneal administration and was found to be in the order: CDTA greater than EDTA greater than DTPA greater than TTHA greater than HEDTA. A possible correlation between the toxicity and molecular structure of the compounds is discussed.

Kinetics of nickel binding in hepatic and renal cytosol of(63)NiCl 2-treated rats.

Elution profiles of nickel-binding protein were investigated in hepatic and renal cytosol of rats at various time intervals (6, 16, 24, and 48 h) after intraperitoneal administration of(63)Ni (1 mg Ni/kg. B. Wt. = 400 µCi as(63)NiCl2). The nickel-binding proteins were characterized in terms of absorbance at 254 nm, sulfhydryl content, and(63)Ni counts. The results demonstrated that in liver it was bound to both high as well as low mol wt sulfhydryl proteins and glutathione-like moiety with a maximum incorporation at 16 h, after which it declined and by 48 h, very little(63)Ni was associated with the bioligands. In kidney the incorporation of(63)Ni was approximately 400-fold higher than liver and most of(63)Ni was associated with the low mol. wt. sulfhydryl moiety. Kidney also exhibited maximum incorporation of(63)Ni at 16 h that was metabolized by 48 h.

Lindane mediated induction of cytochrome P-450 mRNA in rat liver: studies with a nonradioactive cDNA probe.

Effect of lindane on the induction of cytochrome P-450 mRNA in rat liver was studied using a biotinylated cDNA probe. Exposure to lindane resulted in an increase in the levels of P-450 mRNA. The observed increase in mRNA was maximal (5-6-fold) after 18 hr of lindane treatment and lesser (50%) than mediated by phenobarbital for the same duration.

UreC PCR based diagnosis of Helicobacter pylori infection and detection of cag A gene in gastric biopsies.

Polymerase chain reaction assay using ureC gene specific primers for the detection of Helicobacter pylori in gastric biopsy specimens from 116 dyspeptic patients was compared with other routine invasive diagnostic methods (culture, rapid urease test [RUT] and histology). In parallel, gastric biospy specimens from 54 patients and their corresponding Helicobacter pylori isolates were subjected to PCR with cagA targeting primers using standard protocols. Helicobacter pylori were detected in 53%, 43%, 48% and 50% of patients by PCR, RUT, culture and histological examination respectively. Based on histology and culture positive and at least three test positive result, 44 (37%), 46 (39%) and 26 (22%), and 56 (48%), 52 (44%) and 8 (6%) patients were classified as Helicobacter pylori positive, negative and indeterminate respectively. The sensitivity and specificity of PCR assay was the highest-95% and 100% when compared with both culture and histology positive, and at least any three positive results respectively. The result of cagA positivity in 54 gastric biopsy specimens and their corresponding Helicobacter pylori isolates were identical; 18 of 20 (90%) duodenal ulcer patients and 23 of 28 (82%) patients with chronic gastritis and 2 (40%) of 5 patients with portal hypertension and one gastric biopsy specimens from gastric cancer patients were found to be cagA positive. PCR-based method to detect Helicobacter pylori and the virulence gene cag A directly from gastric biopsy specimens appears to be promising and can curtail the lengthy process of culture-based approaches. The procedure proved to be rapid and reliable and could be utilized for diagnostic purposes.

A novel immunocompetent murine model for Candida albicans-promoted oral epithelial dysplasia.

Candida albicans is a common opportunistic pathogen found in the oral mucosa. Clinical observations indicate a significant positive association between oral Candida carriage or infection and oral epithelial dysplasia/neoplasia. The aim of this study was to test whether C. albicans is able to promote epithelial dysplasia or carcinoma in a mouse model of infection where a carcinogen (4 Nitroquinoline 1-oxide [4NQO]) was used as initiator of neoplasia. Mice were divided into four groups: group 1 received 4NQO alone; group 2 received 4NQO followed by C. albicans (ATCC 90234); group 3 received vehicle dimethyl sulfoxide (DMSO) followed by C. albicans and group 4 was untreated. Although 4NQO treated mice did not develop oral lesions, mice exposed to both 4NQO and C. albicans developed oral dysplastic lesions 19 weeks after exposure to 4NQO. Mice challenged with C. albicans only developed hyperplastic lesions. The expression of Ki-67 and p16, two cell-cycle associated proteins that are frequently deregulated in oral dysplasia/neoplasia, was also tested in these lesions. Ki-67 and p16 expression increased from normal to hyperplastic to dysplastic mucosa and was highest in the group exposed to both 4NQO and C. albicans. In conclusion, we showed that C. albicans plays a role in the promotion of oral dysplasia in a mouse model of infection when 4NQO was used as initiator of oral neoplasia.

Regulation of the 5'-flanking region of the human CYP27B1 gene in osteoblast cells.

Synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is catalysed by the enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1). Regulation of CYP27B1 gene expression is poorly understood, particularly in non-renal tissues including bone where 1,25(OH)(2)D(3) is hypothesised to serve autocrine/paracrine roles. Transient transfection of ROS 17/2.8 osteoblast-like cells with reporter gene constructs containing deletions of the 5'-flanking region of the human CYP27B1 gene revealed a proximal promoter, enhancer region and strong upstream repressive region. Putative CCAAT and GC boxes, as well as Ets protein binding sites were shown to contribute to promoter and enhancer activities respectively in common with kidney and prostate cells. Inhibition of basal expression was largely attributed to a palindrome 5'-GTCTCAGAC-3' (-1015/-1007bp) that contains two putative canonical Smad binding elements. We conclude that repression of CYP27B1 gene expression may be a common event but the novel inhibitory elements we have identified may be unique to osteoblasts.

Relative induction of molecular forms of cytochrome P-450 in gamma-hexachlorocyclohexane exposed rat liver microsomes.

The effect of gamma-hexachlorocyclohexane (HCH), (25 mg/kg body weight, i.p., administered for 4 consecutive days) on the induction of types of cytochrome P-450 in rat liver microsomes was studied. Induced proteins were characterized by "Western" blot analysis, using anticytochrome P-450 antibodies. It was observed that HCH is a "mixed-type" inducer and mediates induction of cytochrome P-450 b/e forms by several fold and of cytochrome P-450 c and d forms by nearly three fold.

Regulation of rat cytochrome P450C24 (CYP24) gene expression. Evidence for functional cooperation of Ras-activated Ets transcription factors with the vitamin D receptor in 1,25-dihydroxyvitamin D(3)-mediated induction.

Transcription of the rat CYP24 gene is induced by 1, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) through two vitamin D response elements (VDREs). A functional Ras-dependent Ets-binding site (EBS) was located downstream from the proximal VDRE and was critical to 1,25(OH)(2)D(3)-mediated induction. Cotransfection of Ets-1 and Ets-2 stimulated induction, which was lost when the EBS was mutated. Multiple nuclear-protein complexes from COS-1 cells bound to the EBS in which three complexes were immunologically related to Ets-1. Transcriptional synergy was observed between the proximal VDRE and adjacent EBS as was the attendant formation of a ternary complex between vitamin D receptor- retinoid X receptor (VDR. RXR) and Ets-1. In the absence of 1,25-(OH)(2)D(3) or in the presence of an inactive proximal VDRE, the EBS failed to respond to exogenous Ets-1. However, Ets-1 increased basal expression when cotransfected with a mutant VDR. The inductive action of 1, 25-(OH)(2)D(3) was substantially increased by Ras, which was ablated by mutagenesis of the EBS or by expression of a mutated Ets-1 protein (T38A). EBS contribution to hormone induction was prevented by manumycin A, an inhibitor of Ras farnesylation. A fundamental role was established for transcriptional cooperation between Ras-activated Ets proteins and the VDR.RXR complex in mediating 1, 25-(OH)(2)D(3) action on the CYP24 promoter.

Induction of glutathione-S-transferase isoenzymes by protein A in rat liver.

The administration of Protein A, a cell wall protein of Staphylococcus aureus Cowan I cells, causes an induction of glutathione-s-transferase in rat liver. Proteins, cross reactive with anti human glutathione-s-transferase, acidic (pi), basic (alpha, and neutral (mu) isoenzymes, are induced by 5.8, 2.2 and 6.15 fold respectively. The induction of glutathione -s-transferases, at least in part, might play a role in manifestation of therapeutic properties of Protein A.

Cloning, sequencing and overexpression in Escherichia coli of a xylanase gene, xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor.

A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in lambda ZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70 degrees C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp.

Transcriptional synergism between vitamin D-responsive elements in the rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) promoter.

Transcription of the CYP24 gene is induced by 1,25-(OH)2D3 through a vitamin D receptor-dependent process. The functional activities of three possible vitamin D response elements (VDREs), located on the antisense strand of the rat CYP24 promoter, were investigated by transient expression of native and mutant promoter constructs in COS-1, JTC-12, and ROS 17/2.8 cells. A putative VDRE with a half-site spacing of 6 base pairs at -249/-232 (VDRE-3) did not contribute to 1,25-(OH)2D3 induced expression in the native promoter, although activity has been reported when the element was fused to the heterologous thymidine kinase promoter. Two VDREs with half-site spacings of 3 base pairs at -150/-136 and -258/-244 (VDRE-1 and VDRE-2, respectively), showed transcriptional synergism in COS-1 cells when treated with 1,25-(OH)2D3 (10(-7) to 10(-11) M). The contribution of both VDREs was hormone-concentration dependent from 10(-10) to 10(-12) M, with VDRE-1 demonstrating greatest sensitivity to 1,25-(OH)2D3. Transactivation by VDRE-1 was always greater than VDRE-2, but the converse was observed for the binding of vitamin D receptor-retinoid X receptor complex by each VDRE in gel mobility shift assays. The synergy observed between VDRE-1 and VDRE-2 may have important implications in cellular responses to different circulating levels of 1,25-(OH)2D3.

Bacillus thermoamylovorans sp. nov., a moderately thermophilic and amylolytic bacterium.

A moderately thermophilic, facultatively anaerobic, amylolytic bacterium was isolated from palm wine, a tropical alcoholic beverage that was sampled in Senegal. The cells were gram positive, catalase positive, non-spore forming, rod shaped, and slightly motile with peritrichous flagella. The strain which we examined did not possess cytochrome and produced L-(+)-lactate, acetate, ethanol, and formate but not hydrogen during carbohydrate fermentation. Growth occurred at pH values ranging from 5.4 to 8.5, and optimum growth occurred at around pH 7.0. The optimum temperature for growth was around 50 degrees C, and the upper temperature limit for growth was 58 degrees C. The guanine-plus-cytosine content of the DNA was 38.8 +/- 0.2 mol%. A sequence analysis of the 16S rRNA gene revealed that the new organism is closely related phylogenetically to members of genus Bacillus. Despite the lack of spores, we propose that on the basis of phylogenetic characteristics, the new isolate should be classified as a new Bacillus species, Bacillus thermoamylovorans. The type strain is strain DKP (= Collection of Institut Pasteur CNCM I-1378).