Bril: A novel bone-specific modulator of mineralization

In the course of attempting to define the bone ""secretome"" using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 1.34 arnino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus-mediated Brit overexpression and lentivirus-mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose-dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.

The application of electron microscopy and microanalysis in conjunction with organic petrology to further the understanding of organic-mineral association: examples from Mount Isa and McArthur Basins, Australia

Organic petrology supported by electron microscopical and micro-analytical techniques was applied to organic matter in Proterozoic sediments to better understand hydrothermal processes responsible for ore-grade mineralisation. It was shown that organic maturation was not only closely linked to the geological history of the sediments, but also highlighted heat transfer by convection as differentiated from conduction solely through sediment burial and step-wise subsidence. Water-rock ratios effect organic maturation in hydrothermal systems, and erratic reflectance profiles are indicators of convective heat transfer. Identification and characterisation of organic materials in terms of source rock and migrated hydrocarbons was shown to be a powerful tool in reconstructing the thermal history of sediments, identifying hydrothermal episodes, fluid pathways and heat source in the northern Australian Proterozoic basins. Higher reflectance of organic matter towards the central parts of the Mount Isa Basin and some of the most northerly parts point to proximity to higher heat flow at times, in contrast to relatively low temperatures prevailing in the western parts of the basin, next to the Murphy Inlier. A close correlation shown between peak organic reflectance values and super-sequence boundaries farther highlighted the valuable information to be gained from organic petrology, by allowing the separation of processes responsible for metal dissolution and transportation from those inducing precipitation. (C) 2001 Elsevier Science B.V All rights reserved.

Increasing relevance of pharmacogenetics of drug metabolism in clinical practice

Much of the individual variation in drug response is due to genetic drug metabolic polymorphisms. Clinically relevant examples include acetylator status; cytochrome P450 2D6, 2C9 and 2C19 polymorphisms; and thiopurine methyltransferase deficiency. It is important to be aware of which drugs are subject to pharmacogenetic variability. In the future, population-based pharmacogenetic testing will allow more individualized drug treatment and will avoid the current empiricism.

Use of the web to provide learning support for a large metabolism and nutrition class

To support student learning in a large Metabolism and Nutrition class, we have introduced a web-based package, using a commercially available program, WebCT. The package was developed at a minimal cost and with limited resources. In addition to downloadable (PDF) versions of lecture Powerpoint presentations, tutorial outlines and a practical class exercise, web-based self-directed learning exercises were included to reinforce and extend lecture material in an active learning environment. The web-site also contained a variety of formative and summative assessment tasks that examined both factual recall and higher order thinking Detailed course information, timetables and a bulletin board were also readily accessible. Student usage of the site was generally high, but varied widely between individual students. Students who achieved a high overall score for the course completed on average three times as many formative assessment items and achieved a higher score for all tests than students who did poorly. Student feedback about the site was very positive with the majority of students reporting that the course material and assessment items that were available were useful to their learning. Administration of the course was also facilitated. (C) 2001 IUBMB. Published by Elsevier Science Ltd. All rights reserved.

Pectenotoxins - an issue for public health - A review of their comparative toxicology and metabolism

Pectenotoxins (PTXs) are a group of toxins associated with diarrhetic shellfish poisoning (DSP) and isolated from DSP toxin-producing dinoflagellate algae. Consumption of shellfish contaminated with PTXs has been associated with incidences of severe diarrhetic illness resulting in hospitalisation. Concern has been raised for public health following the discovery that these toxins are not only hepatotoxic and can cause diarrhetic effects in mammals, but that they are potently cytotoxic to human cancer cell lines and have been found to be tumour promoters in animals. With advances in knowledge and technology, more PTXs are being identified, but little is known of their toxicology and the potential impact these toxins may have on public health in the long term. Without such information, adequate health-risk assessments for the consumption of shellfish contaminated with PTXs cannot be performed. This review gives a brief introduction to diarrhetic shellfish toxins, details the known toxicology and metabolism of PTXs in animals, and discusses known incidences of PTX poisoning in humans. (C) 2001 Elsevier Science Ltd. All rights reserved.

Effects of high-dose etidronate treatment on microdamage accumulation and biomechanical properties in beagle bone before occurrence of spontaneous fractures

We recently demonstrated that suppressed bone remodeling allows microdamage to accumulate and causes reductions in some mechanical properties. However, in our previous study, I year treatment with high-dose etidronate (EHDP) did not increase microdamage accumulation in most skeletal sites of dogs in spite of complete remodeling suppression and the occurrence of spontaneous fractures of ribs and/or thoracic spinous processes. This study evaluates the effects of EHDP on microdamage accumulation and biomechanical properties before fractures occur. Thirty-six female beagles, 1-2 years old, were treated daily for 7 months with subcutaneous injections of saline vehicle (CNT) or EHDP at 0.5 (E-low) or 5 mg/kg per day (E-high). After killing, bone mineral measurement, histomorphometry, microdamage analysis, and biomechanical testing were performed. EHDP treatment suppressed intracortical and trabecular remodeling by 60%-75% at the lower dose, and by 100% at the higher dose. Osteoid accumulation caused by a mineralization deficit occurred only in the E-high group, and this led to a reduction of mineralized bone mass. Microdamage accumulation increased significantly by two- to fivefold in the rib, lumbar vertebra, ilium, and thoracic spinous process in E-low, and by twofold in the lumbar vertebra and ilium in E-high. However, no significant increase in damage accumulation was observed in ribs or thoracic spinous processes in E-high where fractures occur following 12 months of treatment. Mechanical properties of lumbar vertebrae and thoracic spinous processes were reduced significantly in both E-low and E-high. These findings suggest that suppression of bone remodeling by EHDP allows microdamage accumulation, but that osteoid accumulation reduces production of microdamage. (Bone 29:271-278; 2001) (C) 2001 by Elsevier Science Inc. All rights reserved.

Growth hormone is permissive for skeletal adaptation to mechanical loading

The Lewis dwarf (DW) rat was used as a model to test the hypothesis that growth hormone (GH) is permissive for new bone formation induced by mechanical loading in vivo. Adult female Lewis DW rats aged 6.2 +/- 0.1 months (187 +/- 18 g) were allocated to four vehicle groups (DW), four GH treatment groups at 32.5 mug/100 g body mass (DWGH1), and four GH treatment groups at 65 mug/100 g (DWGH2). Saline vehicle or GH was injected intraperitoneally (ip) at 6:30 p.m. and 6:30 a.m. before mechanical loading of tibias at 7:30 a.m. A single period of 300 cycles of four-point bending was applied to right tibias at 2.0 Hz, and magnitudes of 24, 29, 38, or 48N were applied. Separate strain gauge analyses in 5 DW rats validated the selection of loading magnitudes. After loading, double-label histomorphometry was used to assess bone formation at the periosteal surface (Ps.S) and endocortical surface (Ec.S) of tibias. Comparing left (unloaded) tibias among groups, GH treatment had no effect on bone formation. Bone formation in tibias in DW rats was insensitive to mechanical loading. At the Ec.S, mechanically induced lamellar bone formation increased in the DWGH2 group loaded at 48N (p < 0.05), and no significant increases in bone formation were observed among other groups. The percentage of tibias expressing woven bone formation (Wo.B) at the Ps.S was significantly greater in the DWGH groups compared with controls (p < 0.05). We concluded that GH influences loading-related bone formation in a permissive manner and modulates the responsiveness of bone tissue to mechanical stimuli by changing thresholds for bone formation.

The ratio of messenger RNA levels of receptor activator of nuclear factor kappa B ligand to osteoprotegerin correlates with bone remodeling indices in normal human cancellous bone but not in osteoarthritis

skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor KB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA, (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.

Self-reported calcium intake and bone mineral content in children and adolescents

Objective: We examined the relationship between self-reported calcium (Cal intake and bone mineral content (BMC) in children and adolescents. We hypothesized that an expression of Ca adjusted for energy intake (El), i.e., Ca density, would be a better predictor of BMC than unadjusted Ca because of underreporting of EI. Methods: Data were obtained on dietary intakes (repeated 24-hour recalls) and BMC (by DEXA) in a cross-section of 227 children aged 8 to 17 years. Bivariate and multivariate analyses were used to examine die relationship between Ca, Ca density, and the dependent variables total body BMC and lumbar spine BMC. Covariates included were height, weight, bone area, maturity age, activity score and El. Results: Reported El compared to estimated basal metabolic rate suggested underreporting of El. Total body and lumbar spine BMC were significantly associated with El, but not Ca or Ca density, in bivariate analyses. After controlling for size and maturity, multiple linear regression analysis revealed unadjusted Ca to be a predictor of BMC in males in the total body (p = 0.08) and lumbar spine (p = 0.01). Unadjusted Ca was not a predictor of BMC at either site in females. Ca density was not a better predictor of BMC at either site in males or females. Conclusions: The relationship observed in male adolescents in this study between Ca intake and BMC is similar to that seen in clinical trials. Ca density did not enable us to see a relationship between Ca intake and BMC in females, which may reflect systematic reporting errors or that diet is not a limiting factor in this group of healthy adolescents.

Effect of supplementation with a by-product of molasses fermentation or a non-protein nitrogen/mineral mix on feed intake and microbial protein supply in sheep consuming chopped oat (Avena sativa) hay

Crossbred ewes, weighing 30-40 kg, were assigned to three groups of six animals. One group of sheep was fed chopped oat hay (control), the second group was fed the control diet plus 30 g per head per day spray dried residue from the fermentation of molasses and the third group was fed the control diet plus 30 g per head per day of a non-protein nitrogen/mineral mix. Voluntary feed intake, digestibility of DM, OM and nitrogen, nitrogen balance and microbial nitrogen flow to the intestines were significantly increased by supplementation but efficiency of microbial protein production was not affected. (C) 2001 Elsevier Science BN. All rights reserved,

Relationships between crude protein and mineral concentrations in alfalfa and value of alfalfa silage as a mineral source for periparturient cows

One hundred and eight samples from three cultivars of alfalfa were obtained from three cuttings in 1996-1998 to evaluate the relationship between crude protein (CP) and mineral concentrations of alfalfa with cutting and maturation. The CP content drastically decreased from 27.9 to 11.4% on DM with maturity. Highly positive correlations were observed between CP and K in the first and the second cutting of alfalfa. The Ca content remained almost constant throughout the growth period. Four multiparous Holstein cows were assigned an alfalfa silage diet or an orchardgrass silage diet from 3 weeks prepartum to 1 week postpartum to examine the effect on the mineral balance. In the prepartum and postpartum diet, the roughage to concentrate ratio was 70:30 and 50:50, with alfalfa being 50 and 100% of the roughage, respectively. The alfalfa contained 1.93% of K. No metabolic disorders occurred, but the body weight decreased drastically from 1 to 6 days postpartum with each diet because of the high milk production immediately after the parturition. Positive retention of N, Ca, P, Mg, and K was observed prepartum, whereas the cows had negative N and mineral retention from 2 to 4 days postpartum. The Ca and P absorption, and Mg retention of cows with the alfalfa diet were higher than with the grass diet. The plasma Ca and inorganic P were not affected by diet, but the plasma PTH at parturition and plasma hydroxyproline from 1 week prepartum to 1 week postpartum were higher with the alfalfa diet. These results suggest that the low K alfalfa is suitable not only to prevent the incidence of milk fever but also to increase Ca, P and Mg utilization of periparturient cows, but the mineral supplementation is needed for the postpartum cows immediately after the parturition. (C) 2001 Elsevier Science B.V. All rights reserved.

Familial Paget's disease of bone: Nonlinkage to the PDB1 and PDB2 loci on chromosomes 6p and 18q in a large pedigree

Paget's disease of bone is a common condition characterized by bone pain, deformity, pathological fracture, and an increased incidence of osteosarcoma. Genetic factors play a role in the pathogenesis of Paget's disease but the molecular basis remains largely unknown. Susceptibility loci for Paget's disease of bone have been mapped to chromosome 6p21.3 (PDB1) and 18q121.1-q22 (PDB2) in different pedigrees, We have identified a large pedigree of over 250 individuals with 49 informative individuals affected with Paget's disease of bone; 31 of whom are available for genotypic analysis. The disease is inherited as an autosomal dominant trait in the pedigree with high penetrance by the sixth decade. Linkage analysis has been performed with markers at PDB1; these data show significant exclusion of linkage with log,, of the odds ratio (LOD) scores < -2 in this region. Linkage analysis of microsatellite markers from the PDB2 region has excluded linkage with this region, with a 30 cM exclusion region (LOD score < -2.0) centered on D18S42, These data confirm the genetic heterogeneity of Paget's disease of bone. Our hypothesis is that a novel susceptibility gene relevant to the pathogenesis of Paget's disease of bone lies elsewhere in the genome in the affected members of this pedigree and will be identified using a microsatellite genomewide scan followed by positional cloning.