Bone turnover markers as determinants of bone density and fracture in men with distal forearm fractures: the pathogenesis examined in the Mr F study.

The pathogenesis for low-trauma wrist fractures in men is not fully understood. This study found that these men had evidence of significantly higher bone turnover compared with control subjects. Bone turnover markers were negative predictors of bone mineral density and were a predictor of fracture. INTRODUCTION: Men with distal forearm fractures have reduced bone density, an increased risk of osteoporosis and of further fractures. The aim of this study was to investigate whether or not men with distal forearm fractures had evidence of altered bone turnover activity. METHODS: Fifty eight men with low-trauma distal forearm fracture and 58 age-matched healthy control subjects were recruited. All subjects underwent a DXA scan of the forearm, both hips, and lumbar spine, biochemical investigations, and health questionnaires. Measurements of beta crosslaps (ßCTX), procollagen type I N-terminal propeptide (PINP), sclerostin, Dickkopf-1 (Dkk1), and fibroblast growth factor 23 (FGF 23) were made. RESULTS: Men with fracture had significantly higher PINP than controls at 39.2 ng/ml (SD 19.5) versus 33.4 ng/ml (SD13.1) (p<0.001). They also had significantly higher ßCTX at 0.45 ng/ml (SD 0.21) versus 0.37 ng/ml (SD 0.17) (p= 0.037). Fracture subjects had significantly lower aBMD and PINP was a negative predictor of aBMD at the total hip and ßCTX a negative predictor of forearm aBMD. Sclerostin was a positive predictor of aBMD at the lumbar spine and hip sites. Sex hormone binding globulin (SHBG) at 37nmol/L (SD 15.0) was lower in fracture cohort compared to 47.9 nmol/L (SD 19.2) (p=0.001) in control. Multiple regression revealed that the best model for prediction of fracture included SHBG, P1NP, and ultra-distal forearm aBMD. The likelihood of distal forearm fracture was decreased by 5.1% for each nmol/L increase in SHBH and by 1.4% for every mg/cm2 increase in ultra-distal forearm aBMD, but increased by 6.1 % for every ng/ml increase in P1NP. Men in the highest quartile of PINP had a significantly greater likelihood of distal forearm fracture than those in the lowest quartile. CONCLUSION: The fracture group had significantly higher PINP and ßCTX compared with the control group, and these markers were negative predictors of aBMD at the total hip and forearm sites, respectively. Sclerostin was a positive predictor of the variance of spinal and hip aBMD. Likelihood of forearm fracture was best predicted by a combination of SHBG, PINP, and ultra-distal forearm aBMD. Findings of such cross-sectional data should be treated with caution, as longitudinal studies would be required to confirm or refute them.

Long-term evaluation of anabolic and anti-resorptive agents in adults with familial osteoporosis due to pro205ala variant of the col1a1 gene.

INTRODUCTION: Osteogenesis imperfecta (OI) is a rare disorder with variable clinical presentation, commonly caused by mutations in collagen type I genes. OI affects both bone quality and density resulting in fractures and deformity. The effectiveness of bisphosphonates in the treatment of adult OI remains unclear. Small, randomised trials have shown increases in BMD, but without fracture rate reduction. AIM: We report the results of BMD of a family harbouring C 613 C>G substitution in exon 8 of Col1A1 gene leading to Pro205Ala missense variant, as well as the results of long term treatment of a mother and daughter with this mutation.

Does regional loss of bone density explain low trauma distal forearm fractures in men (the Mr F study)?

The pathogenesis of low trauma wrist fractures in men is not fully understood. This study found that these men have lower bone mineral density at the forearm itself, as well as the hip and spine, and has shown that forearm bone mineral density is the best predictor of wrist fracture. INTRODUCTION: Men with distal forearm fractures have reduced bone density at the lumbar spine and hip sites, an increased risk of osteoporosis and a higher incidence of further fractures. The aim of this case-control study was to investigate whether or not there is a regional loss of bone mineral density (BMD) at the forearm between men with and without distal forearm fractures. METHODS: Sixty-one men with low trauma distal forearm fracture and 59 age-matched bone healthy control subjects were recruited. All subjects underwent a DXA scan of forearm, hip and spine, biochemical investigations, health questionnaires, SF-36v2 and Fracture Risk Assessment Tool (FRAX). The non-fractured arm was investigated in subjects with fracture and both forearms in control subjects. RESULTS: BMD was significantly lower at the ultradistal forearm in men with fracture compared to control subjects, in both the dominant (mean (SD) 0.386 g/cm2 (0.049) versus 0.436 g/cm2 (0.054), p < 0.001) and non-dominant arm (mean (SD) 0.387 g/cm2 (0.060) versus 0.432 g/cm2 (0.061), p = 0.001). Fracture subjects also had a significantly lower BMD at hip and spine sites compared with control subjects. Logistic regression analysis showed that the best predictor of forearm fracture was ultradistal forearm BMD (OR = 0.871 (0.805-0.943), p = 0.001), with the likelihood of fracture decreasing by 12.9% for every 0.01 g/cm2 increase in ultradistal forearm BMD. CONCLUSIONS: Men with low trauma distal forearm fracture have significantly lower regional BMD at the ultradistal forearm, which contributes to an increased forearm fracture risk. They also have generalised reduction in BMD, so that low trauma forearm fractures in men should be considered as indicator fractures for osteoporosis.

Expression of oxidative stress in metastatic retinoblastoma- a comparative study.

OBJECTIVE: To compare oxidative stress between primary retinoblastoma and retinoblastoma with distant metastasis. PATIENTS AND METHODS: Forty consecutive patients presented with primary retinoblastoma and the same number of patients presented with distant metastasis, attending the outpatient department of our hospital between August 2002 and April 2005. All the patients with retinoblastoma underwent a standard metastasis workup and were subsequently categorized into two groups (without metastasis and with metastasis).Venous blood samples were drawn from each patient. After proper centrifugation, serum was collected and antioxidant enzymes and reactive oxygen species (ROS) were assayed. MAIN OUTCOME MEASURES: Serum collected from the patients was subjected to biochemical assay of the antioxidant enzymes (superoxide dismutase, catalase and peroxidise) and ROS to determine any difference in enzyme activity between the two groups. RESULTS: Antioxidant levels were found to be less in the metastasis group as compared to the primary intraocular retinoblastoma group(p less than 0.05).Mean ROS activity was found to be increased in metastatic group (p less than 0.05). CONCLUSION: The decreased antioxidant enzymes level along with increased ROS activity in patients with metastatic retinoblastoma reflect increased oxidative stress as compared to primary intraocular retinoblastoma patients.

Suppression of bone turnover by B-cell depletion in patients with rheumatoid arthritis.

UNLABELLED: The role of B cells in inflammatory bone formation and resorption is controversial. We investigated this in patients with rheumatoid arthritis (RA) treated with rituximab, a B-cell depleting antibody. We found a significant suppression in bone turnover, possibly a direct effect or as a consequence of a reduction in inflammation and disease activity. INTRODUCTION: RA is the most prevalent inflammatory joint disease, in which B cells play an important role. However, the role of B cells in bone turnover is controversial and RA subjects treated with rituximab, a B-cell depleting monoclonal antibody, provide an ideal model for determining the role of B cells in inflammatory bone resorption. METHODS: Serum from 46 RA patients, collected pre- and post-rituximab therapy, was analysed for biomarkers of bone turnover (procollagen type I amino-terminal propeptide [P1NP], osteocalcin, ß-isomerised carboxy-terminal telopeptide of type 1 collagen [ßCTX] and osteoprotegerin [OPG]). RESULTS: A significant decrease in bone resorption was observed 6 months after rituximab (median change ßCTX -50 ng/L, 95%CI -136, -8 p < 0.001, this equates to -37%; 95%CI -6, -49), mirrored by a reduction in disease activity. Similarly, there was a significant increase in P1NP, a marker of bone formation (median change P1NP 5.0 µg/L, 95%CI -1.0, 11.2, p = 0.02; 13%; 95%CI -3, 39), but no significant change in osteocalcin or OPG levels. The percentage change from baseline of ßCTX in a subgroup of patients (not on prednisolone or bisphosphonate) was significantly correlated with the percentage reduction in DAS28 score (r (s) = 0.570, p = 0.014). CONCLUSIONS: In conclusion, we have found that B-cell depletion increases bone formation and decreases bone resorption in RA patients; this may be a direct effect on osteoblasts and osteoclasts, respectively, and be at least partially explained by the decreased inflammation and disease activity.

Simulation of generation-collection experiments with homogeneous kinetics: application to electrochemical investigation of superoxide radical anion generation by osteoclasts on bone.

We report simulations of electrochemical generation-collection experiments in which the generator is a small disc producing a specified time-dependent flux of the analyte and the collector is a large planar electrode which collects the analyte at the mass transport-controlled rate. This geometry corresponds to many experiments in bioelectrochemistry where a relatively large sensor is used to detect the products of a cell's metabolism at low concentration. In particular, our simulations are motivated by attempts to understand our results on the detection of the superoxide radical anion burst generated by osteoclasts (bone-resorbing cells) in response to various stimuli. Superoxide is present at low levels and disproportionates in aqueous media; however, the homogeneous kinetics are included in our simulations and the results show that it is possible to estimate the magnitude of the flux of superoxide produced by the cells and to accurately determine the time-dependence of the flux in response to stimuli such as injection of parathyroid hormone, vitamin D(3) and pertussis toxin. In all these cases, the superoxide anion flux was successfully modeled as uniform across the cell surface with time-dependence of the form j(0)e(-k(d)t) + j(∞). j(∞) is the sustained flux of superoxide and the first-order rate constant k(d) and the magnitude j(0) describe the transient component of the flux. The simulations indicate that for cell-electrode gaps D approximately < √(D/k(d)), where D is the diffusion coefficient, the value of k(d) can be accurately extracted from the time-dependence of the collector current without detailed knowledge of parameters which are hard to measure during the experiment, e.g., the cell radius a and cell-electrode separation d. In the case of parathyroid hormone, the first-order rate constant describing the decay of the transient component was k(d) = 1.8 ± 0.8 × 10(-1) s(-1), but much slower decays were observed in response to pertussis toxin (k(d) = 1.5 ± 0.5 × 10(-2) s(-1)) and vitamin D(3) (k(d) = 1.1 ± 0.5 × 10(-3) s(-1)).

Plasma vitamin D and cytokines in periodontal disease and postmenopausal osteoporosis.

BACKGROUND AND OBJECTIVE: Osteoporosis and periodontal disease are chronic diseases, in the pathogenesis of which plasma osteoprotogerin (OPG) and RANKL are important. The study aimed to investigate the relationship between periodontal disease and plasma cytokines, vitamin D and bone mineral density in postmenopausal women with and without osteoporosis. MATERIAL AND METHODS: One hundred and eighty-five postmenopausal women with osteoporosis and 185 age- and sex-matched control subjects were recruited. Periodontal disease was subdivided into active or past periodontal disease. Osteoprotegerin, RANKL, 25-hydroxyvitamin D3 (25OHD), biochemical markers of bone turnover (serum C-terminal telopeptide, CTX), anthropometry and bone mineral density were measured. RESULTS: A significantly higher proportion of the women with osteoporosis had active or past periodontal disease or both compared with control subjects (87.6 vs. 37.8%, p < 0.001). Plasma 25OHD was significantly lower (p < 0.001) and RANKL and OPG significantly higher in the women with osteoporosis than in control subjects (p < 0.0001). RANKL, OPG and CTX were significantly higher in women with active periodontal disease than in those without (p < 0.001), as were OPG and CTX in past periodontal disease (p < 0.001). In active and past periodontal disease, 25OHD was significantly lower (p < 0.001). Multiple logistic regression analysis showed that periodontal disease was best predicted by RANKL, 25OHD, C-terminal telopeptide and weight, r² = 10.4%. CONCLUSION: Periodontal disease is more common in women with osteoporosis and is associated with lower vitamin D and higher concentrations of RANKL and OPG. Raised cytokines may provide the underlying mechanism that links these two conditions.

Dispersions of alkyl-capped silicon nanocrystals in aqueous media: photoluminescence and ageing.

Alkyl-capped silicon nanocrystals can be dispersed in aqueous media by shaking or stirring their solutions in organic solvents (DMSO, ether, THF) with excess water. THF is the most straightforward choice with which to prepare stable aqueous dispersions, because the nanocrystals are very soluble in THF and it is also miscible with water. As little as 0.01% v/v tetrahydrofuran is sufficient. DMSO and ether were the preferred choices for subsequent staining of live cells because THF shows some acute toxicity even when very dilute. The luminescence intensity of the aqueous dispersions is linear in particle concentration and independent of pH over the range 5-9. The sols retain their photoluminescence and are stable against flocculation for at least 6 months.

The cell biology of bone metabolism.

Contrary to the commonly held misconception, bone is a relatively dynamic organ that undergoes significant turnover as compared to other organs in the body. This review details how complex intercellular signalling, between the osteoprogenitor cells and mature osteoblasts, osteocytes and osteoclasts, regulates and balances activities of bone cells during remodelling and growth. Both systemic, as well as local autocrine and paracrine factors are discussed. A number of recent important advances in cell biology of bone have led to a new paradigm in understanding of the subject. In this regard, the interaction between the immune system and bone cells is of particular interest, leading to the emergence of a new discipline termed osteoimmunology. The role of lymphocytes and a number of key cytokines in the regulation of osteoclastogenesis and osteoblast function is critically examined. The intracellular signalling regulating key cellular pathways involved in cell differentiation and activity are outlined. The emerging evidence of osteocytes as mechanosensors as well as regulators of mineralisation is discussed.

Vitamin D-binding protein gene microsatellite polymorphism influences BMD and risk of fractures in men.

UNLABELLED: Here we report the results of a vitamin D-binding protein gene microsatellite polymorphism study in 170 men, comprising healthy male subjects and men with osteoporosis-related symptomatic vertebral fractures. We confirm the results of an earlier study in a different cohort, showing relationship between certain genotypes of (TAAAn)-Alu repeats and reduced BMD and vertebral fractures. INTRODUCTION: Vitamin D-binding protein (DBP) plays a critical role in the transport and metabolism of metabolites of vitamin D, including the key calciotropic hormone 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). METHODS: We have investigated intra-intronic variable tandem (TAAA)n-Alu repeat expansion in the DBP gene in 170 men, comprising healthy male subjects and men with idiopathic osteoporosis and low trauma fractures. RESULTS AND CONCLUSIONS: The predominant DBP-Alu genotype in the control subjects was 10/10 (frequency 0.421), whereas the frequency of this genotype in men with osteoporosis was 0.089. DBP-Alu alleles *10, *8 and *9, respectively, were the three commonest in both healthy subjects and men with osteoporosis. Allele *10 was associated with a lower risk of osteoporosis (OR 0.39, 95% CI 0.25-0.64; p < 0.0005), as was allele *11 (odds ratio 0.09, 95% CI 0.01-0.67; p < 0.007). Logistic regression gave similar results, showing that individuals with genotype 10/10 and 19-20 repeats (genotypes 9/10, 9/11, 10/10,) are protected from fracture or osteoporosis. Overall, there was a relationship between DBP Alu genotype and BMD, suggesting that DBP-Alu genotype may influence fracture risk. This effect may be mediated by changes in the circulating concentrations of DBP which influences free concentrations of vitamin D.

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment.

BACKGROUND: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). METHODS: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). RESULTS: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. CONCLUSIONS: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Characterisation of cytosolic FK506 binding protein 12 and its role in modulating expression of Cbfa1 and osterix in ROS 17/2.8 cells.

FK506 is a commonly used immunosuppressant that mediates its action by exclusively interacting with the cytosolic immunophilin, FK506 binding protein 12 (FKBP12). Although FK506-induced acute osteoporosis is now well recognised, its precise mode of action in osteoblasts remains unclear. Therefore, in the present study we characterised FKBP12 in osteoblasts and investigated the role of FK506 in modulating osteoblast-specific transcription factors, core-binding factor alpha1 (Cbfa1) and osterix gene expression in ROS 17/2.8 cells. RT-PCR, immunolocalisation and Western blotting studies were employed to identify and characterise FKBP12 in rat primary osteoblasts and osteoblast-like osteosarcoma ROS 17/2.8 cells. Western blotting extracts of these cells revealed the 12 kDa and hitherto unreported 10 kDa FKBP isoform that were immunolocalised predominantly to the cytosol. The transient exposure of ROS 17/2.8 cells to H2O2 (100 microM) was found to elevate FKBP12 mRNA after 10 min and protein expression after 24 h. Both PTH (10(-9) M) and 1,25 (OH)2D3 (Vitamin D3) (10(-7) M) suppressed FKBP12 protein expression. FK506 in the therapeutic range (25 nmol/L) suppressed expression of Cbfa1 and osterix mRNA. The inhibition of Cbfa1 isoforms II/III expression was evident at 30 min and the extent of inhibition was sustained at 6 h. Osterix inhibition was also seen after 30 min, however, it became maximal after 6 h. The dose-dependant inhibition of osterix in these cells, carried out using 1.25, 12.5 and 125 nmol/L of FK506 was maximal at 1.25 nmol/L. Cbfa1 isoforms II/III were also maximally inhibited at 1.25 nmol/L; interestingly, the inhibition became less marked at higher concentrations of FK506. Similar dose of FK506 was found to inhibit ROS 17/2.8 cell proliferation; the inhibitory effect however was greater in insulin-stimulated cells. The results of this study suggest that immunosuppressant-induced osteoporosis, which is known to involve accelerated bone resorption by increase in osteoclastogenesis, may in fact also be accentuated by the inhibition of osteoblast differentiation and function.

Mechanisms of calcium disposal from osteoclastic resorption hemivacuole.

One of the most remarkable but neglected aspects of osteoclast function is its unique adaptation that allows the cell to function despite its resorbing surface being exposed to extremely high levels of ambient Ca2+. Recently our studies have provided evidence of continuous transcellular Ca2+ disposal, suggesting that osteoclasts are able to prevent Ca2+ accumulation within the resorptive hemivacuole. It has also been shown that matrix protein degradation products that accumulate within the osteoclast resorptive vacuole are also undergoing transcellular transport by transcytosis. However, both experimental evidence and theoretical considerations suggest that transcellular transport of Ca2+ and matrix protein is likely to occur via distinct routes. In light of these considerations, we are able to provide convincing explanations for the apparent anomalies of osteoclast intracellular [Ca2+] responses to a variety of endocrine stimuli. The understanding of the mechanisms involved in Ca2+ handling by osteoclasts indicates the lack of a simple link between osteoclast function and changes in overall cytosolic [Ca2+].

Estimation of tear glucose level and its role as a prompt indicator of blood sugar level.

Tear glucose level was measured by glucose oxidase method to determine the relationship between tear glucose and corresponding blood glucose levels in 200 patients. It was observed that this test had a definite value in finding the hyperglycaemics semiquantitatively.

Mutational and polymorphic analysis of the estradiol receptor-alpha gene in men with symptomatic vertebral fractures.

In view of the importance of estrogens in the maintenance of the skeleton in men, we have carried out mutational analysis of all the exons of the estrogen-receptor-alpha (ER-alpha) gene in 64 men (36 patients with symptomatic vertebral crush fractures and 28 control subjects). Initial screening of the ER-alpha gene, carried out by single-strand conformation polymorphism analysis followed by sequencing, showed conservative mutations in exon 4 which resulted in a single base substitutions producing GGG-->GGC transition in codon 274. We also carried out polymorphic analysis of the ER-alpha gene at the PvuII restriction site in 82 men with a range of bone density measurements (53 with symptomatic vertebral fractures and 29 controls). The frequencies of PP, Pp, and pp genotypes were 20.7%, 48.8%, and 30.5%, respectively. The distribution of the alleles was similar in the patients with symptomatic vertebral crush fractures and male control subjects. There was no association between ER-alpha genotypes and bone mineral density or arthropometric parameters. This relatively small study suggests that mutations in the ER-alpha gene are unlikely to be a common cause of osteoporosis in men with vertebral fractures. Furthermore, polymorphic variation of the ER-alpha gene appears to have little effect on the pathogenesis of osteoporosis in men.

Scanning electrochemical microscopy at the surface of bone-resorbing osteoclasts: evidence for steady-state disposal and intracellular functional compartmentalization of calcium.

Osteoclast resorptive activity occurs despite the presence of extremely high levels of ionized calcium ([Ca2+]) within the osteoclast hemivacuole, which is generated as a by-product of its resorptive activity. Previous in vitro observations have shown that increases in extracellular [Ca2+] ([Ca2+]e) in the surrounding medium can inhibit the osteoclast resorptive activity. Therefore, it has been suggested that the osteoclast acts as a "sensor" for [Ca2+]e, and that high [Ca2+]e leads to an increase in intracellular [Ca2+] ([Ca2+]i), thereby inhibiting osteoclasts in a negative feedback manner. In this report we have carried out an experimental and theoretical analysis of calcium disposal during osteoclast activity to evaluate how in vitro models relate to in vivo osteoclast activity, where it is possible that high [Ca2+]e may be present in the hemivacuole but not over the nonresorbing surface of the cell. Scanning electrochemical microscopy (SECM) studies of [Ca2+] and superoxide anion (O2.-) generation by bone-resorbing osteoclasts on the surface of a bovine cortical bone slice were compared with microspectofluorometric measurements of the levels of [Ca2+]i in single osteoclasts and the effect of [Ca2+]i on various aspects of osteoclast function. The generation of O2.- by the osteoclasts has been shown to be positively correlated with osteoclast resorptive function and can therefore serve as an index of acute changes in osteoclast activity. The SECM of bone-resorbing osteoclasts at the surface of a bone slice revealed a continuous steady-state release of Ca2+. Even after prolonged incubation lasting 3 h the near-surface [Ca2+]e in the solution above the cell remained <2 mM. The SECM real-time measurement data were consistent with the osteoclast acting as a conduit for continuous Ca2+ disposal from the osteoclast-bone interface. We conclude that the osteoclast distinguishes [Ca2+]e in the hemivacuole and in the extracellular fluid above the cell which we denote [Ca2+]e. We found that an increase in [Ca2+]i may be associated with activation; inhibition; or be without effect on O2.- generation, bone-matrix, or bone resorption. Similarly, osteoclast adhesion and bone-resorbing activity was affected by [Ca2+]e' but showed no correlation with [Ca2+]i. The data suggest the existence of functional compartmentalization of [Ca2+]i within the osteoclast, where elevated calcium may have an inhibitory, excitatory, or no effect on the overall osteoclast activity while exerting a selective effect on different functional modalities. These observations lead to the conclusion that far from being inhibited by Ca2+ generated, the osteoclast by virtue of the observed functional compartmentalization is highly adapted at carrying out its activity even when the level of [Ca2+] in resorptive lacunae is elevated.

Ataxica telengiectasia (Louis-Bar syndrome).

A case of ataxia telengiectasia in a 7-year-old girl with symptoms for last 3 years has been reported in detail. The necessary literature has been reviewed with proper references.

Southern analysis of mitochondrial DNA in cortical bone of elderly patients undergoing knee and hip arthroplasty.

The role of mitochondrial DNA deletions (dmtDNA) in involutional bone loss seen in elderly men and women has never been examined. The present investigation was carried out to determine the extent of dmtDNA in cortical bone of elderly patients undergoing knee and hip arthroplasties. The majority of earlier studies have employed the polymerase chain reaction (PCR) to detect and quantify dmtDNA in different body tissues. In the present study, Southern blotting was used to screen bone biopsies from 30 patients undergoing orthopaedic surgery (mean age+/-SD 67.5+/-9.6 years; range 49-87 years). The blotting of PvuII-digested genomic DNA, carried out using mtDNA probes covering the entire span of mtDNA, revealed high levels of deletions in six subjects (mean age+/-SD 63.0+/-10.1 years; range 49-78 years) and moderate to low levels of mutations in another 14 subjects (mean age+/-SD 64.9+/-8.9 years; range 53-87 years). The importance of this rather high prevalence of dmtDNA in the bone of the elderly is discussed in terms of possible involvement of increased production of oxygen-derived free radicals and oxidative stress, and its possible role in the accelerated bone loss leading to osteoporosis.

Triplex-induced recombination in human cell-free extracts. Dependence on XPA and HsRad51.

Triple helix-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner. Triple helix formation has been shown to stimulate recombination in mammalian cells in both episomal and chromosomal targets containing direct repeat sequences. Bifunctional oligonucleotides consisting of a recombination donor domain tethered to a TFO domain were found to mediate site-specific recombination in an intracellular SV40 vector target. To elucidate the mechanism of triplex-induced recombination, we have examined the ability of intermolecular triplexes to provoke recombination within plasmid substrates in human cell-free extracts. An assay for reversion of a point mutation in the supFG1 gene in the plasmid pSupFG1/G144C was established in which recombination in the extracts was detected upon transformation into indicator bacteria. A bifunctional oligonucleotide containing a 30-nucleotide TFO domain linked to a 40-nucleotide donor domain was found to mediate gene correction in vitro at a frequency of 46 x 10(-)5, at least 20-fold above background and over 4-fold greater than the donor segment alone. Physical linkage of the TFO to the donor was unnecessary, as co-mixture of separate TFO and donor segments also yielded elevated gene correction frequencies. When the recombination and repair proteins HsRad51 and XPA were depleted from the extracts using specific antibodies, the triplex-induced recombination was diminished, but was either partially or completely restored upon supplementation with the purified HsRad51 or XPA proteins, respectively. These results establish that triplex-induced, intermolecular recombination between plasmid targets and short fragments of homologous DNA can be detected in human cell extracts and that this process is dependent on both XPA and HsRad51.

An unusual case of a retained metallic arrowhead in the orbit and sphenoidal sinus.

A case of retained metallic arrowhead in orbit and sphenoidal sinus through an unusual route is reported. The eyeball was removed because of a possible risk of sympathetic ophthalmia.