Deficiency in VHR/DUSP3, a suppressor of focal adhesion kinase, reveals its role in regulating cell adhesion and migration.

Vaccinia H1-related phosphatase (VHR/DUSP3) is a member of the dual-specificity phosphatase family. Deregulation of VHR is observed in various malignant diseases. We identified focal adhesion kinase (FAK) as a VHR-interacting molecule. Over-expression of VHR decreased tyrosine phosphorylation of FAK and decreasing VHR promoted FAK tyrosine phosphorylation. In vitro assays proved that recombinant VHR directly dephosphorylated FAK and paxillin. VHR-knockout mice did not have obvious abnormality; however, VHR-knockout cells showed decreased expression of integrins and FAK but stronger FAK and paxillin phosphorylation upon attachment to fibronectin. Additionally, VHR-knockout fibroblast and lung epithelial cells had elevated ligand-induced epidermal growth factor receptor (EGFR) phosphorylation. Inducible expression of VHR suppressed directional cell migration, and VHR deficiency resulted in a higher cell migratory ability. VHR-knockout cells have stronger FAK phosphorylation in cell adhesions, long-lasting trailing ends and slower turnover of focal adhesions. These collective data indicate that VHR is a FAK phosphatase and participates in regulating the formation and disassembly of focal adhesions.

Visualizing caveolin-1 and HDL in cholesterol-loaded aortic endothelial cells.

Caveolae are vesicular invaginations of the plasma membranes that regulate signal transduction and transcytosis, as well as cellular cholesterol homeostasis. Our previous studies indicated that the removal of cholesterol from aortic endothelial cells and smooth muscle cells in the presence of HDL is associated with plasmalemmal invaginations and plasmalemmal vesicles. The goal of the present study was to investigate the location and distribution of caveolin-1, the main structural protein component of caveolae, in cholesterol-loaded aortic endothelial cells after HDL incubation. Confocal microscopic analysis demonstrated that the caveolin-1 appeared to colocalize with HDL-fluorescein 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) conjugates on the cell surface. No free HDL-DiI conjugates were revealed in the cytoplasm. Immunoelectron microscopy further demonstrated that caveolin-1 gold (15 nm) conjugates colocalized with HDL gold (10 nm) conjugates in the plasmalemmal invaginations. These morphological results indicated that caveolae are the major membrane domains facilitating the transport of excess cholesterol to HDL on the cell surface of aortic endothelial cells.

Effects of high-cholesterol diet on the interendothelial clefts and the associated junctional complexes in rat aorta.

The arterial endothelial intercellular cleft (AEC) and its associated junctional complex (JC) are the determinants of permeability to macromolecules. This study analyzed frequencies of AEC and JC profile types in the rat thoracic aorta at 1 and 12 months after feeding the animals with a normal or a high-cholesterol diet. Rats on either a normal diet or high-cholesterol diet for 12 months showed more of the simple 'end to end' or 'overlap' types (P < 0.01) but fewer complex 'interdigitating' type (P < 0.01) of AEC compared to the 1 month group. With regard to JC, the frequencies of gap junctions were decreased (P < 0.01) while the tight junctions and the normal junctionless complex were increased (P < 0.01) after 12 months of normal diet as compared with 1 month on the normal diet. These changes in frequencies for gap junction and tight junction were even greater for the high-cholesterol diet than for the normal diet treatment. Moreover, the incidence of open junctions was also noticeably increased after 12 months of high-cholesterol diet. These findings suggest that the proportions of the AEC and JC were highly responsive to aging whereas those of JC were more susceptible to the high-cholesterol diet treatment.

Use of low intensity roentgen ray imaging for determination of tube and catheter placement in the young primate: implication for use of the Lixiscope in the surgical neonate.

The new technology of low intensity roentgen ray imaging offers promise in rapid bedside location of tube and catheter placement in the surgical neonate. Using the Lixiscope we have been able to accurately detect in an animal model the exact location of various tubes and catheters used routinely in pre and postoperative neonatal care. Minimal training is required to be able to use the device. We think the Lixiscope offers detection of the positions of standard tubes and catheters with increased speed, as well as a reduction in the radiation exposure for patients and staff in the neonatal unit.

Effect of cyclic AMP on collagen production by corneal fibroblasts.

Subcultured bovine corneal fibroblasts accumulated cyclic AMP intracellularly and in the culture media in response to incubation with 3-isobutyl-1-methylxanthine, isoproterenol, cholera toxin or forskolin. The duration of the intracellular cyclic AMP effect was rather short using isoproterenol (less than three hours) but cholera toxin and forskolin maintained cyclic AMP stimulations for 24 hours at greater than 3-fold and 27-fold over control, respectively. Agents which stimulated adenylate cyclase by different mechanisms or mimicked the action of cyclic AMP decreased collagen secretion by these cells. In general, the degree of inhibition of collagen production was consistent with the magnitude and duration of each drug's cyclic AMP response.

Adenosine regulation of cyclic AMP in corneal endothelium.

Adenosine receptor agonists increased cyclic AMP in cultures of bovine corneal endothelium up to 12-fold over control. Effects were dose-dependent between 1 microM and 0.5 mM adenosine. N6-methyladenosine produced a greater maximal effect but was approximately 10-fold less potent. Isoproterenol and N6-methyladenosine produced an additive response. Propranolol blocked the isoproterenol, but not the adenosine effect on cyclic AMP. Adenine-9-beta-D-arabinofuranoside had no effect on cyclic AMP alone, but inhibited stimulations by N6-methyladenosine, isoproterenol and forskolin. These data indicate the presence of specific adenosine receptors which stimulate adenylate cyclase in cultured bovine corneal endothelium.

Desensitization of beta-adrenergic receptors in corneal endothelium.

Corneal endothelial cells in primary culture responded to isoproterenol (Iso), epinephrine (Epi), nor-epinephrine (NE), Prostaglandins E1 and E2 (PGE1, PGE2) and forskolin by increasing cyclic AMP 61, 52, 28, 14, 10, and 176-fold, respectively, over control within a 10 minute incubation period. However, when cells were preincubated with Iso for one hour, they lost 76% of their responsiveness to a subsequent Iso challenge. Iso preincubation also decreased the effectiveness of other beta-adrenergic agonists (Epi and NE), but responses to PGE1, PGE2 and forskolin remained unchanged. The decreased cyclic AMP response was associated with a loss of beta-adrenergic binding sites. The affinity of the remaining receptors was unchanged as determined by propranolol displacement of [125I] pindolol binding. This study shows that corneal endothelial cells not only respond to beta-adrenergic stimuli, but can also regulate the magnitude and duration of their response to the hormone.

Characterization of beta-adrenergic receptors in fresh and primary cultured bovine corneal endothelium.

Homogenates of fresh bovine corneal endothelium and of cells from primary cultures exhibited high affinity binding of [3H]-dihydroalprenolol, a specific beta-adrenergic antagonist. The binding was rapid and reversible. Specific binding of the radioligand in each preparation was saturable with half-maximal binding occurring at 0.5 nM. Homogenates of fresh tissue consistently showed a higher maximal binding capacity than did those from cell cultures. Both homogenates bound adrenergic agents in a manner consistent with the labelling of beta-adrenergic receptors. The relative affinities of epinephrine and norepinephrine indicated that beta-adrenergic receptors in both preparations are of the beta 2 subtype. The beta-adrenergic agonist, isoproterenol, increased the cAMP content of intact, cultured endothelial cells 27-fold over control. This effect was completely blocked by the beta-adrenergic antagonist, propranolol. Bovine corneal endothelial cells in primary culture contain beta-adrenergic receptors which are essentially identical to those from in vivo sources and appear to be linked functionally to cAMP synthesis in these cells.

Characteristics of beta-adrenergic receptors in bovine corneal epithelium: comparison of fresh tissue and cultured cells.

The characteristics of the beta-adrenergic receptors in homogenates of fresh tissue and cultured bovine corneal epithelium were compared using [3H]dihydroalprenolol. High affinity, specific binding sites were observed in both preparations. Fresh tissue exhibited a higher binding site density (165 fmol/mg protein) than did cells in culture (57 fmol/mg protein). Studies with various beta-adrenergic agonists and antagonists indicated that binding characteristics were typical of beta-adrenergic receptors, predominantly of the beta 2 subtype. These results demonstrate that beta-adrenergic receptors exist in both fresh and cultured bovine corneal epithelium and that these receptors are qualitatively and quantitatively similar.

Trisomy 6q22 leads to 6qter due to maternal 6;21 translocation. Case report review of the literature.

Partial trisomy for the long arm of chromosome 6, involving 6q22 leads to 6qter, was observed in a 2-month-old male infant. The mother was 6q;21p translocation carrier. A review of the previously published cases with trisomies of different 6q segments suggests that the critical segment responsible for the clinically recognizable phenotype of 6q trisomy seems to be limited to bands 6q26 and/or 6q27.