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Biomedical subjects

D A O'Rourke

Publications and source records attributed to D A O'Rourke.

At least 19 recordsLinked to original sources
PubMed2000

Differential MAPK pathways utilized for HGF- and EGF-dependent renal epithelial morphogenesis.

Cells derived from the inner medullary collecting duct undergo in vitro branching tubulogenesis to both the c-met receptor ligand hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF) receptor ligands. In contrast, many other cultured renal epithelial cells respond in this manner only to HGF, suggesting that these two receptors may use independent signaling pathways during morphogenesis. We have therefore compared the signaling pathways for mIMCD-3 cell morphogenesis in response to EGF and HGF. Inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway with the mitogen-activated protein kinase kinase (MKK1) inhibitor PD98059 (50 microm) markedly inhibits HGF-induced cell migration with only partial inhibition of EGF-induced cell motility. Similarly, HGF-dependent, but not EGF-dependent, branching morphogenesis was more greatly inhibited by the MKK1 inhibitor. Examination of EGF-stimulated cells demonstrated that extracellular-regulated kinase 5 (ERK5) was activated in response to EGF but not HGF, and that activation of ERK5 was only 60% inhibited by 50 microm PD98059. In contrast, the MKK inhibitor U0126 markedly inhibited both ERK1/2 and ERK5 activation and completely prevented HGF- and EGF-dependent migration and branching process formation. Expression of dominant negative ERK5 (dnBMK1) likewise inhibited EGF-dependent branching process formation, but did not affect HGF-dependent branching process formation. Our results indicate that activation of the ERK1/ERK2 signaling pathway is critical for HGF-induced cell motility/morphogenesis in mIMCD-3 cells, whereas ERK5 appears to be required for EGF-dependent morphogenesis.

Cell Line, Transformed↗
PubMed1999

Expression of c-ret promotes morphogenesis and cell survival in mIMCD-3 cells.

c-Ret, a protein tyrosine kinase receptor, and its ligand glial-derived neurotropic factor (GDNF) are critical for early regulation of ureteric bud development and nephrogenesis. To address whether c-ret directly initiates epithelial cell morphogenesis, the c-ret receptor was expressed in murine inner medullary collecting duct cells (mIMCD-3, a cell line of ureteric bud origin, which has no detectable endogenous c-ret expression). Stable expression of wild-type c-ret was found to yield a constitutively tyrosine-phosphorylated receptor, with no change after the addition of GDNF. Examination of mRNA from these cells demonstrated the message for endogenous GDNF, suggesting that c-ret was potentially being constitutively activated by an autocrine mechanism. When mIMCD-3 cells stably expressing the phosphorylated c-ret receptor were cultured in a type I collagen matrix, they exhibited little GDNF-independent or -dependent branching process formation at early time points compared with the known morphogen hepatocyte growth factor (HGF) (48 h; control, 0.33 +/- 0.33; GDNF, 1.0 +/- 0.58, P = nonsignificant; and HGF, 6.33 +/- 0.33 processes/20 cell clusters, P < 0.001), whereas extended culture (7 days) under serum-free conditions revealed a marked increase in cell survival and the spontaneous development of rudimentary branching process formation. Extended culture (7 days) of c-ret-expressing clones in type I collagen with the epithelial morphogens HGF and/or epidermal growth factor (EGF) resulted in the development of complex three-dimensional spiny cysts, whereas parental mIMCD-3 cells died under these conditions. We conclude that activated c-ret appears to mediate epithelial morphogenesis by prolonging cell survival and, in conjunction with activation of the morphogenic receptors c-met and the EGF receptor, initiates the events required for very early branching morphogenesis.

Animals↗
PubMed1995

Cloning of the porcine D1A dopamine receptor gene expressed in renal epithelial LLC-PK1 cells.

The porcine renal epithelial cell line LLC-PK1 expresses a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. The molecular identity of this receptor is unknown. We isolated a partial cDNA from LLC-PK1 poly(A)+ RNA by the reverse transcription-polymerase chain reaction procedure with degenerate D1 receptor oligonucleotide primers and used the partial cDNA to screen a porcine genomic library. One such genomic clone (lambda PGD1A.1) contained an open-reading frame that encoded a 446-amino-acid protein that is 95% identical to the human D1A receptor. The functional properties of the genomic clone transiently transfected into COS-7 cells were consistent with expression of a D1 receptor. RNA hybridization analyses with LLC-PK1 poly(A)+ RNA were positive. Primer extension analysis indicated that the primary transcription initiation site of the porcine D1A gene expressed in LLC-PK1 cells is 1,033 nucleotides upstream from the translation start site. The 5'-flanking region of the gene lacks a TATA and CAAT box but is high in GC content (68%) and contains multiple Sp1 binding sites. There is a 97-bp intron within the 5'-noncoding region, separating exons 1 and 2. These results add support to the view that the D1A receptor is the major D1 receptor expressed in kidney and further suggest that LLC-PK1 cells may be a useful model for study of the regulation of the renal D1A receptor gene.

Amino Acid Sequence↗
PubMed1995

Cloning of the dopamine-1A (D1A) receptor gene expressed in porcine renal epithelial cells.

We sought to determine the molecular identify of the dopamine-1 (D1) receptor expressed in the porcine renal epithelial cell line LLC-PK1. We first isolated a partial cDNA by the reverse transcription-polymerase chain reaction procedure and then used the partial cDNA to isolate positive overlapping clones from a porcine genomic DNA library. Sequence analysis of the gene revealed that the longest open-reading frame encoded a 446 amino acid protein that was 95% identical to the human D1A receptor. Expression studies in mammalian cells were also consistent with the clones encoding a D1 receptor. Northern blot hybridizations with LLC-PK1 poly (A+) RNA were strongly positive. The porcine D1A gene has two exons and a short intron in the 5' untranslated region. The 5' flanking region lacks a TATA and CAAT box but is high in GC content (68%) and contains multiple Sp1 binding sites. The 5' flanking region also contains numerous other cis-acting elements for transcription factors. These results indicate that the D1A receptor is the major D1 receptor expressed in LLC-PK1 cells and further suggest that LLC-PK1 cells may be a useful model to study the regulation of renal D1A receptor gene transcription.

Amino Acid Sequence↗
PubMed1994

Aberrant snacking patterns and eating disorders in patients with obsessive compulsive disorder.

BACKGROUND: Appetitive symptoms, particularly carbohydrate craving, have been shown to occur in patients whose conditions responded to treatment with drugs that enhance serotonin-mediated neurotransmission. This suggested that patients with obsessive compulsive disorder (OCD) who also frequently respond to serotonergic drugs also might have similar distributions of appetitive and eating patterns. METHOD: A survey study of 170 OCD patients and 920 controls was conducted using a questionnaire that inquired about snacking behavior, including food preference, mood changes after eating, and previous diagnosis of eating disorders. The frequency responses in the two groups were tested for statistical significance. RESULTS: Significant differences were found between the OCD and control groups with respect to the reported incidence of eating disorders, snacking patterns, and mood response to food. CONCLUSION: This finding of different snacking patterns in OCD mirrors that found in other disorders that have been shown to be responsive to serotonergic drugs. The high incidence of carbohydrate snacking among OCD patients compared with the control group provides additional evidence that brain serotonin may be involved in this disorder.

Adult↗
PubMed1985

A puborectal sling in the management of anal incontinence and rectal prolapse.

A review is presented of 11 years experience in the use of a synthetic puborectal sling in the treatment of rectal prolapse and faecal incontinence. This perineal approach operation has been performed on 24 patients from 1973 to 1984. In over 60% of the patients rectal prolapse and anal incontinence were controlled.

Adult↗
PubMed1976

Three score and ten.

Excluding infant and child mortality, the average "natural" life span seems to be about 70 years. It has remained so for several thousand years. It shows little sign of change. Hard work and stress appear to increase rather than decrease longevity.

Adult↗