INTESTINAL TRANSPORT OF CALCIUM AND PHOSPHATIDYLINOSITOL

Project: Research project

Project Details

Description

The principal investigator's interest has been the subject of calcium and
phosphate transport across the intestinal epithelium during maturation.
Initially, the P.I. utilized in vivo perfusion and in vitro everted gut
sacs to define the overall picture of transport. These studies were
extended to the plasma membranes (brush border and basolateral membranes)
and then to the subcellular organelles (mitochondria, golgi, endoplasmic
reticulum). The role of vitamin D in these processes was defined. The current proposal will extend knowledge acquired during the tenure of
the grant to explore the Na-phosphate transporter located at the brush
border membrane of the intestine of the rat. Specifically, the intestinal
Na-phosphate transporter will be expressed in Xenopus laevis oocytes by
injecting poly(A)+ RNA into Xenopus laevis oocytes. This will be followed
by size fractionation of poly(A)+ RNA to determine which size selected
fraction encoding for a functional Na-phosphate transporter by screening
for expression in Xenopus laevis oocytes. A cDNA library will be
constructed from appropriate size selected mRNA fragments. A 32P labelled
cDNA encoding for the transport protein will be synthesized and used to
determine the amount and size of homologous transcripts in RNA isolated
from intestinal and renal cortical homogenates by Northern blot analysis.
The restriction maps of the gene as well as its assignment of its locus to
the X-chromosome will be determined by analyzing genomic DNA from mice and
humans. The protein encoding the Na-phosphate transporter cDNA will be
incorporated into proteoliposomes and examined for the transport properties
of this protein in reconstituted proteoliposomes. This extensive knowledge will then be applied for the investigation of the
defect in Na-phosphate transport in the hypophosphatemic mouse which is a
murine homologue of human X-linked hypophosphatemic rickets. These studies
will first define the defect in phosphate transport in the intestine and
kidney and its regulation by vitamin D and its active metabolite and
second, will utilize our cDNA to measure and compare sequence homologies in
RNA isolated from the hypophosphatemic mouse (Hyp/y) and its genetically
age matched controls (+/y) by Northern blot analysis. The cloned probe
will also be used to study genomic DNA from the (Hyp/y) by digesting their
DNA with a variety of restriction endonucleases, performing Southern blots
and hybridizing the blotted DNA to cloned cDNA probes. This will permit
identification of gene deletions and genetic polymorphisms linked to the
genes probed. Sequencing the affected genes will delineate the genetic
defect in the hypophosphatemic mice. These studies will pioneer work on
the Na-phosphate transporter and will shed light on the defect in phosphate
transport in the (Hyp/y) mouse and eventually in man with hypophosphatemic
rickets.
StatusFinished
Effective start/end date12/1/832/28/18

Funding

  • National Institutes of Health: $340,875.00
  • National Institutes of Health: $314,489.00
  • National Institutes of Health: $226,791.00
  • National Institutes of Health: $314,489.00
  • National Institutes of Health: $356,025.00
  • National Institutes of Health
  • National Institutes of Health: $267,231.00
  • National Institutes of Health: $317,666.00
  • National Institutes of Health
  • National Institutes of Health: $347,659.00
  • National Institutes of Health: $209,681.00
  • National Institutes of Health: $340,875.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $356,025.00
  • National Institutes of Health: $275,247.00
  • National Institutes of Health: $340,875.00
  • National Institutes of Health: $337,577.00
  • National Institutes of Health
  • National Institutes of Health: $320,875.00
  • National Institutes of Health
  • National Institutes of Health: $194,827.00
  • National Institutes of Health: $340,875.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $191,100.00
  • National Institutes of Health: $605,250.00
  • National Institutes of Health: $356,025.00
  • National Institutes of Health: $289,448.00
  • National Institutes of Health
  • National Institutes of Health: $320,875.00
  • National Institutes of Health

ASJC

  • Medicine(all)

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