Screening of novel nuclear receptor agonists by a convenient reporter gene assay system using green fluorescent protein derivatives.

Nuclear receptors represent a very good family of protein targets for the prevention and treatment of diverse diseases. In this study, we screened natural compounds and their derivatives, and discovered ligands for the retinoic acid receptors (RARs) and the farnesoid X receptor (FXR). In the reporter assay systems of nuclear receptors presented here, two fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), were used for detection of a ligand-based induction and as an internal control, respectively. By optimizing the conditions (e.g., of hormone response elements and promoter genes for reporter plasmids), we established a battery of assay systems for ligands of RARs, retinoid X receptor (RXR) and FXR. The screening using the reporter assay system can be carried out without the addition of co-factors or substrates. As a result of screening of more than 140 compounds, several compounds were detected which activate RARs and/or FXR. Caffeic acid phenylethyl ester (CAPE), known as a component of propolis from honeybee hives, and other derivatives of caffeic acid up-regulated the expression of reporter gene for RARs. Grifolin and ginkgolic acids, which are non-steroidal skeleton compounds purified from mushroom or ginkgo leaves, up-regulated the expression of the reporter gene for FXR.

Initial experience of percutaneous renal cryosurgery under the guidance of a horizontal open MRI system.

OBJECTIVE: The aim of this study was to obtain preliminary results of cryoablation of renal tumors by using a percutaneous approach guided by a horizontal open MRI system, and to assess the safety and efficacy of this procedure. MATERIALS AND METHODS: Four patients with renal tumors underwent percutaneous cryosurgery with local anesthesia using a horizontal open MRI system (AIRIS II, Hitachi Medical Corp., Tokyo, Japan). The size of the mass was radiographically documented as 4 cm or less in diameter. A 2- or 3-mm cryoprobe was advanced into the renal mass under real-time MR monitoring. Growth of the iceball during cryoablation was monitored by two-dimensional MR images. Follow-up dynamic CT and physical examination were done after two weeks and six weeks. RESULT: MR imaging demonstrated the iceballs as sharply marginated regions of signal loss that expanded and engulfed the renal mass with clear contrast between the iceball and surrounding tissue. Cryoablated tumors resolved, and there were no serious complications and no clinically significant changes during the procedures and follow-up study. CONCLUSION: In this limited clinical trial of percutaneous renal tumor surgery, cryoablation demonstrated its feasibility with minimal morbidity. Intraprocedual MR-guided cryosurgery can be used as a safe modality, although further studies are necessary to determine the long-term efficacy of this procedure.

Correction of completely buried penis using the modified preputial island pedicle flap method.

The buried penis is a rare congenital abnormality caused by a deficiency of penile shaft skin and abnormal attachments of the dartos fascia to Buck's fascia. The basis for surgical correction is directed at freeing the penile shaft from abnormal dartos attachments, refixing dartos fascia to Buck's fascia to prevent retraction of the penis, and providing adequate shaft skin coverage with the inner preputial skin. The authors report a modified preputial island pedicle flap method for correcting the completely buried penis performed on 2 patients (ages 9 months and 1 year) at Nagoya City University Medical School.

Perfluorooctanoic acid, a peroxisome-proliferating hypolipidemic agent, dissociates apolipoprotein B48 from lipoprotein particles and decreases secretion of very low density lipoproteins by cultured rat hepatocytes.

The hypolipidemic effect is evoked by various peroxisome proliferators. Modulation of gene transcription via peroxisome proliferator-activated receptor (PPAR) is generally responsible for this effect. In addition, we have found a PPAR-independent mechanism in which fibrates, known peroxisome proliferators, decrease hepatic secretion of very low density lipoproteins (VLDL) through inhibition of phosphatidylcholine synthesis via methylation of phosphatidylethanolamine (PE) (T. Nishimaki-Mogami et al., Biochim. Biophys. Acta 1304 (1996) 21-31). In the present study, we show a novel mechanism by which perfluorooctanoic acid (PFOA), a potent peroxisome proliferator and inhibitor of PE methylation, exerts its hypolipidemic effect. PFOA (100 microM) added to the medium rapidly decreased the secretion of triglyceride by cultured rat hepatocytes, which was independent of the activity of cellular PE methylation. Analysis of the density of apoB secreted into the medium showed that PFOA decreased apoB48 in VLDL, but increased apoB48 in the bottom d>1.21 fraction. This lipid-poor apoB48 was also generated by incubating medium that had been harvested from control cells with PFOA, indicating that PFOA has the ability to dissociate apoB48 from lipoprotein particles. Exposure of cells to PFOA for 2 h prior to the experiment was sufficient to generate lipid-poor apoB48, indicating that PFOA exerted its effect intracellularly. Taken together, the data suggest that a strong interaction of PFOA with apoB48 disturbs the association of apoB48 with lipids in the process of intracellular VLDL assembly, thereby inhibiting VLDL secretion. This study shows that the mechanisms of hypolipidemic effect caused by various classes of peroxisome proliferators are diverse.

[A clinical study on virilization of external genitalia in congenital adrenal hyperplasia].

PURPOSE: In the newborn female with congenital adrenal hyperplasia (CAH), there are variable degrees of clitoral enlargement and virilization of the urethra. In order to investigate the factors that cause these variations, the degree of musculinization of the external genitalia in 44 patients with CAH was studied retrospectively. PATIENTS AND METHODS: This study includes a total of 44 patients with CAH aged 2 months to 12 years and 11 months old who had consulted Nagoya City University Hospital from 1977 to 1994. The length of clitoris on the first consultation was compared with the age and its change was followed. Also, the relationship among the length of clitoris on the first consultation, the degree of urethral virilization, the clinical forms i.e. salt-losing or simple-virilization, and the glucocorticoid hormonal condition were investigated with each other. In 5 pairs of sisters, the length of clitoris on the first consultation was compared within each pairs, and also the degree of urethral virilization was compared. RESULTS: The clitoral length on the first consultation was significantly correlated with the age (y = 1.8 x + 1.8, r = 0.7, p < 0.0001). The mean clitoral length at birth calculated from these data was 18 mm, which is as large as that of adult female one, and the clitoral enlargement rate was 1.8 mm/yr. The correlation was not clear between the clitoral length and the degree of urethral virilization, the clinical forms, and the glucocorticoid hormonal condition, either. On the other hand, the degree of urethral virilization was comparable within clitoral length was not comparable within them. CONCLUSIONS: The urethral virilization was suggested to be determined by the internal hormonal environment during embryonal stage which was shared by the each pair of sisters. On the Other hand, the clitoral length was depend on the patient's age though the factor which determined the length was not apparent. These facts indicated that, not only the severely musculinizing cases, but also the new born female with mildly virilizing urethra should be followed strictly not to undergo the clitoral enlargement.

49,XXXXY syndrome with hydronephrosis caused by intravesical ureterocele.

A 1-month-old boy was referred to our hospital with right hydronephrosis. Excretory urography showed poor visualization of the right kidney and a filling defect in the bladder. Chromosomal analysis of peripheral blood revealed a karyotype of 49,XXXXY, and a diagnosis of 49,XXXXY Klinefelter s syndrome associated with hydronephrosis caused by intravesical ureterocele was made. 49,XXXXY Klinefelter s syndrome with anomalies of the urinary tract is extremely rare, and only 2 cases have been reported so far.

[Operative methods for severe hypospadias].

BACKGROUND: Pediatric urologists tend to use one-stage procedures for the repair of hypospadias. As there are various types of hypospadias, we cannot repair this disease with a single modality. It is difficult to estimate the exact length of the neourethra in cases of severe hypospadias before surgery. METHODS: After a circumferential incision is made about the coronal sulcus and the chordee is completely released, the distance between the glans tip and the retreated native meatus is measured to determine the length of the neourethra. Urethroplasty with the method of Transverse Preputial Island Flap (TPIF) is selected when the distance ranges from 3 to 4 cm, while urethroplasty using with modified OUPF IV (Koyanagi) is selected in cases of more than 4 cm. RESULTS: We performed surgery on 14 hypospadiac patients with chordee between April 1996 and April 1997. Eight patients underwent urethroplasty using the TPIF method and 6 underwent urethroplasty with the method of the modified OUPF IV. With the TPIF methods, 7 to 8 patients underwent repair successfully and one experienced urethrocutaneous fistula, while 5 of 6 treated by the modified OUPF IV method has successful repairs and meatal stenosis occurred in one patient. CONCLUSION: Even if we encounter severe hypospadias, we can treat these patients with one stage repair alternatively. A relatively high success rate was obtained with both methods to repair severe hypospadias.

Malignant mesodermal mixed tumor of the ureter.

Malignant mesodermal mixed tumor comprised of epithelial and nonepithelial components is rarely encountered in the urological field. Only 6 cases of malignant mesodermal mixed tumor of the ureter have previously been reported in the literature. We recently encountered a 7th case of malignant mesodermal mixed tumor of the ureter and describe the case in detail. The etiology of malignant mesodermal mixed tumor is also discussed.

Results of closure of urethrocutaneous fistulas after hypospadias repair.

BACKGROUND: Urethrocutaneous fistulas are one of the major causes of morbidity after hypospadias repair. METHODS: During the last 2.5 years, 26 patients underwent repair of 41 urethrocutaneous fistulas. These fistulas were repaired by a 3-layered closure method, by using meticulous surgical techniques aided by optical magnification. In large fistulas, a dermal subcutaneous flap was created and brought over the surgically repaired urethral fistula. RESULTS: Twenty-four of the 26 patients with urethrocutaneous fistulas after hypospadias repair had fistula closure, with a 92% success rate. CONCLUSION: A high success rate was obtained with a multilayered closure using meticulous techniques to repair urethrocutaneous fistulas.

[Transurethral incision for ureterocele in children].

Transurethral incision (TUI) was performed as the initial treatment in 10 children with ureteroceles. Three patients had ureteroceles associated with a single ureter. TUI relieved hydronephrosis and preserved renal function in all 3 cases. Urinary tract infection developed in no patients. However, all the patients required an antireflux operation because of postoperative vesicoureteral reflux (VUR). Seven children had a total of 8 ureteroceles associated with a duplex system. TUI resulted in preservation of the upper pole function in 6 of the 8 ureteroceles. Urinary tract infections and VUR developed in 3 and 7 patients, respectively. Common sheath reimplantation was performed in 2 ureteroceles. TUI relieves obstruction before the onset of devastating infections although it carries the risk of postoperative VUR. We recommend TUI as the initial treatment for ureteroceles associated with both single and duplex systems.

[Usefulness of 99mTc-MIBI SPECT in the metastatic lesions of thyroid cancer].

99mTc-MIBI is widely used as a tumor-seeking agent for parathyroid tumor, lung cancer, etc. We tried to detect the metastatic lesions of thyroid cancer by 99mTc-MIBI SPECT and evaluated 131I-uptake in the region of 99mTc-MIBI accumulation. Twenty-seven cases of thyroid cancer (23 papillary adenocarcinoma, 3 follicular adenocarcinoma, 1 unknown) were examined by 99mTc-MIBI. All cases were confirmed by surgery. Thyroglobulin was measured in all cases before 99mTc-MIBI SPECT. 131I-therapy (4.5-5.5 GBq) was performed on 24 patients and whole body scintigram was taken 7-10 days after. Thirty minutes after an injection of 99mTc-MIBI (740 MBq), SPECT with a three-head gamma camera was performed. Abnormal accumulation of 99mTc-MIBI was noted in 14 patients, all cases detected on CT or MRI. In 11 of the 13 cases in which accumulation was not visualized, no metastasis was detected. Most cases of abnormal accumulation on 99mTc-MIBI showed a high level of thyroglobulin. Metastatic lesions of follicular adenocarcinoma in three patients (right humerus, axillary region and sternum) showed strong accumulation of 99mTc-MIBI, and 131I was strongly taken up in these lesions. 131I was also taken up in the metastatic lesions of papillary adenocarcinoma with marked accumulation of 99mTc-MIBI. 99mTc-MIBI did not accumulate in a case with diffuse pulmonary uptake of 131I. 99mTc-MIBI SPECT could detect occult metastatic lesions in the soft tissue more clearly than CT or MRI. In conclusion, 99mTc-MIBI study might be useful as a follow-up study of patients after surgery for thyroid cancer.

Bezafibrate and clofibric acid are novel inhibitors of phosphatidylcholine synthesis via the methylation of phosphatidylethanolamine.

The effects of bezafibrate and clofibric acid, fibrate hypolipidemic agents, on phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway were studied. In cultured rat hepatocytes, bezafibrate and clofibric acid added to the medium rapidly and markedly reduced the conversion of ethanolamine-labeled PE to PC (IC50 30 and 150 microM, respectively). Furthermore, the methylation of PE derived from serine was also blocked by bezafibrate, as was the secretion of PC derived from either serine or ethanolamine. The microsomal activity of PE N-methyltransferase was inhibited by these agents. Perfluorooctanoic acid but not DCQVA, though both are potent peroxisome proliferators comparable to fibrates, produced this inhibition. The inhibitory effects produced by these agents were diminished by dithiothreitol (DTT) added to the assay or alkaline pH assay condition. Inhibition by oleic acid was also attenuated under these conditions, suggesting a common mechanism of inhibition. However, unlike fatty acids, fibrates did not have rapid stimulatory effects on the CDP-choline pathway in hepatocytes. These results suggest that fibrates may mimic fatty acids in regulating PC synthesis from the PE methylation pathway but not the CDP-choline pathway.

The role of phosphatidylethanolamine methylation in the secretion of very low density lipoproteins by cultured rat hepatocytes: rapid inhibition of phosphatidylethanolamine methylation by bezafibrate increases the density of apolipoprotein B48-containing lipoproteins.

The role of phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway in the secretion of very low density lipoproteins (VLDL) by cultured rat hepatocytes has been investigated by determining the effects of inhibitors. We have shown that bezafibrate and clofibric acid, known hypolipidemic agents, are potent inhibitors of PE methylation (see accompanying paper by Nishimaki-Mogami et al. (1996) Biochim. Biophys. Acta 1304). In hepatocytes incubated with ethanolamine, which maintained cellular PE levels and PE methylation activity, bezafibrate (200 microM) decreased the secretion of triacylglycerol (TG), PC, apolipoproteins B48, and E in VLDL by 50-75%. In contrast, bezafibrate at this concentration had marginal effect on VLDL secretion (83-115% of control) by hepatocytes that had been cultured in the absence of ethanolamine. In these cells PE levels and PE methylation activity had decreased by approx. 40%. VLDL secretion was decreased at concentrations similar to those required to inhibit PE methylation, and was accompanied by an increase in cellular TG levels. The same ethanolamine-dependent effects were produced by clofibric acid and also by 3-deazaadenosine (DZA), an inhibitor of cellular methylation reactions. These results indicate that PC synthesis via the PE methylation pathway plays a significant role in VLDL secretion in rat hepatocytes if the pathway is maintained at levels comparable to those in vivo. The reductions of PE methylation by bezafibrate and DZA did not affect the total amount of apolipoprotein B48 secreted into the medium. The decrease in apolipoprotein B48 in VLDL caused by bezafibrate was accompanied by an increase in apolipoprotein B48 in the HDL density range. In contrast, the amount of apolipoprotein B100 in VLDL and density determined by sequential flotation were unaffected. These findings indicate that rapid reduction of PC synthesis via PE methylation does not affect the secretion of apolipoprotein B48- or B100-containing lipoprotein particles, but does impair the lipidation of particles containing apolipoprotein B48, but not apolipoprotein B100.

Transinguinal laparoscopy for nonpalpable testis.

BACKGROUND: About 20% of all undescended testes are nonpalpable. Although surgical exploration was previously the method of choice for management, laparoscopy from the inferior edge of the umbilicus has been established as a useful method of examining a testis that is nonpalpable. However, conventional subumbilical laparoscopy is unnecessary when the testis or its remnant is located below the internal inguinal ring. We evaluated the efficacy of transinguinal laparoscopy after inguinal exploration performed for nonpalpable testis. METHODS: Twelve children with 14 nonpalpable testes (2 children had bilateral nonpalpable testes) were considered for a novel method of laparoscopy. We performed laparoscopy through the internal inguinal ring after opening the inguinal canal. RESULTS: Inguinal exploration revealed the testis or its remnant in 5 patients, and thus prevented unnecessary laparoscopy in these individuals. Transinguinal laparoscopy identified the anatomy in 7 patients. Four of the latter were found to have blind-ending vas deferens and spermatic vessels, while in 3 patients, the testes were located in the peritoneal cavity. CONCLUSION: Transinguinal laparoscopy may become the accepted method for managing nonpalpable testes, as inguinal exploration can eliminate the need for laparoscopy in some cases, and the visualization achieved with transinguinal laparoscopy is just as good as that with conventional laparoscopy.

[One-stage urethroplasty with the modified OUPF IV for proximal hypospadias].

BACKGROUND: We previously performed one-stage urethroplasy with OUPF IV in 17 patients with proximal hypospadias and experienced meatal stenosis in 3 and meatal regression in 2. METHODS: Because these complications seemed to be caused by diminished vascularity, we modified the technique to preserve the vascularity to the peripheral site of the neourethra. The modified OUPF IV (Koyanagi) was used to treat 6 patients with proximal hypospadias during the last 3 months. RESULTS: Urethrocutaneous fistulas developed in 2 patients but meatal stenosis and meatal regression did not occur. CONCLUSION: We think this modification is suitable for severe proximal hypospadias.

Detection of sex-determining region in a male with chromosomal abnormality (46,X+mar) and hypospadias.

A 1-year-old boy was referred to us because of abnormal external genitalia; that is, his penis was kinked toward the perineal region. We diagnosed hypospadias. Chromosomal examination was performed on a sample of peripheral blood, the karyotype was 46,X+mar. Detailed karyotypic analysis revealed that the short arm of the marker chromosome originated on the ninth chromosome, but we could not determine whether the long arm was part of the Y chromosome, even with the use of quinacrine mustard. The sex-determining region of the Y was detected using the polymerase chain reaction method, which suggested that the long arm of the marker chromosome was delivered from the short arm of the Y chromosome.

[Experience with one stage repair of hypospadias and chordee without hyposradias using free graft of prepuce].

We treated 14 patients with moderately severe hypospadias and chordee without hypospadias using a free graft of prepuce by a modified Devine-Horton technique, between September 1993 and April 1995. There were 12 primary cases and 2 secondary cases in which prior operations had already been done. Four (33%) of the primary cases required a second procedure; 3 for urethrocutaneous fistula and 1 for urethral shrinkage. Both of the secondary cases needed further procedures; 1 for meatal stenosis and 1 for urethral stricture and diverticula. Although the need for reoperation is unfortunately high, six of the 7 recently treated patients experienced no complications. Thirteen of the 14 patients (93%) achieved excellent functional and cosmetic results with 1 or 2 procedures. We believe the use of free grafts allows a better functional and cosmetic outcome because the secondary torsion and bulkiness of the penile shaft caused by a vascular pedicle are eliminated.

[A case of adult pure yolk sac tumor of the testis achieving pathological complete response by chemotherapy].

We report a case of pure yolk sac tumor of the left testis in a 22-year-old male. He consulted a physician with left back pain and induration of his left scrotal content in December, 1992. Intravenous pyelography (IVP) revealed left hydronephrosis. Computerized tomography (CT) revealed para-aortic lymph node swelling and lung metastases. Left high inguinal orchiectomy was performed. Histopathological diagnosis was pure yolk sac tumor. After two courses of "COMPE" chemotherapy consisting of cisplatin, vincristine, methotrexate, peplomycin and etoposide, two courses of "high dose "COMPE" chemotherapy and three courses of "high dose COME" chemotherapy without peplomycin, he achieved a partial response (the regression rate of the pulmonary metastases and the retroperitoneal lymph node metastasis were 100% and 96.0% on CT, respectively) and the residual masses in the retroperitoneum were removed. Necrosis and xanthogranulomatous fibrosis were found in the resected material. The patient showed no evidence of disease two years after chemotherapy.

Activation induces dephosphorylation of cofilin and its translocation to plasma membranes in neutrophil-like differentiated HL-60 cells.

We suggested that a cytosolic 21-kDa phosphoprotein played an important role in opsonized zymosan-trigered activation of superoxide-generating enzyme in neutrophil-like HL-60 cells through dephosphorylation (Suzuki, K., Yamaguchi, T., Oshizawa, T., Yamamoto, Y., Nishimaki-Mogami, T., Hayakawa, T., and Takahashi, A (1995) Biochim. Biophys. Acta 1266, 261-267). In the present study, we characterized the phosphoprotein and studied changes in it localization upon activation of phagocytes. The 21-kDa phosphoprotein was rapidly dephosphorylated upon activation not only wit opsonized zymosan but also with formyl-Met-Leu-Phe and arachidonic acid. The peptide fragments derived from the 21-kDa phosphoprotein were found to have the same amino acid sequences as those of cofilin, an actin-binding protein. The phosphoprotein reacted exclusively with anti-cofilin antibody on two dimensional immunoblots. Accordingly, together with its apparent molecular weight, isoelectric point, and detection of phosphoserine as a phosphoamino acid, we concluded that the 21-kDa phosphoprotein was a phosphorylated form of cofilin. The amount of cofilin in membranous fractions was increased upon activation. Furthermore, confocal laser scanning microscopy showed that cofilin existed diffusely in the cytosol and nuclear region of the resting cells, while in the activated cells, it was accumulated at the plasma membrane area, forming ruffles or endocytic vesicles on which O2.- should be produced. These results suggested that in resting cells cofilin exists as a soluble phosphoprotein in the cytosol and nuclei, while upon stimulation a large portion of cofilin is dephosphorylated and translocated to the plasma membrane regions.