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Biomedical subjects

G E Parker

Publications and source records attributed to G E Parker.

11 recordsLinked to original sources
PubMed2001

Role of the LIM domains in DNA recognition by the Lhx3 neuroendocrine transcription factor.

LIM homeodomain transcription factors regulate many aspects of development in multicellular organisms. Such factors contain two LIM domains in their amino terminus and a DNA-binding homeodomain. To better understand the mechanism of gene regulation by these proteins, we studied the role of the LIM domains in DNA interaction by Lhx3, a protein that is essential for pituitary development and motor neuron specification in mammals. By site selection, we demonstrate that Lhx3 binds at high affinity to an AT-rich consensus DNA sequence that is similar to sequences located within the promoters of some pituitary hormone genes. The LIM domains reduce the affinity of DNA binding by Lhx3, but do not affect the specificity. Lhx3 preferentially binds to the consensus site as a monomer with minor groove contacts. The Lhx3 binding consensus site confers Lhx3-dependent transcriptional activation to heterologous promoters. Further, DNA molecules containing the consensus Lhx3 binding site are bent to similar angles in complexes containing either wild type Lhx3 or Lhx3 lacking LIM domains. These data are consistent with Lhx3 having the properties of an architectural transcription factor. We also propose that there are distinct classes of LIM homeodomain transcription factors in which the LIM domains play different roles in modulating interactions with DNA sites in target genes.

AT Rich Sequence↗
PubMed2001

LHX3 transcription factor mutations associated with combined pituitary hormone deficiency impair the activation of pituitary target genes.

The Lhx3 LIM homeodomain transcription factor is critical for pituitary gland formation and specification of the anterior pituitary hormone-secreting cell types. Two mutations in LHX3, a missense mutation changing a tyrosine to a cysteine and an intragenic deletion that results in a truncated protein lacking the DNA-binding homeodomain, have been identified in humans. These mutations were identified in patients with retarded growth and combined pituitary hormone deficiency and also abnormal neck and cervical spine development. For both the LHX3a and LHX3b isoforms, we compared the ability of wild type and mutant LHX3 proteins to trans-activate pituitary genes, bind DNA recognition elements, and interact with partner proteins. The tyrosine missense mutation inhibits the ability of LHX3 to induce transcription from selected target genes but does not prevent DNA binding and interaction with partner proteins such as NLI and Pit-1. Mutant LHX3 proteins lacking a homeodomain do not bind DNA and do not induce transcription from pituitary genes. These studies demonstrate that mutations in the LHX3 isoforms impair their gene regulatory functions and support the hypothesis that defects in the LHX3 gene cause complex pituitary disease in humans.

Animals↗
PubMed2001

Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits.

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged alpha- or beta-subunits of the epithelial Na(+) channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominantly subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na(+) transport. The basal level of Na(+) transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na(+) transport was 64% greater than control in both the alpha- and beta-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na(+) transport.

Animals↗
PubMed2000

NP/NMP4 transcription factors have distinct osteoblast nuclear matrix subdomains.

The mechanisms underlying the coupling of type I collagen and matrix metalloproteinase (MMP) expression to cell structure and adhesion are poorly understood. We propose that nuclear matrix architectural transcription factors link cell structure and transcription via their association with nuclear matrix subdomains and by their capacity for altering promoter geometry. NP/NMP4 are nuclear matrix proteins that contain from five to eight Cys(2)His(2) zinc fingers. Some NP/NMP4 isoforms bind to the rat type I collagen alpha1(I) polypeptide chain promoter in the manner of architectural transcription factors and alter basal transcription in osteoblast-like cells (Thunyakitpisal et al. in review). Certain isoforms of NP/NMP4 are identical to CIZ, Cas-interacting zinc finger protein, a nucleocytoplasmic shuttling protein that associates with focal adhesions and regulates MMP expression [Nakamoto et al. (2000): Mol Cell Biol 20:1649-1658]. To better understand the role of subnuclear architecture in collagen and MMP expression, we mapped the osteoblast nuclear distribution of NP/NMP4 proteins and identified the functional motifs necessary for nuclear localization and nuclear matrix targeting. Immunofluorescence microscopy was used to determine the cellular and subnuclear distribution of native NP/NMP4 proteins and green fluorescent protein (GFP)-NP/NMP4 fusion proteins in osteoblast-like cells. All GFP-NP/NMP4 fusion proteins localized to the nucleus, but accumulated in distinct nuclear matrix subdomains. The zinc finger domain was necessary and sufficient for nuclear import and matrix targeting. We conclude that the arrangement of the NP/NMP4 zinc fingers largely determines the subnuclear location of these isoforms.

Amino Acid Motifs↗
PubMed2000

The homeodomain coordinates nuclear entry of the Lhx3 neuroendocrine transcription factor and association with the nuclear matrix.

LIM homeodomain transcription factors regulate development in complex organisms. To characterize the molecular signals required for the nuclear localization of these proteins, we examined the Lhx3 factor. Lhx3 is essential for pituitary organogenesis and motor neuron specification. By using functional fluorescent derivatives, we demonstrate that Lhx3 is found in both the nucleoplasm and nuclear matrix. Three nuclear localization signals were mapped within the homeodomain, and one was located in the carboxyl terminus. The homeodomain also serves as the nuclear matrix targeting sequence. No individual signal is alone required for nuclear localization of Lhx3; the signals work in combinatorial fashion. Specific combinations of these signals transferred nuclear localization to cytoplasmic proteins. Mutation of nuclear localization signals within the homeodomain inhibited Lhx3 transcriptional function. By contrast, mutation of the carboxyl-terminal signal activated Lhx3, indicating that this region is critical to transcriptional activity and may be a target of regulatory pathways. The pattern of conservation of the nuclear localization and nuclear matrix targeting signals suggests that the LIM homeodomain factors use similar mechanisms for subcellular localization. Furthermore, upon nuclear entry, association of Lhx3 with the nuclear matrix may contribute to LIM homeodomain factor interaction with other classes of transcription factors.

Amino Acid Sequence↗
PubMed1999

Characterization of the porcine Lhx3/LIM-3/P-Lim LIM homeodomain transcription factor.

Lhx3/LIM-3/P-Lim is a LIM homeodomain transcription factor which is essential in mice for the development of anterior and intermediate lobes of the pituitary gland. We report the cloning and characterization of porcine Lhx3. The porcine Lhx3 protein exhibits strong similarity to murine Lhx3 within the amino terminal LIM domains and the homeodomain, however, it is diverged in regions outside these motifs. Expression vectors for porcine Lhx3 activated murine and porcine alpha-glycoprotein reporter genes in transfection assays, and recombinant porcine Lhx3 protein specifically bound to a target site within the porcine alpha-glycoprotein gene upstream sequence. In addition, porcine Lhx3 synergistically induced transcription from prolactin enhancer/promoter reporter genes in cooperation with the Pit-1 pituitary transcription factor. Porcine Lhx3 protein interacted with Pit-1 protein in solution and also with the LIM domain-binding protein NLI/Lbd1/CLIM. Together, these data indicate that many aspects of Lhx3 function in the mammalian pituitary are conserved and that Lhx3 may be involved in the activation of trophic hormone genes during early and late stages of pituitary organogenesis. Divergence in the Lhx3 amino acid sequence between mammalian species may suggest distinct activities for this protein in some species and may help identify important functional domains of this key developmental transcription factor.

Amino Acid Sequence↗
PubMed1999

Differential activation of pituitary hormone genes by human Lhx3 isoforms with distinct DNA binding properties.

Lhx3 is a LIM homeodomain transcription factor essential for pituitary development and motor neuron specification in mice. We identified two isoforms of human Lhx3, hLhx3a and hLhx3b, which differ in their ability to trans-activate pituitary gene targets. These factors are identical within the LIM domains and the homeodomain, but differ in their amino-terminal sequences preceding the LIM motifs. Both isoforms are localized to the nucleus and are expressed in the adult human pituitary, but gene activation studies demonstrate characteristic functional differences. Human Lhx3a trans-activated the alpha-glycoprotein subunit promoter and a reporter construct containing a high-affinity Lhx3 binding site more effectively than the hLhx3b isoform. In addition, hLhx3a synergized with the pituitary POU domain factor, Pit-1, to strongly induce transcription of the TSHbeta-subunit gene, while hLhx3b did not. We demonstrate that the differences in gene activation properties between hLhx3a and hLhx3b correlate with their DNA binding to sites within these genes. The short hLhx3b-specific amino-terminal domain inhibits DNA binding and gene activation functions of the molecule. These data suggest that isoforms of Lhx3 may play distinct roles during development of the mammalian pituitary gland and other neuroendocrine systems.

Amino Acid Sequence↗
PubMed1997

A human resource planning model for hospital/medical technologists: an analytic hierarchy process approach.

This paper deals with the development of a human resource planning (HRP) model for hospital laboratory personnel. External and organizational factors impacting the demand and supply of clinical laboratory personnel in an urban academic health center are identified by a Delphi process. These factors are structured into a hierarchy for the application of the Analytic Hierarchy Process (AHP). Computer software, Expert Choice, was used in model hierarchy development and judgmental elicitation process. The model result can be applicable to eliciting the perceptions, insights, and understanding of health-care decision-makers for strategic human resource planning.

Academic Medical Centers↗
PubMed1992

Technetium-99m-medronate uptake in hepatic necrosis associated with Budd-Chiari syndrome.

A 99mTc-MDP bone scan performed on a 52-yr-old female for possible bone metastasis revealed prominent hepatic uptake. Subsequently, a 99mTc-SC scan revealed tracer uptake in the caudate lobe with diminished uptake in the remainder of the liver. Further imaging with Doppler ultrasound and hepatic venography confirmed a diagnosis of Budd-Chiari syndrome. Hepatic necrosis, demonstrated on CT imaging, was secondary to Budd-Chiari syndrome and was felt to be the cause of 99mTc-MDP hepatic uptake in this patient.

Budd-Chiari Syndrome↗