[A Case of Venous Malformation that Occurred in the Retroperitoneum].

A 70-year-old man complaining of pain in his right leg presented to the Department of Orthopedics in our hospital. X-ray findings revealed calcifications around the left kidney. He was referred to our department for further examination. Computed tomography revealed a tumor 3 cm in diameter with calcifications and an obscure border that was located on the caudal side of the pancreas, anterior to the left iliopsoas muscle and at the left side of the aorta. Magnetic resonance imaging showed that the tumor had comparatively low intensity in diffusion-weighted images and the cell density was not high. The contrast of the tumor by enhanced computed tomography was weak, and we had difficulty judging whether the tumor was benign or malignant. Each tumor marker, immunity factor, and hormone-like catecholamine were within the normal range. We considered the retroperitoneal tumor with calcifications as Castleman disease or tumor of nerve origin. It is believed that most retroperitoneal tumors are malignant. We performed laparoscopic surgery to resect the retroperitoneal tumor. Histopathological diagnosis was a primary retroperitoneal venous malformation. Vascular malformation derived from the retroperitoneum is rare. Furthermore, very few cases of venous malformation in the retroperitoneum have been reported.

Target gene knockdown by 2',4'-BNA/LNA antisense oligonucleotides in zebrafish.

Gene knockdowns using oligonucleotide-based approaches are useful for studying gene function in both in vitro cell culture systems and in vivo animal models. We evaluated the efficacy of 2',4'-bridged nucleic acids (BNA)-modified antisense oligonucleotides (AONs) for gene knockdown in zebrafish. We used the tcf7l1a gene as a model for testing the knockdown efficacy of 2',4'-BNA AONs and examined how the target sites/affinity and RNase H induction activity of 2',4'-BNA AONs affect knockdown efficacy. We found that tcf7l1a gene function was knocked down by 2',4'-BNA AONs that target the start codon and induce RNase H activity. Although nonspecific p53-mediated developmental defects were observed at higher doses, the effective dose of the 2',4'-BNA AONs for tcf7l1a is much lower than that of morpholino oligonucleotides. Our data thus show a potential application for 2',4'-BNA AONs in the downregulation of specific genes in zebrafish.

[Long-term survival of metastatic clear cell adenocarcinoma of the female urethra by multidisciplinary treatment: a case report].

We report a case of clear cell adenocarcinoma of the female urethra. A 57-year-old woman presented with complaint of gross hematuria. Abdominal ultrasonography, cystourethroscopy, computed tomography (CT) and magnetic resonance imaging (MRI) revealed the urethral tumor was invasive to bladder neck. Clinical stage was determined as cT3N1M0, then anterior pelvic exenteration and ileal conduit formation were performed. The pathological diagnosis was clear cell adenocarcinoma of urethra and the stage was pT3N1. The patient received TS-1 and cisplatin for postoperative recurrence, but she died from multiple lung metastasis 54 months after the operation. Clear cell adenocarcinoma of the female urethra is rare case in the Japanese literatures. Pathogenesis and management of this rare condition are discussed.

Synthesis and evaluation of novel caged DNA alkylating agents bearing 3,4-epoxypiperidine structure.

Previously, we reported that the 3,4-epoxypiperidine structure, whose design was based on the active site of DNA alkylating antitumor antibiotics, azinomycins A and B, possesses prominent DNA cleavage activity. In this report, novel caged DNA alkylating agents, which were designed to be activated by UV irradiation, were synthesized by the introduction of four photo-labile protecting groups to a 3,4-epoxypiperidine derivative. The DNA cleavage activity and cytotoxicity of the caged DNA alkylating agents were examined under UV irradiation. Four caged DNA alkylating agents showed various degrees of bioactivity depending on the photosensitivity of the protecting groups.

Characteristics of aggressive variants in T1a renal cell carcinoma.

PURPOSE: To explore factors associated with metastasis and prognosis in T1a renal cell carcinoma (RCC). METHODS: We retrospectively reviewed 451 cases of sporadic T1aRCC among 1,060 patients admitted to the Department of Urology at Hamamatsu University Hospital and affiliated hospitals between 1978 and 2007. Clinicopathological factors were analyzed for metastatic and prognostic risks. RESULTS: We identified 32 RCC patients with metastatic disease, 22 with synchronous and 10 with metachronous metastatic RCC. Patients with metastatic disease had a significantly higher incidence of symptomatic cancer, as well as greater tumor size, C-reactive protein (CRP) level, sarcomatoid component ratio, histological grade 3 and microvascular invasion than those without metastasis. Among the 32 patients with metastasis, there is no significant difference in clinicopathological factors. The most common site of metastasis was bone. Among patients with metastatic T1aRCC, findings at diagnosis of a symptomatic cancer, CRP level of 0.4 mg/dL or more, tumor size of 3.0 cm or greater, histological grade 3, a sarcomatoid component and microvascular invasion appeared to be significant and independent risk factors. Significant independent risk factors with metachronous metastatic RCC were a symptomatic cancer and a sarcomatoid component at diagnosis. A CRP level of 0.4 mg/dL or more was also an independent prognostic factor for overall survival. CONCLUSION: RCC patients with findings at diagnosis of a symptomatic cancer, a sarcomatoid component and CRP level of 0.4 mg/dL or more require intensive follow-up.

Arteriosclerosis related factors had no clinical significant correlation with resistive index in symptomatic benign prostatic hyperplasia.

OBJECTIVE: To investigate whether the resistive index (RI) in symptomatic benign prostatic hyperplasia (BPH) could be used as a surrogate index of the severity of lower urinary tract symptoms (LUTS) due to BPH, and whether arteriosclerosis-related factors were associated with the RI in LUTS due to BPH. METHODS: From January 2005 to April 2008, a total of 625 men with LUTS due to BPH were prospectively enrolled. Patients with heart failure, liver cirrhosis, prostatic cancer, neurogenic bladder, acute prostatitis, acute urinary retention, urethral stenosis, history of transurethral resection or any drug treatment for BPH, or currently under drug treatment for type 2 diabetes mellitus or dyslipidemia were excluded. Variables analyzed included estimated smoking status, blood pressure, body mass index (BMI), serum fasting glucose (FBS), lipid profile (low-density lipoprotein-cholesterol, high density lipoprotein-cholesterol and triglyceride), serum prostate-specific antigen, International Prostatic Symptom Score (IPSS), quality of life score, maximum urinary flow rate (Q(max.)), and postvoid residual urine volume (PVR). We also measured total prostate volume, transition zone (TZ) index, and RI using transrectal ultrasonography. Correlations among parameters were statistically examined. RESULTS: RI was significantly correlated with IPSS, Q(max.), and PVR, but not with blood pressure, BMI, or FBS. On multiple regression analysis, RI was a significant independent variable of IPSS, TZ index, and PVR. CONCLUSIONS: These findings suggest that RI might represent a surrogate index of the severity of LUTS due to BPH, and that RI might have no clinically significant relationship with arteriosclerosis-related factors.

Resistive index as risk factor for acute urinary retention in patients with benign prostatic hyperplasia.

OBJECTIVES: To examine the usefulness of several parameters obtained by transrectal ultrasonography in predicting acute urinary retention (AUR). METHODS: The present study consecutively enrolled 1962 men with a complaint of lower urinary tract symptoms. Of these men, 245 were found to have AUR on examination at our clinic. We assessed the International Prostate Symptom Score (IPSS), maximal urinary flow rate, and postvoid residual urine volume and measured the total prostate volume, transition zone index (TZI), and resistive index (RI) using transrectal ultrasonography. To compare the usefulness of these indexes for predicting AUR, we calculated the area under the receiver operating characteristic curve for each index and for age. RESULTS: In patients without AUR, age, prostate-specific antigen level, IPSS, maximal urinary flow rate, and postvoid residual urine volume were significantly correlated with both the TZI and the RI (P < .001). Multiple regression analysis demonstrated that age, maximal urinary flow rate, postvoid residual urine volume, and TZI were significant independent determinants of the RI (P < .001). Patients with AUR were, on average, older and had an elevated prostate-specific antigen level, increased IPSS, and greater TZI and RI than patients without AUR (P < .001). The area under the receiver operating characteristic curve was 0.640 (95% confidence interval [CI] 0.618-0.662) for age, 0.674 (95% CI 0.653-0.695) for prostate-specific antigen level, 0.787 (95% CI 0.768-0.805) for total prostate volume, 0.821 (95% CI 0.803-0.838) for IPSS, 0.860 (95% CI 0.844-0.875) for TZI, and 0.867 (95% CI 0.851-0.882) for RI. CONCLUSIONS: The RI and TZI obtained using transrectal ultrasonography correlated with the incidence of AUR and are useful predictors of AUR in patients with benign prostatic hyperplasia.

RNA interference with 2',4'-bridged nucleic acid analogues.

In this study, a number of 2',4'-BNA- and 2',4'-BNA(NC)-modified siRNAs were designed and synthesized. Their thermal stability, nuclease resistance and gene silencing properties against cultured mammalian cells were evaluated and compared with those of natural siRNAs. The 2',4'-BNA- and 2',4'-BNA(NC)-modified siRNAs (named siBNA and siBNA(NC), respectively) showed very high T(m) values, were remarkably stable in serum sample and showed promising RNAi properties equal to those exhibited by natural siRNAs. Thermally stable siBNAs composed of slightly modified sense and antisense strands were capable of suppressing gene expression equal to that of natural siRNA. A number of modifications on the sense strand by 2',4'-BNA or 2',4'-BNA(NC), either consecutively or separated by natural RNA nucleotides, is tolerable in RNAi machinery. Modifications at the Argonauate (Ago2) cleavage site of the sense strand (9-11th positions from the 5'-end of the sense strand) produced variable results depending on siRNA composition. Mostly, modification at the 10th position diminished siRNA activity. In moderately modified siRNAs, modification at the 11th position displayed usual RNAi activity, while modification at the 9th position showed variable results depending on siRNA composition.

Resistive index: a newly identified predictor of outcome of transurethral prostatectomy in patients with benign prostatic hyperplasia.

OBJECTIVE: To examine the usefulness of several preoperative parameters obtained through transrectal ultrasonography in predicting the outcome of transurethral resection of the prostate (TURP). METHODS: A total of 572 men aged 51-85 years scheduled to undergo TURP for benign prostatic hyperplasia were prospectively enrolled, and 560 were ultimately evaluated. We preoperatively assessed International Prostate Symptom Score (IPSS), quality of life (QOL) score, maximum urinary flow rate (Q(max)), and postvoid residual urine volume (PVR), and measured total prostate volume (TPV), transition zone (TZ) index, and resistive index (RI) using transrectal ultrasonography. To compare the usefulness of the latter 3 indices, we calculated the area under the receiver operating characteristic (ROC) curve for each index and for IPSS. RESULTS: IPSS (total, postmicturition symptoms, storage symptoms, voiding symptoms), QOL score, Q(max), and PVR were significantly improved after TURP. Significant differences between the effective and noneffective groups were observed with regard to age, IPSS (total, postmicturition symptoms, storage symptoms, voiding symptoms), QOL score, TPV, TZ index, RI, Q(max), and PVR. The area under the ROC curve was 0.663 for IPSS, 0.691 for TPV, 0.719 for the TZ index, and 0.845 for the RI. CONCLUSIONS: The RI is a useful predictor of an effective outcome after TURP in patients with benign prostatic hyperplasia and may be useful for determining suitability for surgical intervention.

Biologically active 2-5A analogs containing 3'-O,4'-C-propylene adenosine as potent RNase L agonists.

Novel 2-5A analogs composed of 3'-O, 4'-C-alkylene adenosine were synthesized as potent RNase L activators. When we examined their properties, including their RNase L activating ability and their stability to exonuclease, we found that these 2-5A analogs showed high RNase L activation and high resistance to enzymatic degradation without the modification of an essential adenosine at the second position.

Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides.

In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2',4'-bridged nucleotides and three types of 2',4'-bridged nucleoside-5'-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates containing 2'-O,4'-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min. Although the Vent(exo-) DNA polymerase also read through these modified templates, kinetic study indicates that the KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides. When either of the DNA polymerase was used, the presence of 2',4'-bridged nucleotides on a template strand substantially decreased the reaction rates of nucleotide incorporations. The modified templates containing sequences of seven successive 2',4'-bridged nucleotides could not be completely transcribed by any of the DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars. Successive incorporation of 2',4'-bridged nucleotides into extending strands using 2',4'-bridged nucleoside-5'-triphospates was much more difficult. These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification.

Inhibition of bcl-xL expression by antisense oligonucleotides containing various bridged nucleic acids (BNAs).

Recently, we described the antisense activity of 2',4'-BNA oligonucleotides in living cells.(1) Here, we examine the antisense effect of 2',4'-BNA, 2',4'-(COC) and 3'-amino-2',4'-BNA oligonucleotides targeting the bcl-xL gene. The results showed that while S-oligo had a slightly inhibiting effect on bcl-xL expression and natural DNA had no effect, an antisense oligonucleotide (AON) containing six 2',4'-(COC) nucleotides reduced the bcl-xL mRNA level to 64% of the untreated level. Interestingly, several types of AON-those containing nine 2',4'-BNA(COC) nucleotides, 2',4'-BNA and 3'-amino-2',4'-BNA- completely inhibited expression of the target gene. These data show that, compared with the corresponding natural DNA and S-oligo, BNA-based AONs are efficient inhibitors of bcl-xL expression.

Antisense activity of 2',4'-BNA targeted to PPAR gamma in THP-1 and HCT116 cells.

2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) is a nucleic acid analogue that has high affinity binding to its complementary RNA. Peroxisome proliferator-activated receptor (PPAR) y belongs to the nuclear hormone receptor superfamily and is a drug target in the treatment of type 2 diabetes. Here, we show the antisense effects of 2',4'-BNA oligonucleotides against PPARy in the human monocytic leukaemia cell line THP-1 and the human colorectal tumor cell line HCT116.

Antisense activity of 2',4'-BNA targeted to bcl-xL gene in HepG2 cell.

Introduction of the 2',4'-BNA monomer into oligonucleotides significantly enhanced binding affinity toward ssRNA and resistance to nuclease degradation. Here, we evaluated the antisense activity of 2',4'-BNA oligonucleotides against bcl-xL in HepG2 cells. Our data revealed that 2',4'-BNA antisense oligonucleotides remarkably inhibited the expression of bcl-xL in HepG2 cells compared to the natural DNA antisense oligonucleotide. The inhibitory effect of 2',4'-BNA antisense oligonucleotide was dose-dependent and highly sequence-specific.

Role of CCR1 and CCR5 in homing and growth of multiple myeloma and in the development of osteolytic lesions: a study in the 5TMM model.

Multiple myeloma (MM) is a plasma cell malignancy, characterized by the localization of the MM cells in the bone marrow (BM), where they proliferate and induce osteolysis. The MM cells first need to home or migrate to the BM to receive necessary survival signals. In this work, we studied the role of CCR1 and CCR5, two known chemokine receptors, in both chemotaxis and osteolysis in the experimental 5TMM mouse model. A CCR1-specific (BX471) and a CCR5-specific (TAK779) antagonist were used to identify the function of both receptors. We could detect by RT-PCR and flow cytometric analyses the expression of both CCR1 and CCR5 on the cells and their major ligand, macrophage inflammatory protein 1alpha (MIP1alpha) could be detected by ELISA. In vitro migration assays showed that MIP1alpha induced a 2-fold increase in migration of 5TMM cells, which could only be blocked by TAK779. In vivo homing kinetics showed a 30% inhibition in BM homing when 5TMM cells were pre-treated with TAK779. We found, in vitro, that both inhibitors were able to reduce osteoclastogenesis and osteoclastic resorption. In vivo end-term treatment of 5T2MM mice with BX471 resulted in a reduction of the osteolytic lesions by 40%; while TAK779 treatment led to a 20% decrease in lesions. Furthermore, assessment of the microvessel density demonstrated a role for both receptors in MM induced angiogenesis. These data demonstrate the differential role of CCR1 and CCR5 in MM chemotaxis and MM associated osteolysis and angiogenesis.

Inhibition of VEGF mRNA by 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides and their influence on off-target gene expressions.

We investigated 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides (AONs) for vascular endothelial growth factor (VEGF) in human lung carcinoma A549 cells. An ENA/DNA gapmer AON with RNase H-mediated activity was virtually stable in rat plasma and exhibited more than 90% inhibition of VEGF mRNA production. Moreover, 22 genes that are likely to bind to the AON were found in the GenBank database by BLAST and CLUSTAL W searches. Three of these genes were actually inhibited by the ENA AON. In shorter ENA AONs with fewer matched sequences of these genes, inhibitiory activities were decreased and off-target effects were improved. These results indicate that ENA AONs act in a sequence-specific manner and could be used as effective antisense drugs.

Effects of the 2',4'-BNA modification on the sequence specificity of molecular beacons.

Molecular beacons (MBs) are stem-loop hairpin oligonucleotide probes with an internally quenched fluorophore. These probes recognize their targets with higher specificity than conventional linear probes. To further enhance sequence-specificity of MBs, we have designed and synthesized MBs having 2',4'-BNA modification. Thermodynamic analysis revealed that the MBs with the 2',4'-BNA-modified stem region have high sequence-specificity. In addition, the 2',4'-BNA modification in the loop region was also found to be efficient for discrimination of one base mismatch.

2'-O,4'-C-ethylene-bridged nucleic acid (ENA) for effective antisense formation.

ENA antisense oligonucleotides for vascular endothelial growth factor (VEGF) mRNA were synthesized and evaluated in A549 lung cancer cells. It was found that the VEGF ENA-antisense inhibited not only the expression of VEGF, but also the expression of three genes, which were found in Genbank by BLAST and Clustal W search and considered likely to bind to the VEGF ENA-antisense. These results indicate that ENA-antisense oligonucleotides act in a sequence-specific manner, and could be used as effective antisense drugs.

Total synthesis of an antitumor antibiotic, Fostriecin (CI-920).

The total synthesis of an antitumor antibiotic, fostriecin (CI-920), via a highly convergent route is described. A characteristic feature of the present total synthesis is that the synthesis was achieved via a coupling procedure of three segments A, B, and C. The unsaturated lactone moiety of fostriecin, corresponding to segment A, was constructed from a known Horner-Emmons reagent, and the stereochemistry of the C-5 position was introduced by asymmetric reduction with (R)-BINAl-H. Segment B having a series of stereogenic centers was synthesized from (R)-malic acid and the stereogenic centers at the C-8 and C-9 positions were prepared by a combination of Wittig reaction and Sharpless asymmetric dihydroxylation reaction. The conjugated Z,Z,E-triene moiety of fostriecin, corresponding to segment C, was eventually constructed by Wittig reaction and Stille coupling reaction. The phosphate moiety, which is known to be essentially important for the antitumor activity, was introduced via two routes: (i) direct phosphorylation of the monohydroxyl derivative in which other hydroxyl groups are protected with silyl groups; (ii) cyclic phosphorylation and selective cleavage of the cyclic phosphate derivative. Although the former route is basically the same as those reported by other groups, the latter route is novel and more effective than the former one. The present total synthesis would serve as a versatile synthetic route to not only fostriecin, but also its various analogues including stereoisomers.