ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN.

Overexpression of MYCN is a hallmark of neuroblastoma (NB). ALK(R1275Q), an activating mutation of ALK (anaplastic lymphoma kinase), has been found in sporadic and familial NB patients. In this report, we demonstrated that ALK(R1275Q) knock-in, MYCN transgenic compound mice developed NB with complete penetrance. Transcriptome analysis revealed that ALK(R1275Q) globally downregulated the expression of extracellular matrix (ECM)- and basement membrane (BM)-associated genes in both primary neuronal cells and NB tumors. Accordingly, ALK(R1275Q)/MYCN tumors exhibited reduced expression of ECM/BM-related proteins as compared with MYCN tumors. In addition, on MYCN transduction, ALK(R1275Q)-expressing neuronal cells exhibited increased migratory and invasive activities. Consistently, enhanced invasion and metastasis were demonstrated in ALK(R1275Q)/MYCN mice. These results collectively indicate that ALK(R1275Q) confers a malignant potential on neuronal cells that overexpress MYCN by impairing normal ECM/BM integrity and enhancing tumor growth and dissemination. Moreover, we found that crizotinib, an ALK inhibitor, almost completely inhibited the growth of ALK(R1275Q)/MYCN tumors in an allograft model. Our findings provided insights into the cooperative mechanism of the mutated ALK and overexpressed MYCN in the pathogenesis of NB and demonstrated the effectiveness of crizotinib on ALK(R1275Q)-positive tumors.

Ink4a and Arf are crucial factors in the determination of the cell of origin and the therapeutic sensitivity of Myc-induced mouse lymphoid tumor.

The cell of origin of tumors and the factors determining the cell of origin remain unclear. In this study, a mouse model of precursor B acute lymphoblastic leukemia/lymphoma (pre-B ALL/LBL) was established by retroviral transduction of Myc genes (N-Myc or c-Myc) into mouse bone marrow cells. Hematopoietic stem cells (HSCs) exhibited the highest susceptibility to N-Myc-induced pre-B ALL/LBL versus lymphoid progenitors, myeloid progenitors and committed progenitor B cells. N-Myc was able to induce pre-B ALL/LBL directly from progenitor B cells in the absence of Ink4a and Arf. Arf was expressed higher in progenitor B cells than Ink4a. In addition, N-Myc induced pre-B ALL/LBL from Arf(-/-) progenitor B cells suggesting that Arf has a predominant role in determining the cell of origin of pre-B ALL/LBL. Tumor cells derived from Ink4a/Arf(-/-) progenitor B cells exhibited a higher rate of proliferation and were more chemoresistant than those derived from wild-type HSCs. Furthermore, the Mdm2 inhibitor Nutlin-3 restored p53 and induced massive apoptosis in mouse pre-B ALL/LBL cells derived from Ink4a/Arf(-/-) cells and human B-ALL cell lines lacking Ink4a and Arf expression, suggesting that Mdm2 inhibition may be a novel therapeutic approach to the treatment of Ink4a/Arf(-/-) B-ALL/LBL, such as is frequently found in Ph(+) ALL and relapsed ALL. Collectively, these findings indicate that Ink4a and Arf are critical determining factors of the cell of origin and the therapeutic sensitivity of Myc-induced lymphoid tumors.

[Pulmonary embolectomy for acute massive pulmonary thromboembolism after failure of thrombolytic therapy].

Acute massive pulmonary thromboembolism (PTE) is associated with an exceptionally high mortality rate and results in death if not diagnosed early and treated properly. We observed 3 cases of acute massive PTE. One of the patients had undergone a surgery for femoral neck fracture. Ten days postoperatively, she developed severe dyspnea with hypoxia, and computed tomography (CT) pulmonary angiography confirmed the PTE diagnosis. She then had cardiac arrest when catheter examination. Although emergency surgical thrombectomy was successful with good postoperative hemodynamic stability and oxygenation, the patient did not recover from the unconsciousness caused by preoperative ischemic brain damage. Subsequently, she died 6 months after surgery. Of the 3 patients, 2 suffered from right ventricular dysfunction without hemodynamic instability. They underwent open thrombectomy after the failure of conservative treatment with a systemic injection of urokinase. Both patients demonstrated a good clinical course and were discharged from hospital in a good general condition 22 and 28 days postoperatively. Herein, we review the current literature on PTE treatment. We concluded that an aggressive surgical intervention might be preferred to thrombolytic therapy for PTE patients with massive thrombosis and progressive right ventricular dysfunction.

Oral administration of pulverized Konjac glucomannan prevents the increase of plasma immunoglobulin E and immunoglobulin G levels induced by the injection of syngeneic keratinocyte extracts in BALB/c mice.

BACKGROUND: Immunoglobulin (Ig) E plays a key role in the pathogenesis of atopic diseases, such as asthma, atopic dermatitis and allergic rhinitis. Oral administration of pulverized Konjac glucomannan (PKGM) has recently been demonstrated to prevent both plasma IgE elevation and developing dermatitis in NC/Nga mice, a model of atopic dermatitis. OBJECTIVE: To clarify the direct effect of PKGM on the increase of plasma IgE, we employed the system of BALB/c mouse that increases IgE level without developing dermatitis in response to continuous injection of the extract of syngeneic keratinocytes, PAM 212 cells (PAM extract). METHODS: Three weeks after the start of feeding with either control or PKGM diet, mice were injected subcutaneously with PAM extract bi-weekly for 10 weeks. The levels of plasma Igs were measured by enzyme-linked immunosorbent assay every 2 weeks after the injection. The levels of epsilon germline transcription and the amounts of mRNA for IL-4, IFN-gamma, GATA-3 and T bet gene in the spleen were evaluated by real-time RT-PCR at the end of the experiment. RESULTS: On the one hand, PKGM prevented the increase of plasma IgE and IgG (IgG1, IgG2b) induced by PAM extract, and on the other hand, it enhanced the levels of plasma IgG3. However, it did not affect the level of plasma IgM. PKGM also reduced the levels of plasma ovalbumin (OVA)-specific IgE in OVA-sensitized mice. Moreover, PKGM attenuated the induction of epsilon germline transcription and expression levels of mRNA for IL-4, IFN-gamma and GATA-3 in the spleen of PAM extract-injected mice. PKGM also attenuated the induction of epsilon germline transcription and mRNA for IFN-gamma and T bet in the spleen of phosphate-buffered saline-injected control mice. CONCLUSIONS: These results suggested that oral administration of PKGM prevents the elevation of plasma IgE by suppressing IgE class switching in B cells and/or the commitment development of naive lymphocytes to both T-helper type 1 (Th1) and Th2.

Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase.

The antioncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell-cycle checkpoint regulation. Here we show that Chk2 associates with the oncogenic protein Wip1 (wild-type p53-inducible phosphatase 1) (PPM1D), a p53-inducible protein phosphatase. Phosphorylation of Chk2 at threonine68 (Thr68), a critical event for Chk2 activation, which is normally induced by DNA damage or overexpression of Chk2, is inhibited by expression of wild-type (WT), but not a phosphatase-deficient mutant (D314A) of Wip1 in cultured cells. Furthermore, an in vitro phosphatase assay revealed that Wip1 (WT), but not Wip1 (D314A), dephosphorylates Thr68 on phosphorylated Chk2 in vitro, resulting in the inhibition of Chk2 kinase activity toward glutathione S-transferase-Cdc25C. Moreover, inhibition of Wip1 expression by RNA interference results in abnormally sustained Thr68 phosphorylation of Chk2 and increased susceptibility of cells in response to DNA damage, indicating that Wip1 acts as a negative regulator of Chk2 in response to DNA damage.

A case of inflammatory fibroid polyp causing small-bowel intussusception in which retrograde double-balloon enteroscopy was useful for the preoperative diagnosis.

We showed a newly developed method, retrograde double-balloon enteroscopy, to be useful for preoperative diagnosis in a case of inflammatory fibroid polyp accompanied by small-bowel intussusception. A 64-year-old woman was admitted to our hospital with small-bowel intussusception. Results of radiographic and ultrasonographic examination were suggestive of a small-bowel mass. Retrograde double-balloon enteroscopy was performed in an attempt to make a preoperative diagnosis. Endoscopic observation, in combination with histological findings derived from endoscopic biopsy, was suggestive of an inflammatory fibroid polyp. The patient then underwent laparotomy with minimal incision, which revealed a polypoid mass leading to a jejunojejunal intussusception, without bowel necrosis, and a partial small-bowel resection was performed. The pathological diagnosis was an inflammatory fibroid polyp.

Galacto-oligosaccharide production using a recycling cell culture of Sterigmatomyces elviae CBS8119.

Addition of small amounts of Fe2+, Zn2+, Cu2+ and thiamine-HCL to the culture medium was required for promoting the galactooligosaccharide (Gal-OS)-producing activity of Sterigmatomyces elviae CBS8119, when the concentration of yeast extract in the medium was lowered to 0.1 g l-1. Galactooligosaccharide production using a recycling cell culture was performed in a medium containing 360 mg ml-1 of lactose supplemented with optimal concentrations of Fe2+ (1.5 mg l-1 of FeSO4 x 7H2O), Zn3+ (15 mg l-1 of ZnSO4 x 7H2O), Cu2+ (0.5 mg l-1 of CuSO4 x 5H2O) and thiamine-HCL (1 mg l-1). Galacto-oligosaccharide production was maintained at high levels during six cycles of production, with the amount of Gal-OS produced in each cycle being more than 216 mg ml-1 (weight yield of more than 60%.

Galacto-oligosaccharide production from lactose by Sirobasidium magnum CBS6803.

Galacto-oligosaccharide (Gal-OS) was produced from lactose by a yeast, Sirobasidium magnum CBS6803. With toluene-treated resting cells, 136 mg ml-1 of Gal-OS was produced from 360 mg ml-1 of lactose at 50 degrees C for 42 h. Then, the yield of Gal-OS was increased by a culture method in which cell growth followed the enzymatic reaction: 224 mg ml-1 of Gal-OS was produced at 30 degrees C for 60 h. Finally, combination of the toluene-treated resting cells and glucose oxidase plus catalase was applied to improve productivity by the removal of a by-product, glucose, which inhibits the Gal-OS production, from the reaction mixture. In this case, 242 mg ml-1 of Gal-OS was produced at 50 degrees C for 42 h without cell growth. The structure of the major product was identified as 4'-galactosyl-lactose.

Production of Galacto-Oligosaccharide from Lactose by Sterigmatomyces elviae CBS8119.

Volume 61, no. 11, p. 4022, column 2, line 10: "K(inf2)HSO(inf4)" and "KH(inf2)SO(inf4)" should read "K(inf2)HPO(inf4)" and "KH(inf2)PO(inf4)," respectively. Line 13: "K(inf2)HSO(inf4)" should read "K(inf2)HPO(inf4)." Line 14: "KH(inf2)SO(inf4)" and "0.2 g of CaCO(inf3)" should read "KH(inf2)PO(inf4)" and "1 g of CaCO(inf3)," respectively. [This corrects the article on p. 4022 in vol. 61.].

Purification and Properties of a Novel Thermostable Galacto-Oligosaccharide-Producing (beta)-Galactosidase from Sterigmatomyces elviae CBS8119.

Volume 61, no. 11, p. 4028, column 1, line 37: "p-nitrophenyl-(beta)-d-galactopyranoside" should read "p-nitrophenyl-(beta)-d-glucopyranoside." Page 4028, column 1, line 38: "p-nitrophenyl-(beta)-l-arabinopyranoside" should read "p-nitrophenyl-(alpha)-l-arabinopyranoside." Page 4028, column 2, Table 2, last line: "PNP-(beta)-l-arabinopyranoside" should read "PNP-(alpha)-l-arabinopyranoside." Page 4028, column 2, Table 2, footnote a, line 4: "PNP-(alpha)-l-arabinopyranoside" should read "PNP-(beta)-l-arabinopyranoside." [This corrects the article on p. 4026 in vol. 61.].

Production of galacto-oligosaccharide from lactose by Sterigmatomyces elviae CBS8119.

Our stock cultures were screened for microorganisms that can produce galacto-oligosaccharide (Gal-OS) from lactose. Of the 574 strains of bacteria and yeasts tested, Sterigmatomyces elviae CBS8119, Rhodotorula minuta IFO879, and Sirobasidium magnum CBS6803 were found to be efficient producers of Gal-OS from lactose and S. elviae CBS8119 was selected as a representative, high-level producing strain. With toluene-treated resting S. elviae CBS8119 cells, 135 mg of Gal-OS per ml was produced from 360-mg/ml lactose. During this reaction, the by-product glucose was found to inhibit Gal-OS production. Therefore, in order to remove the glucose from the reaction mixture, a culture method in which cell growth followed the enzymatic reaction was devised, which increased the yield of Gal-OS considerably because of the consumption of glucose for cell growth. Under such conditions, 232 mg of Gal-OS per ml was produced from 360-mg/ml lactose after incubation at 30 degrees for 60 h. The structure of the major product was identified as O-beta-D-galactopyranosyl-(1-->4)-O-beta-D-galactopyranosyl-(1-->4)-D- glucopyranose (4'-galactosyl-lactose) by 13C nuclear magnetic resonance spectroscopy.

Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119.

A thermostable beta-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-beta-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85 degrees C. It was stable at temperatures up to 80 degrees C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri- and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.

Reduced amoxicillin uptake into human gastric mucosa when gastric juice pH is high.

Amoxicillin when administered with gastric acid suppressors has been shown to be effective in eradication of Helicobacter pylori in 50 to 80% of subjects. The aim of this investigator-blind crossover study was to determine if gastric mucosal amoxicillin uptake was affected by increasing gastric juice pH. Fifteen male subjects (7 H. pylori positive and 8 H. pylori negative) were randomized to receive 150 mg of ranitidine twice a day, 300 mg of ranitidine twice a day, or no drug for 2 days prior to upper endoscopy. The last dose of ranitidine was given 60 min prior to upper endoscopy, and amoxicillin (500 mg) was given 30 min prior to upper endoscopy. The amoxicillin concentrations in mucosal biopsy samples, gastric juice, and serum were determined by a standard microbiological bioassay technique. Mean amoxicillin levels were greater in samples of antrum, fundus, and duodenum for volunteers who received no ranitidine than in those receiving 300 mg of ranitidine (P < 0.05) and those receiving 150 mg of ranitidine (P < 0.05 except for fundus). Amoxicillin levels in the antrum, fundus, and duodenum were negatively correlated with gastric juice pH (P < 0.005 for antrum; P < 0.001 for fundus and duodenum). There was no correlation between gastric juice pH and amoxicillin levels in either gastric juice or serum. The amoxicillin concentration in gastric juice was significantly higher with 300 mg of ranitidine than with no ranitidine (P < 0.05). Thus, lower gastric juice pH is associated with a higher rate of mucosal uptake of amoxicillin.

[Immunohistochemical study of PCNA, EGFR, c-erbB-2 and p53 in carcinomas of large intestine].

Immunohistochemical stainings for EGFR, c-erbB-2, p53 and PCNA were performed on 36 and 30 cases of intramucosal and advanced carcinomas of large intestine. Positive rate was 58.3%, 41.6%, 58.3% and 60.6% for EGFR, c-erbB-2, p53 and PCNA in the intramucosal cases, and 66.7%, 50%, 66.7% and 72.6% in the advanced ones, respectively. Relationship between EGFR and c-drbB-2 was more significant in the advanced carcinomas than that in the intramucosal ones. It seemed likely that relationship between p53 and c-erbB-2 was more significant than that between p53 and EGFR. Positive rate of PCNA was of intimate relationship among that of EGFR, c-erbB-2 and p53, and the positive rate increased in the advanced carcinomas.

[A case of 48 XXYY Klinefelter's syndrome].

We observed a patient with 48 XXYY Klinefelter syndrome who visited our hospital because of a short penis as chief complaint. The patient was a 21-year-old, tall and obese man. He had gynecomastia. The penis was short and bilateral testes were underdeveloped. Endocrinologically the LH and FSH showed high level and the testosterone was low. A diagnosis of very rare 48 XXYY Klinefelter was made based of the chromosomal analysis.

[Transurethral teflon paste injection for vesicoureteral reflux in neurogenic bladder dysfunction].

We experienced transurethral teflon paste injection for 12 refluxing ureters of 7 patients with neurogenic bladder dysfunction. Preoperative assessment of cystometry showed hypoactive bladder function with normal bladder compliance in 4 patients, and low compliance bladder (< 10 ml/cmH2O) in 1. Voiding cystography revealed grade 1 reflux in 2 ureters, grade 2 in 3, grade 3 in 2, grade 4 in 2, and grade 5 in 2. One ureter did not show reflux. Zero point two to 1.6 ml of teflon paste was injected on each ureter under cystoscopic observation. These patients were followed for a mean of 25.1 months. Reflux disappeared immediately after the first operations in all patients, however recurrence was observed in 2 ureters, in which improvement of reflux (grade 5 to 2) was achieved in 1 ureter but no improvement (grade 2 to 2) in another. Pyelonephritis was not encountered in any patients after injection. No complication was observed through the follow up period. In conclusion, we advocate that endoscopic teflon paste injection is a useful alternative to ureteroneocystostomy in the treatment of reflux in patients with neurogenic bladder dysfunction.

[Immunohistochemical study of growth factors and oncogenes in gastric carcinomas].

Immunohistochemical staining for EGF, EGFR, c-erbB-2, p53, K-ras and PCNA was performed on the formalin-fixed, paraffin embedded sections of resected gastric carcinomas. A relatively high positive rate was observed for EGFR and c-erbB-2 in the well-differentiated adenocarcinomas and p53 in the poorly-differentiated adenocarcinomas. The positive rate of these factor was higher in the advanced cases than in the early cases, and also in the deep invasive area than the superficial area. According to the PCNA staining, a relatively high positive rate was observed in the well-differentiated adenocarcinomas compared with the early cases of poorly-differentiated adenocarcinomas, but the positive rate was markedly higher in the advanced cases of the latter. Typical signet-ring cell carcinomas showed the lowest positivity rate compared with the other histological types of gastric carcinomas.

[Immunohistochemical study for growth factor and oncogene on atypical epithelium of the stomach].

Almost all atypical epithelial lesions of the stomach consist of atypical cells in the superficial part of the glands and nonatypical cells in the deeper portion of the glands. A transition zone was formed between the superficial atypical gland cells and the deeper nonatypical gland cells. Positive cells were widely demonstrated with immunohistochemical stains for PCNA in the superficial atypical glands and transition zone. The rate of PCNA positivity was 37.7%. However, a small number of positive cells for EGFR (8.5%), c-erbB-2(11.3%), p53(11.3%) and c-K-ras(1.7%) were found in ATP. The incidence of positivity for these factors was low compared with that for carcinomas. The percentages of positive cells for EGFR(1.5%) and c-erbB-2(4.5%) were very low in intestinal metaplasia.

[Immunohistochemical study of the lymph-follicles and lymphocytes in the gastric mucosa].

Immunohistochemical stainings according to ABC method (UCHL-1,L-26,MT-1,MB-1,IgG,IgA,IgD,IgM, kappa, lambda, LN-1 and LN-2) for the lymphocytes in the germinal center, mantle zone and infiltrative lymphocytes in the gastric mucosa of 30 cases of chronic gastritis and 10 cases of reactive lymphoreticular hyperplasia (RLH) were performed. Lymphocytes in the germinal center and mantle zone consisted usually of B-cells positively stained by L-26 and MB-1. However, in the interstitially infiltrative cells,T-cells positively stained by UCHL-1 and MT-1 were not infrequently contained. Immunoglobulin stainings revealed marked positivity for IgG,IgA,IgD,IgM, kappa and lambda in the interstitial lymphocytes and plasma cells. As to the RLH, small number of T-cells scattered in the germinal center and surrounding area of lymph follicles, and large number of T-cells were found among the follicles, where B-cells were more infrequently found than in the interstitial area of propria mucosae. Confusion of enlarged germinal centers and monotonous proliferation of lymphocytes among the lymph follicles showing monotonous positivity for the stains of heavy and light chains in the cases of RLH were suggestive of malignant change.