No description available.

No description available.

No description available.

Phosphate (Pi) is essential for healthy bone growth as well as normal fracture repair. Studies have shown that when animals are phosphate deficient normal fracture healing is interrupted. Although phosphate deficiency has been shown to impair fracture healing, it is unknown how different genetic factors interact with phosphate deficiency to disrupt healing. Furthermore, it is unknown if upon replenishing phosphate in the diet healing will be re-initiated or if the deficiencies will persist irreversibly to prolong the healing of the bones. To assess how genetic factors interact with phosphate deficiency, fractures were generated in three genetically distinct strains of mice that had previously been shown to have different patterns of endochondral bone formation. Phosphate deficiency was initiated two days prior to fracture and was then maintained for a 15 day period covering the normal duration of endochondral bone development. To assess if replenishing phosphate could rescue genetic expression of deficient healing, normal phosphate was re-introduced into the diet after 15 days of deficiency and bone healing was allowed to continue until 35 days post fracture. Messenger RNA expression for marker genes for cartilage and bone formation was assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis over this time course of healing. Structural properties, callus mineralization and cartilage contents were assessed by micro-computed tomography and contrast agent enhanced micro- computed tomography (CECT). Torsional mechanical testing was used to measure bone strength. To assess if replenishing phosphate could rescue mineralization and biomechanical properties of deficient healing, normal phosphate was re-introduced after 15 days of deficiency and bone healing was allowed to continue until 21 days post fracture. The biological assessment of fracture healing showed that all three genetic strains had impaired expression of both cartilage and bone associated genes during the period of phosphate deficiency. Once phosphate was returned to the diet, however, the osteogenesis genes showed a burst of late expression in all three strains. Interestingly, torsional testing of the bones showed that phosphate deficient/replenished groups were all stronger but also more brittle than the bones of control mice. Micro-computed tomography demonstrated that bone mineral density was slightly higher in the phosphate deficient mice but the bone mineral density standard deviation in the calluses were also higher indicating that the mineralization within the healing calluses was unevenly distributed in the phosphate deficient/replenished group. Lastly, contrast agent enhanced computed tomography data showed that the overall callus tissue mineral density was lower in phosphate deficient/replenished calluses due to the greater cartilage in the phosphate deficient/replenished calluses. These results suggest that the increase strength in the phosphate deficient/replenished calluses is due to the burst of expression in osteogenesis genes that led to the rapid mineralization of the fracture gap in order to compensate for fracture instability due to the phosphate restriction. They also show that a gross metabolic alteration supersedes all other aspects of genetic variably in endochondral development. Finally, they show that even though fracture healing may be greatly delayed by phosphate deficiency, replacement of phosphate after deficiency leads to rapid regain in function. Future studies need to be carried out to determine if longer time lengths of phosphate deficiency can be rescued upon reintroduction of phosphate.

No description available.

This thesis includes three studies examining the role of the type I (NPT1) and type II (NPT2) renal sodium (Na+)-phosphate (Pi) cotransporter genes in inherited hypophosphatemias. In the first study, the chromosomal locations of the NPT1 and NPT2 genes in human and rabbit are determined by physical mapping techniques. The NPT1 and NPT2 genes map respectively to human chromosomes 6p22 and 5q35 and to rabbit chromosomes 12p11 and 3p11. The localization of the two cotransporter genes to autosomes excludes them as candidate genes for X-linked hypophosphatemia. In addition, these assignments agree with the previously reported homology between rabbit chromosome 12 and human chromosome 6 and provide the basis for the establishment of a conserved syntenic group between rabbit chromosome 3 and human chromosome 5. / The goal of the second study was to clone, sequence and characterize the structure of the human NPT2 gene in order to design intronic primers to amplify NPT2 exons from patient DNA. Parallel experiments were performed on the mouse Npt2 gene, so that a vector could be designed to knockout the mouse Npt2 gene. In both species, the type II renal Na+-Pi cotransporter gene is approximately 16kb in length and is comprised of 13 exons and 12 introns. This work provides a basis for the study of the regulation of NPT2 transcription and facilitates the screening of DNA samples from patients with autosomally inherited disorders of renal Pi reabsorption for mutations in the NPT2 gene. / In the third study, polymorphic markers flanking the NPT1 and NPT2 genes were typed in members of a Bedouin kindred segregating the autosomal disorder Hereditary hypophosphatemic rickets with hypercaloiuria (HHRH). Genotype data were examined for excess homozygosity and allele sharing among affected pedigree members. Data did not reveal excess allele sharing on either chromosome 6 or 5, where the NPT1 and NPT2 genes are located, but suggested chromosome 3p as a site for further investigation. Identification of a HHRH locus is the first step toward identifying a gene involved in the pathophysiology of this disorder.

No description available.

The expectation for a gene dose effect in an X-linked phenotype is that the corresponding metrical trait in heterozygous females will lie between values for affected hemizygous males and unaffected males and females. I made sequential measurements (at 30, 60, 90, 120 and 150 days) of serum phosphate concentration and tail length in mice with X-linked hypophosphatemia (mutant genotypes: Hyp/+, Hyp/Y and Hyp/Hyp) and in their normal littermates (genotypes: +/+ +/Y). I also measured renal mitochondrial 24-OHase activity in mice fed control and low phosphate diets and representing all five genotypes. I further studied serum AP activity and vertebral bone histomorphometry in the five genotypes. The mutant animals all had uniformly and significantly different values than unaffected littermates. There was no evidence of a gene dose effect because values were not significantly different among the three mutant genotypes. / I also studied the influence of gamete of origin on serum phosphate, tail length, renal mitochondrial 24-OHase activity, serum AP activity and vertebral bone histomorphometry in the Hyp/+ offspring of affected males (Hyp/Y) or affected females (Hyp/+ or Hyp/Hyp). I found no effect on the distribution of trait values. / I conclude that parental origin of the mutant allele does not explain the absence of a gene dose effect in Hyp mice.

The X-linked Hyp mutation is characterized by a specific defect in phosphate (Pi) transport at the renal brush-border membrane (BBM). To understand the mechanism for the 50% decrease in Vmax of the high affinity Pi transport system in BBM of Hyp mice, we compared the effects of external Na$ sp+$ concentration, membrane potential, external pH, and Pi transport inhibitors on Pi uptake in BBM vesicles (BBMV) prepared from normal mice and Hyp littermates. / The apparent affinity for Na$ sp+$, the Na$ sp+{:}$Pi stoichiometry, the response to membrane potential and the response to external pH are similar in BBMV from both normals and mutants. / The Ki for phosphonoformic acid (PFA) inhibition of Na$ sp+$-Pi cotransport is lower in BBMV prepared from Hyp mice when compared to normal mice but not different in BBMV from Pi-deprived mice which are characterized by an increase in Vmax of the high affinity Na$ sp+$-Pi cotransport system. / We conclude that the decrease in Vmax of the high affinity Na$ sp+$-Pi cotransport system in the Hyp mouse is not the result of an inappropriate response of the transport system to Na$ sp+$, membrane potential or pH.

Na$ sp+$-Pi cotransport across the brush border membrane is the rate limiting step in renal Pi reabsorption. To determine its precise role in the maintenance of Pi homeostasis, we cloned and characterized the renal-specific Na$ sp+$-Pi cotransporter/Npt2 gene and generated a gene targeting vector for the creation of a knockout mouse. The gene was cloned by screening a genomic DNA library with a rat Npt2 cDNA probe. The Npt2 gene is approximately 17kb and contains 13 exons and 12 introns. A targeting construct was generated by inserting 5$ sp prime$ and 3$ sp prime$ homologous arms of 2.1 and 2kb, respectively, into the pPNT vector and replacing 7.7kb of Npt2 with a neomycin resistance gene. The vector also contained the herpes simplex virus thymidine kinase gene for negative selection. After electroporation into embryonic stem cells, clones were picked following selection in G418 and FIAU. Of 100 doubly-resistant clones that were screened by Southern analysis, 6 positive clones were detected giving a targeting frequency of 6%.