Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

The effect of implantation on scaffoldless three-dimensional engineered bone constructs

Our laboratory has previously developed scaffoldless engineered bone constructs (EBC). Bone marrow stromal cells (BMSC) were harvested from rat femur and cultured in medium that induced osteogenic differentiation. After reaching confluence, the monolayer of cells contracted around two constraint points forming a cylinder. EBCs were placed in small diameter (0.5905 × 0.0625 in.) or large diameter (0.5905 × 0.125 in.) silicone tubing and implanted intramuscularly in the hind limb of a rat. Bone mineral content (BMC) of the EBC was analyzed before implantation and at 1 and 2 mo following implantation and compared to that of native femur bone at different stages of development. Negligible BMC was observed in E-20 femur or EBCs prior to implantation. One-month implantation in both small and large tubing increased BMC in the EBC. BMC of EBC from large tubing was greater than in 14 d rat neonatal femurs, but was 2% and 3% of BMC content in adult bone after 1 and 2 mo of implantation, respectively. Alizarine Red and osteopontin staining of the EBCs before and after implantation confirmed increased bone mineralization in the implanted EBCs. Implanted EBCs also had extensive vascularization. Our data suggest that BMSC can be successfully used for the generation of scaffoldless EBC, and this model can be potentially used for the generation of autologous bone transplants in humans.     

Histidine ligand protonation and redox potential in the rieske dioxygenases: role of a conserved aspartate in anthranilate 1,2-dioxygenase

The Rieske dioxygenase, anthranilate 1,2-dioxygenase, catalyzes the 1,2-dihydroxylation of anthranilate (2-aminobenzoate). As in all characterized Rieske dioxygenases, the catalytic conversion to the diol occurs within the dioxygenase component, AntAB, at a mononuclear iron site which accepts electrons from a proximal Rieske [2Fe-2S] center. In the related naphthalene dioxygenase (NDO), a conserved aspartate residue lies between the mononuclear and Rieske iron centers, and is hydrogen-bonded to a histidine ligand of the Rieske center. Engineered substitutions of this aspartate residue led to complete inactivation, which was proposed to arise from elimination of a productive intersite electron transfer pathway [Parales, R. E., Parales, J. V., and Gibson, D. T. (1999) J. Bacteriol. 181, 1831-1837]. Substitutions of the corresponding aspartate, D218, in AntAB with alanine, asparagine, or glutamate also resulted in enzymes that were completely inactive over a wide pH range despite retention of the hexameric quaternary structure and iron center occupancy. The Rieske center reduction potential of this variant was measured to be approximately 100 mV more negative than that for the wild-type enzyme at neutral pH. The wild-type AntAB became completely inactive at pH 9 and exhibited an altered Rieske center absorption spectrum which resembled that of the D218 variants at neutral pH. These results support a role for this aspartate in maintaining the protonated state and reduction potential of the Rieske center. Both the wild-type and D218A variant AntABs exhibited substrate-dependent rapid phases of Rieske center oxidations in stopped-flow time courses. This observation does not support a role for this aspartate in a facile intersite electron transfer pathway or in productive substrate gating of the Rieske center reduction potential. However, since the single turnovers resulted in anthranilate dihydroxylation by the wild-type enzyme but not by the D218A variant, this aspartate must also play a crucial role in substrate dihydroxylation at or near the mononuclear iron site.     

Identifiability in biobanks: models,measures, and mitigation strategies

The collection and sharing of person-specific biospecimens has raised significant questions regarding privacy. In particular, the question of identifiability, or the degree to which materials stored in biobanks can be linked to the name of the individuals from which they were derived, is under scrutiny. The goal of this paper is to review the extent to which biospecimens and affiliated data can be designated as identifiable. To achieve this goal, we summarize recent research in identifiability assessment for DNA sequence data, as well as associated demographic and clinical data, shared via biobanks. We demonstrate the variability of the degree of risk, the factors that contribute to this variation, and potential ways to mitigate and manage such risk. Finally, we discuss the policy implications of these findings, particularly as they pertain to biobank security and access policies. We situate our review in the context of real data sharing scenarios and biorepositories.     

VEGF in biological control

Vascular endothelial growth factor A (VEGF-A) belongs to a family of heparin binding growth factors that include VEGF-B, VEGF-C, VEGF-D, and placental-like growth factor (PLGF). First discovered for its ability to regulate vascular endothelial cell permeability, VEGF is a well-known angiogenic factor that is important for vascular development and maintenance in all mammalian organs. The development of molecular tools and pharmacological agents to selectively inhibit VEGF function and block angiogenesis and/or vascular permeability has led to great promise in the treatment of various cancers, macular degeneration, and wound healing. However, VEGF is also important in animals for the regulation of angiogenesis, stem cell and monocyte/macrophage recruitment, maintenance of kidney and lung barrier functions and neuroprotection. In addition to its role in regulating endothelial cell proliferation, migration, and cell survival, VEGF receptors are also located on many non-endothelial cells and act through autrocrine pathways to regulate cell survival and function. The following review will discuss the role of VEGF in physiological angiogenesis as well as its role in non-angiogenic processes that take place in adult organs.     

Isolation of the MurineNbr1Gene Adjacent to the MurineBrca1Gene

The humanNBR1cDNA has previously been identified using polyclonal sera to CA125, an ovarian tumor antigen used in monitoring ovarian cancer. The gene was mapped to theBRCA1region on chromosome 17q21 and subsequently found to lie in close proximity to the recently identifiedBRCA1gene. The NBR1 protein has a B-box motif but the function of the protein is as yet unknown. To investigate the function and importance of this gene, we have studied the conservation of this gene in other species and in particular in the mouse. We have isolated murineNbr1cDNA and genomic clones. Translation of the cDNA sequence indicates that the protein is highly conserved, being 89% similar and 84% identical to the human. Analysis of the murineNbr1genomic clones indicates that it maps less than 1 kb from theBrca1gene and that, unlike that in human, this region is not duplicated.     

Structure,function, aging and turnover of aggrecan in the intervertebral disc

Background

Aggrecan is the major non-collagenous component of the intervertebral disc. It is a large proteoglycan possessing numerous glycosaminoglycan chains and the ability to form aggregates in association with hyaluronan. Its abundance and unique molecular features provide the disc with its osmotic properties and ability to withstand compressive loads. Degradation and loss of aggrecan result in impairment of disc function and the onset of degeneration.

Scope of review

This review summarizes current knowledge concerning the structure and function of aggrecan in the normal intervertebral disc and how and why these change in aging and degenerative disc disease. It also outlines how supplementation with aggrecan or a biomimetic may be of therapeutic value in treating the degenerate disc.

Major conclusions

Aggrecan abundance reaches a plateau in the early twenties, declining thereafter due to proteolysis, mainly by matrix metalloproteinases and aggrecanases, though degradation of hyaluronan and non-enzymic glycation may also participate. Aggrecan loss is an early event in disc degeneration, although it is a lengthy process as degradation products may accumulate in the disc for decades. The low turnover rate of the remaining aggrecan is an additional contributing factor, preventing protein renewal. It may be possible to retard the degenerative process by restoring the aggrecan content of the disc, or by supplementing with a bioimimetic possessing similar osmotic properties.

General significance

This review provides a basis for scientists and clinicians to understand and appreciate the central role of aggrecan in the function, degeneration and repair of the intervertebral disc.     

Hyperelastic Material Properties of Mouse Skin under Compression

The skin is a dynamic organ whose complex material properties are capable of withstanding continuous mechanical stress while accommodating insults and organism growth. Moreover, synchronized hair cycles, comprising waves of hair growth, regression and rest, are accompanied by dramatic fluctuations in skin thickness in mice. Whether such structural changes alter skin mechanics is unknown. Mouse models are extensively used to study skin biology and pathophysiology, including aging, UV-induced skin damage and somatosensory signaling. As the skin serves a pivotal role in the transfer function from sensory stimuli to neuronal signaling, we sought to define the mechanical properties of mouse skin over a range of normal physiological states. Skin thickness, stiffness and modulus were quantitatively surveyed in adult, female mice (Mus musculus). These measures were analyzed under uniaxial compression, which is relevant for touch reception and compression injuries, rather than tension, which is typically used to analyze skin mechanics. Compression tests were performed with 105 full-thickness, freshly isolated specimens from the hairy skin of the hind limb. Physiological variables included body weight, hair-cycle stage, maturity level, skin site and individual animal differences. Skin thickness and stiffness were dominated by hair-cycle stage at young (6–10 weeks) and intermediate (13–19 weeks) adult ages but by body weight in mature mice (26–34 weeks). Interestingly, stiffness varied inversely with thickness so that hyperelastic modulus was consistent across hair-cycle stages and body weights. By contrast, the mechanics of hairy skin differs markedly with anatomical location. In particular, skin containing fascial structures such as nerves and blood vessels showed significantly greater modulus than adjacent sites. Collectively, this systematic survey indicates that, although its structure changes dramatically throughout adult life, mouse skin at a given location maintains a constant elastic modulus to compression throughout normal physiological stages.     

1,25-Dihydroxyvitamin D and the Vitamin D Receptor Gene Polymorphism Apa1 Influence Bone Mineral Density in Primary Hyperparathyroidism

Objective

Parathyroid hormone (PTH) and vitamin D are the most important hormones regulating calcium metabolism. In primary hyperparathyroidism (PHPT) excessive amounts of PTH are produced. Bone turnover is enhanced, leading to reduced bone mineral density and elevated levels of serum calcium. The aim of this study was to investigate relations between serum levels of 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)₂D) and bone mineral density, as well as known genetic polymorphisms in the vitamin D receptor and enzymes metabolising vitamin D in patients with PHPT.

Design/Subjects

We conducted a cross-sectional study of 52 patients with PHPT.

Results

Mean level of 25(OH)D was 58.2 nmol/L and median 1,25(OH)₂D level was 157 pmol/L. Among our patients with PHPT 36.5% had 25(OH)D levels below 50 nmol/L. Serum 1,25(OH)₂D was inversely correlated to bone mineral density in distal radius (p = 0.002), but not to bone mineral density at lumbar spine or femoral neck. The vitamin D receptor polymorphism Apa1 (rs7975232) was associated with bone mineral density in the lumbar spine.

Conclusions

The results suggest that PHPT patients with high blood concentrations of 1,25(OH)₂D may have the most deleterious skeletal effects. Randomized, prospective studies are necessary to elucidate whether vitamin D supplementation additionally increases serum 1,25(OH)₂D and possibly enhances the adverse effects on the skeleton in patients with PHPT.