PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways.

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg(-/-) mouse ES cells and human cancer cell lines. Parg(-/-) mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg(-/-) mouse ES cells. Parg(-/-) ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.

Renal transplantation in patients with human T-cell lymphotropic virus type 1.

BACKGROUND: Renal transplantation (RTx) in carriers of human T-cell lymphotropic virus type 1 (HTLV-1) has a risk of developing overt leukemia upon immunosuppression. Although there have been a few reports of such cases, it is unclear HTLV-1 carrier if patients on the modern immunosuppressants would develop HTLV-1-associated myelopathy or adult T-cell leukemia lymphoma. METHODS: We retrospectively reviewed the clinical outcomes of RTx in nine HTLV-1 carriers to assess a risk of developing leukemia from 2002 to 2011 using immunosuppression with a calcineurin inhibitor, mycophenolate mofetil (MMF), and steroid. The anti-CD25 monoclonal antibody basiliximab was used for induction. In two cases of ABO-incompatible RTx, the rituximab was also administered before RTx. RESULTS: The ratio of male to female subjects was 2 to 7 with an overall mean recipient age of 54.3 ± 8.1 years. We prescribed cyclosporine (n = 5) or tacrolimus (n = 4). There was only one graft loss due to the death caused by aspiration pneumonia with a functioning graft. No one developed overt leukemia with combined treatment with MMF, basiliximab and rituximab. CONCLUSION: We concluded that RTx in HTLV-1 carriers could be performed using a modern immunosuppressive regimen, without the risk of developing leukemia.

Poly(ADP-ribose) Glycohydrolase deficiency sensitizes mouse ES cells to DNA damaging agents.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme for degradation of poly(ADP-ribose) by splitting ribose-ribose bonds. Parg-deficient (Parg(+/-) and Parg(-/-)) mouse ES cell lines have been established by disrupting both alleles of Parg exon 1 through gene-targeting. A transcript encoding a full length isoform of Parg was eliminated and only low amounts of Parg isoforms were detected in Parg(-/-) embryonic stem (ES) cells. Poly(ADP-ribose) degradation activity was decreased to one-tenth of that in Parg(+/+) ES cells. Parg(-/-) ES cells exhibited the same growth rate as Parg(+/+) ES cells in culture. Sensitivity of Parg(-/-) ES cells to various DNA damaging agents, including an alkylating agent dimethyl sulfate, cisplatin, gemcitabine, 5-fluorouracil, camptothecin, and gamma-irradiation was examined by clonogenic survival assay. Parg(-/-) ES cells showed enhanced lethality after treatment with dimethyl sulfate, cisplatin and gamma-irradiation compared with wild-type (Parg(+/+)) ES cells (p<0.05, respectively). In contrast, a sensitization effect by Parg-deficiency was not observed with gemcitabine and camptothecin. These results suggest the possibility that functional inhibition of Parg leads to sensitization of tumor cells to some chemo- and radiation therapies.

The rotational effect of pronation and supination osteotomies of the forearm in a cadaver model: a comparison of osteotomy sites on the radius and the ulna.

The purpose of this study was to investigate the effect on forearm rotation of rotation osteotomies at the distal and proximal levels of the radius and the ulna. Rotation osteotomies of 15 degrees and 30 degrees were made in the distal and proximal one-third of the radius and the ulna in ten cadaver specimens and changes of forearm arc of rotation were compared after osteotomy at the four sites. This study identifies the proximal ulna as the best of these sites for rotation osteotomy because of the high gain in the rotated direction and minimal loss in the opposite direction.

Gastrointestinal stromal tumor of the stomach: report of a case.

We report herein the case of a 70-year-old woman found to have a gastrointestinal stromal tumor (GIST) of the stomach. Preoperative X-ray and endoscopic examination revealed a hemispheric submucosal tumor with central depression in the anterior wall of the gastric fornix. The tumor, which was 3 cm in diameter, was resected by a laparoscopy-assisted procedure. Histologic examination revealed that it was composed of spindle-shaped cells with elongated nuclei, and few mitoses. Most of the tumor cells showed immunoreactivity for vimentin and CD34, but not for alpha-smooth muscle actin, desmin, or S-100 protein. The PCNA index was 40.5%. Thus, the GIST did not show differentiation toward smooth muscle or neural cells. A gastrectomy was not performed because the small size of the tumor, and the paucity of the mitoses indicated that it was benign. Nevertheless, careful and long-term follow-up is needed to monitor for signs of possible local recurrence or distant metastases.

Synthesis and antifungal activities of novel 1,3-beta-D-glucan synthase inhibitors. Part 1.

Highly potent 1,3-beta-D-glucan synthase inhibitors 10, 11 and 13 have been identified by the chemical modification of the fungicidal macrocyclic lipopeptidolactone, RO-09-3655 (1), isolated from the cultured broth of Deuteromycotinia spp. D-Ornithine derivative (10) showed improved antifungal activity in the systemic candidiasis model in mice and reduced hepatotoxicity in vitro, as compared with 1.

Diagnostic ultrasonography of the ulnar nerve in cubital tunnel syndrome.

Thirty-two elbows in 31 patients diagnosed as having cubital tunnel syndrome underwent ultrasonographic examination to assess morphological changes in the ulnar nerve and its surrounding tissues. On longitudinal images, the site of constriction due to the fibrous band and proximal swelling of the nerve were observed by ultrasonography and were confirmed intraoperatively. On axial images, the lengths of the major axis [7.2 (SD 1.6) mm] and the minor axis [3.7 (0.9) mm] of the nerve at the medial epicondyle were greater than those in normal subjects. There was a correlation between the stage of ulnar nerve palsy and the diameter of the major axis. Preoperatively, ganglia were detected by ultrasonography in the cubital tunnel in three cases and an anconeus epitrochlearis muscle in two.

Morphology and dynamics of the ulnar nerve in the cubital tunnel. Observation by ultrasonography.

We examined 200 normal elbows to assess the usefulness of ultrasonography in examining the ulnar nerve in the cubital tunnel. On longitudinal images in elbow extension, the nerve changed its course at the fibrous band region 11.5 (SD 2.8) mm distal to the medial epicondyle. On axial images, the diameter of the major axis of the nerve was 3.1 (0.5) mm and that of the minor axis was 1.9 (0.4) mm in men. The respective values were 2.7 (0.4) mm and 1.8 (0.4) mm in women. Dynamic studies showed that in 53 elbows (27%), the nerve moved on to the tip of the epicondyle with the elbow flexed and in 39 elbows (20%), the nerve dislocated anteriorly. The diameters of the hypermobile nerves were significantly larger than nerves that did not displace.

Argyrophilic nucleolar organizer regions (AgNORs) in mucosal epithelium under experimental denture bases in rats.

The purpose of this study was to evaluate changes in the argyrophilic nucleolar organizer region (AgNOR) counts in mucosal epithelium induced by continuous or intermittent compressive pressure exerted through experimental denture bases and to examine the relationships between the AgNOR count, histopathological changes and the intensity of the pressure under denture bases. Continuous or intermittent compressive pressure exerted through the denture bases was applied to the hard palate of the molar region in rats. A morphometric analysis of AgNORs was performed in denture-supporting tissue 3 days and 1, 2, 4, 8, 12 and 20 weeks after the denture insertion. From the results of this study, it was found that non-pressure contact of the denture bases with palatal tissues did not change the AgNOR count. The AgNOR count was decreased by continuous or intermittent compressive pressure, and then recovered to almost the same level as with the non-pressure contact at 20 weeks following a decrease of the pressure. The AgNOR counts in the epithelium under the denture bases were revealed to be related to the histopathological changes in the denture-supporting tissues and the intensity of the pressure under the denture bases.

[Usefulness of percutaneous sclerosing treatment for low-flow vascular lesions].

Peripheral vascular lesions may occur as a result of various clinical problems, cosmetic or dysfunctional causes, or bleeding. In severe cases, coagulopathy and congestive heart failure may occur. Although the efficacy of transarterial embolization (TAE) for arterial vascular lesions is well known, TAE has no effect on low-flow vascular lesions (venous malformations, venous angiomas, and venous components of arteriovenous malformation). Therefore, in such cases, a percutaneous approach is the best method, and we consider sclerotherapy to be the most useful conservative treatment. The primary objective of this study was to confirm the efficacy of percutaneous sclerosing treatment for peripheral low-flow vascular lesions. Lesions were classified on the basis of state of blood flow and morphologic features, and infiltration was classified on the basis of angiographic features and magnetic resonance imaging (MRI) findings. In sclerosing treatment, we used 5% solutions of polidocanol, absolute ethanol, and N-butyl-2-cyanoacrylate (NBCA) as sclerosing agents. Each type of lesion, static or slow-flow, cystic, or localized, showed remarkable improvement after sclerosing treatment with only polidocanol. However, for moderate-to-fast-flow lesions, another sclerosing agent (absolute ethanol/NBCA) was needed. With diffuse infiltrative lesions, surgical repair might be needed, but we recognize the usefulness of sclerosing treatment for functional or cosmetic improvement in these cases.

Case of gonadoblastoma in a 9-year-old boy without physical abnormalities.

BACKGROUND: A 9-year-old boy was admitted to Jikei University Hospital complaining of gradual enlarging of the left scrotal contents. METHODS/RESULTS: Physical examination was significant for bilateral descended testicles. No abnormalities were detected in the testicles or along the spermatic cords. Scrotal ultrasound showed that hyperechoic shadows were recognized in the central area of the left testicle. Subsequent testicular biopsy and histopathological examination showed intratubular malignant germ cells in the testicular tubules. One week later, left orchiectomy was performed. CONCLUSIONS: Histopathological evaluation revealed gonadoblastoma. Gonadoblastoma, a rare gonadal neoplasm, is composed of germ cells and sex cord derivatives and usually occurs in phenotypically female patients with gonadal dysgenesis. To date, only three cases of gonadoblastoma have been reported in anatomically normal male patients with scrotal testicles. We report on a case of gonadoblastoma unaccompanied by a germ cell tumor in a physically normal male.

[Experimental study of intraarterial injection of polidocanol as an embolic agent].

The treatment of hemangiomas and vascular malformations in the soft tissue presents several difficult problems. Transarterial embolization and/or percutaneous sclerosing therapy are useful for such lesions, but the effectiveness of these therapies is often partial, and serious problems like ulceration and tissue necrosis may occur. Therefore, we examined the efficacy of intraarterial injection of polidocanol solution as an embolic agent for hemangiomas and vascular malformations using the rabbit kidney. Three embolic agents were compared with polidocanol solution (polidocanol 3%, n = 5; absolute ethanol, n = 5; n-butyl-2 cyanoacrylate: NBCA, n = 5; polyvinyl alcohol: PVA, n = 5). All embolizations were followed by angiography and resection after a week. Results showed that absolute ethanol (n = 5), NBCA (n = 4) and PVA (n = 1) embolized completely. In the specimens, this led to cell necrosis throughout the kidney. In contrast, polidocanol (n = 5) obstructed neither the main trunk of the renal artery nor the peripheral capillary arteries following angiography. In the specimens, the inner medulla of the kidney suffered necrosis. However, residual tissue with massive fibrotic change was seen. These results suggest the efficacy of "embolosclerosing" treatment for capillary vascular lesions and the possibility of alleviating complications from such therapy.

Isolation of anti-glutathione antibodies from a phage display library.

We have isolated anti-glutathione antibodies from a human synthetic phage antibody scFv library (Nissim,A., Hoogenboom,H.R., Tomlinson,I.M., Flynn,G., Midgley,C., Lane,D. and Winter,G., 1994, EMBO J., 13, 692-698). Glutathione (GSH) conjugates with carrier proteins, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) and human lysozyme (LZM), were used as antigens. After four cycles of panning and affinity chromatography, clones that recognized GSH-conjugated proteins, but not BSA, KLH or LZM, were isolated. The isolated phage antibodies and the soluble scFv fragments were characterized by immunoblotting, and the nucleotide sequences of the VH segments of selected clones were determined. The binding of several isolates to GSH-BSA was competitively inhibited by GSH in an ELISA. These observations have demonstrated that antibodies against GSH, a tripeptide, can be isolated from the library. We constructed the tertiary models of several scFv fragments and discussed the mechanism of antigen binding sites.

[Intraoperative interstitial microwave therapy for recurrent hepatocellular carcinoma of the caudate lobe: a case report].

Microwave tissue coagulation (MTC) therapy was given patients with recurrent hepatocellular carcinoma (HCC) of the caudate lobe of the liver, in which radical surgery for deteriorated liver function was impossible. A total of 40 MTC sessions was applied to two tumors under laparotomy. MTC was administered for 20 seconds in each session. Microwave energy output was 70 watts for 15 mm needle-electrodes and 100 watts for 30 mm electrodes. Alpha-fetoprotein levels in serum had decreased after surgery. Abdominal computed tomography showed no blood flow whatsoever in tumors undergoing MTC. There are fewer limits to the sites and angles for insertion of electrodes under laparotomy. Thus, the surgical approach provides access to all parts of the liver for treatment. We conclude that intraoperative MTC is highly effective in tumor necrosis, and can be a useful local treatment for nonresectable HCC.

MRI in carcinomatous encephalitis.

We report a rare case of miliary brain metastases presenting with symptoms similar to encephalitis ("carcinomatous encephalitis"). Contrast-enhanced MRI demonstrated miliary metastases more distinctly than other imaging methods and reproduced the pathological features.

Enhanced induction of apoptosis of human gastric carcinoma cells after preoperative treatment with 5-fluorouracil.

BACKGROUND: It has been reported that anticancer drugs induce apoptosis, thus it is considered that apoptosis may be important in cancer chemotherapy. The authors examined whether the administration of 5-fluorouracil (5-FU) enhanced apoptosis of gastric carcinoma cells, and investigated the relationship between apoptosis and the expression of Ki-67 and the Bax gene. METHODS: Twenty patients with gastric carcinoma were divided into 2 groups. Ten patients received continuous intravenous 5-FU at 500 mg/body weight/day for 7 days preoperatively whereas the other 10 did not receive any chemotherapy or radiotherapy, and served as controls. For detection of apoptotic cells, apoptotic indices were examined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling (TUNEL) method. The expression of Ki-67 and the Bax gene were examined by immunohistochemical staining. RESULTS: Apoptosis of gastric carcinoma cells increased significantly to 6.1 +/- 3.6% in the 5-FU-treated group compared with the controls (3.2 +/- 1.8%; P < 0.05). The level of expression of Ki-67, a marker of cell proliferation, was inversely correlated with the extent of TUNEL staining that was specific for apoptosis. The percentage of cells that were immunopositive for Ki-67 fell to 50.8 +/- 7.3% of the apoptotic area in the 5-FU-treated group compared with 57.5 +/- 6.9% in the controls (P < 0.05). There was no significant correlation between frequency of apoptotic cells and the level of expression of the Bax gene. CONCLUSIONS: The authors demonstrated enhanced apoptosis in gastric tumors after continuous intravenous infusion of 5-FU for 7 days. This result suggests that it may be possible to evaluate the effects of chemotherapy by detecting apoptotic cells.

Flow cytometric examination of p53 protein in primary tumors and metastases to the liver and lymph nodes of colorectal cancer.

PURPOSE AND METHODS: To confirm prognostic significance of overexpression of p53 in cases of colorectal cancer, expression of p53 protein was examined by flow cytometry in 113 cases of colorectal cancer and its metastasis to the liver and lymph nodes. RESULTS: Overexpression of p53 was found in 44 (39 percent) of the 113 primary tumors. There were no significant correlations among the level of p53 protein in the primary tumor, clinicopathologic features, and prognosis of colorectal cancer. Overexpression of p53 protein was detected in 72 percent (18/25) of liver metastases and in 40 percent (10/25) of lymph node metastases. Frequency of samples that were positive for p53 was significantly higher for liver metastases than for primary tumors and lymph node metastases (P < 0.01). By comparing overexpression of p53 in primary tumors with that in corresponding secondary tumors, a decrease of more than 5 percent in the fluorescence index, compared with primary tumor, was not found in liver metastasis but was found in 20 percent of lymph node metastases. Incidence of cases with lower level expression of p53, compared with primary tumor, was significantly higher in lymph node metastases (32 percent) than in liver metastases (8 percent; P < 0.05). CONCLUSIONS: From these results, it seems possible that overexpression of p53 may not be a good prognostic indicator of colorectal cancer and may be influenced by environments of the tumor.

Apoptotic cell death and its relationship to carcinogenesis in colorectal carcinoma.

BACKGROUND: Apoptotic cell death plays an important role in the proliferation and turnover of cells in various tumors. The relationship between apoptosis and cell proliferation was studied to determine each of their roles in colorectal carcinogenesis. METHODS: Apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method. The occurrence of apoptosis was examined in colorectal cancer that had invaded the submucosa. Specimens were obtained from 38 cases of cancer with adenoma and 29 cases of cancer de novo. Apoptotic indices (AIs) as percentages of TUNEL-positive cells relative to the number of tumor cells and Ki-67 labeling indices (PI) were investigated. The relationship between the frequency of apoptosis and the expression of p53 and c-myc proteins was also investigated. RESULTS: In cancer with adenoma, the ratio of AI/PI in adenoma cells was significantly higher than that of cancer cells (P < 0.0001). Mean AIs of cancer with adenoma were significantly higher than those of cancer de novo particularly in the flat-type cancers (P < 0.05). Among p53 negative tumors, the ratio of AI/PI for cancer de novo was significantly lower than that for cancer with adenoma (P < 0.05). AI of cancer de novo was lower than that of cancer with adenoma in cases with overexpression of c-myc protein (P < 0.05), whereas there was no significant difference in the ratio of AI/PI between cancer de novo and cancer with adenoma. CONCLUSIONS: Colorectal carcinogenesis is related to the inhibition of apoptosis and to the augmentation of proliferative activity both in cancer with adenoma and in cancer do novo. A reduction of the rate of apoptosis as compared with that of cell proliferation might explain the rapid-growing nature of cancer de novo particularly in cases with the flat-type appearance.

Lengthening of the forearm by callus distraction.

Ten patients aged 3 to 13 years (mean, 9 years and 7 months) underwent forearm lengthening by callotasis. The indications for lengthening were shortening and/or deformity of the forearm due to exostosis of the distal ulna in five cases, enchondroma of the distal ulna in one, growth disturbance after fracture of the distal radius in one, radial club hand in one, congenital amputation of the forearm in one and congenital dislocation of the radial head in one. Four had lengthening of the ulna, one of the radius and five of both the radius and the ulna. The average lengthening achieved was 30 mm. Complications encountered were pin track discharge in three cases, callus fracture in five, delayed consolidation of the callus in one and no callus formation in one. Review after 1 to 7 years follow-up (with a mean of 4 years and 9 months) showed satisfactory improvement in appearance and function especially in patients who had tumorous conditions or traumatic epiphyseal arrest.