PET imaging of the dopamine transporter with 18F-FECNT: a polar radiometabolite confounds brain radioligand measurements.

18F-2beta-Carbomethoxy-3beta-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane (18F-FECNT), a PET radioligand for the dopamine transporter (DAT), generates a radiometabolite that enters the rat brain. The aims of this study were to characterize this radiometabolite and to determine whether a similar phenomenon occurs in human and nonhuman primate brains by examining the stability of the apparent distribution volume in DAT-rich (striatum) and DAT-poor (cerebellum) regions of the brain. METHODS: Two rats were infused with 18F-FECNT and sacrificed at 60 min. Extracts of brain and plasma were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometric (LC-MS) techniques. Two human participants and 3 rhesus monkeys were injected with 18F-FECNT and scanned kinetically, with serial arterial blood analysis. RESULTS: At 60 min after the injection of rats, 18F-FECNT accumulated to levels about 7 times higher in the striatum than in the cortex and cerebellum. The radiometabolite was distributed at equal concentrations in all brain regions. The LC-MS techniques identified N-dealkylated FECNT as a major metabolite in the rat brain, and reverse-phase HPLC detected an equivalent amount of radiometabolite eluting with the void volume. The radiometabolite likely was 18F-fluoroacetaldehyde, the product expected from the N-dealkylation of 18F-FECNT, or its oxidation product, 18F-fluoroacetic acid. The distribution volume in the cerebellum increased up to 1.7-fold in humans between 60 and 300 min after injection and 2.0 +/- 0.1-fold (mean +/- SD; n = 3) in nonhuman primates between 60 and 240 min after injection. CONCLUSION: An 18F-fluoroalkyl metabolite of 18F-FECNT originating in the periphery confounded the measurements of DAT in the rat brain with a reference tissue model. Its uniform distribution across brain regions suggests that it has negligible affinity for DAT (i.e., it is an inactive radiometabolite). Consistent with the rodent data, the apparent distribution volume in the cerebellum of both humans and nonhuman primates showed a continual increase at late times after injection, a result that may be attributed to entry of the radiometabolite into the brain. Thus, reference tissue modeling of 18F-FECNT will be prone to more errors than analysis with a measured arterial input function.     

Big mitogen-activated protein kinase 1 (BMK1)/extracellular signal regulated kinase 5 (ERK5) is involved in platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell migration

Big mitogen-activated protein kinase 1 (BMK1), also known as extracellular signal-regulated kinase 5 (ERK5), is a newly identified member of the mitogen-activated protein (MAP) kinase family. Recently, several studies have suggested that BMK1 plays an important role in the pathogenesis of cardiovascular disease. To clarify the pathophysiological significance of BMK1 in the process of vascular remodeling, we explored the molecular mechanisms of BMK1 activation in vascular smooth muscle cells (VSMCs). From the results of co-immunoprecipitation and immunoblotting analyses, it was found that platelet-derived growth factor (PDGF), a known potent mitogen, activated BMK1 and triggered the Gab1-SHP-2 interaction in rat aortic smooth muscle cells (RASMCs). The abrogation of SHP-2 phosphatase activity by transfection of the SHP-2-C/S mutant suppressed PDGF-stimulated BMK1 activation. Infection with an adenoviral vector expressing dominant-negative MEK5alpha, which can suppress PDGF-stimulated BMK1 activation to the control level, inhibited PDGF-induced RASMC migration. Moreover, we observed an increase of BMK1 activation in injured mouse femoral arteries. From these findings, it is suggested that BMK1 activation leads to VSMC migration induced by PDGF via Gab1-SHP-2 interaction, and that BMK1-mediated VSMC migration may play a role in the pathogenesis of vascular remodeling.     

The participation of adenosine receptors in the adenosine 5''-triphosphate-induced relaxation in the isolated rabbit corpus cavernosum penis

AIM: To investigate the participation of adenosine receptors in the adenosine 5'-triphosphate (ATP)-induced relaxation in the corpus cavernosum penis (CCP) of rabbits. METHODS: The ATP-induced relaxation was assessed on the noradrenaline precontracted CCP of rabbits in the presence and absence of 8-(3-chlorostyryl)caffeine (CSC); an adenosine A(2A) receptor antagonist; alloxazine and MRS1754; adenosine A(2B) receptor antagonists; and ARL67156, an inhibitor of ecto-nucleoside triphosphate diphosphohydrolases. RESULTS: Adenosine and ATP relaxed the noradrenaline precontracted CCP of rabbits in a concentration-dependent manner. The adenosine- and ATP-induced relaxations were suppressed by alloxazine and MRS1754, but not by 8-(3-chlorostyryl)caffeine. ARL67156 potentiated the ATP-induced relaxation but not the adenosine-induced one. MRS1754 suppressed the ATP-induced relaxation potentiated by ARL67156. CONCLUSIONS: The above results suggest that, in the CCP of rabbits, the adenosine receptor mediating adenosine-induced relaxation is of the A(2B) receptor and the ATP directly causes relaxation through the A(2B) receptor on the CCP.     

Up-regulation of urinary UPIII mRNA levels in vesicoureteral reflux patients: Potential application as a screening test for vesicoureteral reflux

OBJECTIVES: Vesicoureteral reflux (VUR) is the most common congenital urinary tract anomaly. This disease can pose a major threat to the kidneys as twenty percent of patients with endstage renal disease are reported to have VUR. Although genetic studies for uroplakin III (UPIII) have been reported recently, no study has focused on UPIII gene expression in VUR patients. We describe here the up-regulation of UPIII mRNA in exfoliated urinary cells from primary VUR patients. METHODS: A real-time RT-PCR for UPIII mRNA was performed on exfoliated urothelial cells from 18 primary VUR and 38 control samples. UPIII mRNA copies were calculated for each sample. The statistical differences were assessed by the Mann-Whitney U test. Receiver operator characteristic curves were constructed for analysis of the diagnostic values. RESULTS: UPIII mRNA was found to be up-regulated to a greater extent in VUR than in control exfoliated urinary cells (mean +/- SE: 497.0 +/- 178.5 copies vs. 69.0 +/- 10.0 copies, respectively, P < 0.001). In evaluating the measurement of urinary UPIII mRNA as a screening test for VUR, the sensitivity was 77.8% and the specificity was 76.3% by the best diagnostic cutoff point. CONCLUSIONS: This is the first report demonstrating up-regulation of UPIII in mRNA levels in VUR patients. We submit that the quantitative measurement of urinary UPIII mRNA has a potential of developing into the first non-invasive screening test for VUR.     

Granular cell tumors of the breast: A report of two cases

Granular cell tumors (GCT) are rare neoplasms, and only 173 cases of benign GCT of the breast have been documented. We report herein the cases of two patients with this tumor and discuss the methods of diagnosis and treatment. The first patient was a 60-year-old woman who presented with a firm ill-defined mass in her left breast. Mammography showed a dense shadow with spicula and skin thickness, and ultrasonography revealed a hypoechoic mass with an irregular border. Radical mastectomy was performed under the wrong preoperative diagnosis of breast cancer. The second patient was a 31-year-old woman who presented with an elastic-hard mass in her left breast. Mammography showed a well-demarcated dense mass, and ultrasonography revealed a well-defined hypoechoic mass with a large depth-width ratio. Fine-needle aspiration cytology (FNAC) showed a large number of histiocytic cells with abundant granular cytoplasm. An excisional biopsy was performed, and histological examination confirmed a diagnosis of GCT. GCT is benign, but often misdiagnosed as breast cancer both clinically and radiologically. Therefore, histological examination is essential for making a correct diagnosis, while FNAC is also useful. Local resection is still the treatment of choice, and surgeons should do their utmost to avoid performing needless radical mastectomy.     

Pressure overload-induced transient oxidative stress mediates perivascular inflammation and cardiac fibrosis through angiotensin II.

Oxidative stress is implicated in the pathogenesis of various cardiovascular diseases. We have shown that in Wistar rats with a suprarenal aortic constriction (AC), pressure overload-induced transient perivascular inflammation (monocyte chemoattractant protein-1 [MCP-1] induction and macrophage accumulation) in the early phase is the determinant of reactive myocardial fibrosis and resultant diastolic dysfunction in the late phase. Thus, we investigated the role of reactive oxygen species production in cardiac remodeling in AC rats. Superoxide production and the footprint of lipid peroxidation were assessed using dihydroethidium staining and immunohistostaining against 4-hydroxy-2-nonenal (4-HNE), respectively. In sham rats, dihydroethidium and 4-HNE signals were scarcely found in the heart. At day 3, AC rats showed dihydroethidium signals mainly in the intramyocardial arterial wall, whereas modest 4-HNE staining was observed diffusely in the myocardium. These signals declined to lower levels by day 14 despite sustained hypertension. Chronic administration of a subdepressor dose of an angiotensin II type 1 receptor blocker candesartan reduced the pressure overload-induced dihydroethidium and 4-HNE signals at day 3. Moreover, candesartan decreased MCP-1 induction and macrophage infiltration at day 3 and prevented myocardial fibrosis at day 14, without affecting left ventricle and myocyte hypertrophy. In conclusion, acute pressure overload induced self-limited superoxide production mainly in the vascular wall. The reactive oxygen species production would contribute to the perivascular inflammation and subsequent myocardial fibrosis. Angiotensin II was suggested to have a pressure-independent effect on the reactive oxygen species production.     

Sensitive Detection of p53 Gene Mutations in Esophageal Endoscopic Biopsy Specimens by Cell Sorting Combined with Polymerase Chain Reaction Single-strand Conformation Polymorphism Analysis

For the rapid and sensitive detection of p53 gene mutations in esophageal endoscopic biopsy specimens, we combined cell sorting with the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis. Mutations in exons 5–8 of the p53 gene were investigated by FCR-SSCF analysis using 10³ sorted nuclei obtained from each endoscopic biopsy specimen of 16 patients with esophageal cancer. DNAs extracted from their respective surgical specimens were investigated by a conventional method of PCR-SSCP analysis. Mutations in the biopsy specimens were detected in 6 of the 12 aneuploid tumors but in none of the 4 diploid tumors. After tumor cell enrichment by cell sorting, one mutation in exon 8 became apparent, which could not be detected from the surgical specimen by a conventional method of PCR-SSCP analysis. This method should improve the sensitivity of detecting p53 gene mutations, and provides additional information concerning the DNA ploidy pattern in the tumors.     

Reactivity of hydroxybenzoic acids in the hydrogen-isotope exchange reaction.

The hydrogen-isotope exchange reaction (T-for-H exchange reaction) between tritiated water vapor (HTO vapor) and 3-hydroxy-4-methoxybenzoic acid (and it's analog; 4-hydroxy-3-methoxybenzoic acid) were observed at 50 and 70 degrees C in a gas-solid system to reveal the reactivity of a functional group in an aromatic compound having two substituents in the aromatic ring. Further, it was shown that (a) the reactivity of the compounds used in this work follows the Hammett's rule, and (b) the reactivity of trisubstituted aromatic compound could be analyzed by applying the additive property of the Hammett's rule even if the compound contains a substituent at the ortho-position.