NMR characterization of the interaction between the PUB domain of peptide:N-glycanase and ubiquitin-like domain of HR23.

PUB domains are identified in several proteins functioning in the ubiquitin (Ub)-proteasome system and considered as p97-binding modules. To address the further functional roles of these domains, we herein characterized the interactions of the PUB domain of peptide:N-glycanase (PNGase) with Ub and Ub-like domain (UBL) of the proteasome shuttle factor HR23. NMR data indicated that PNGase-PUB exerts an acceptor preferentially for HR23-UBL, electrostatically interacting with the UBL surface employed for binding to other Ub/UBL motifs. Our findings imply that PNGase-PUB serves not only as p97-binding module but also as a possible activator of HR23 in endoplasmic reticulum-associated degradation mechanisms.

Fbs1 protects the malfolded glycoproteins from the attack of peptide:N-glycanase.

Fbs1 is a cytosolic lectin putatively operating as a chaperone as well as a substrate-recognition subunit of the SCF(Fbs1) ubiquitin ligase complex. To provide structural and functional basis of preferential binding of Fbs1 to unfolded glycoproteins, we herein characterize the interaction of Fbs1 with a heptapeptide carrying Man3GlcNAc2 by nuclear magnetic resonance (NMR) spectroscopy and other biochemical methods. Inspection of the NMR data obtained by use of the isotopically labeled glycopeptide indicated that Fbs1 interacts with sugar-peptide junctions, which are shielded in native glycoprotein, in many cases, but become accessible to Fbs1 in unfolded glycoproteins. Furthermore, Fbs1 was shown to inhibit deglycosylation of denatured ribonuclease B by a cytosolic peptide:N-glycanase (PNGase). On the basis of these data, we suggest that Fbs1 captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol.

A case of pulmonary type of ovarian small cell carcinoma.

Small cell carcinoma is a rare form of ovarian cancer with a poor prognosis. It is divided into two types, the hypercalcemic and the pulmonary type, of which the latter is extremely rare. A 49-year-old woman presented with an acute abdomen and was suspected to have torsion of a left ovarian tumor, which was followed up with an emergency operation. Postoperative pathological examination gave a diagnosis of the pulmonary type of ovarian small cell carcinoma. Six courses of paclitaxel and carboplatin therapy were given as adjuvant chemotherapy. The patient has survived for 36 months without recurrence. Here we present an extremely rare patient with the pulmonary type of ovarian small cell carcinoma.

Angiogenesis and developmental expression of vascular endothelial growth factor in rat lingual papillae.

We used an embryological approach to investigate development and microvasculature of lingual papillae, and expression of vascular endothelial growth factor (VEGF) in the rat tongue. Temporal changes in the rat tongue at each developmental stage from embryonic day 13 (E13) to postnatal day 7 (P7) were observed by intravascular injection of India ink and immunohistochemistry using a VEGF antibody. At E13, the primordium of circumvallate papilla was observed among various lingual papillae. VEGF was widely expressed at E16 on the proliferated epithelium and the connective tissue core of circumvallate papilla. Invasion by capillary sprouts forming the lingual papillae was observed at E17. The primordium of fungiform papillae was observed at E14. VEGF was strongly expressed around the basal cells of proliferated epithelial tissues of fungiform papillae at E17. At E18, blind-ended capillary sprouts invaded into connective tissue cores from subepithelial sinusoidal capillaries by sprout angiogenesis. At P1, the invading capillary sprouts formed loops by vascular remodeling. The primordium of foliate papillae was observed at E16. VEGF was slightly expressed, but uniformly at E17 on the epithelium, muscle cells, and fibroblasts of foliate papillae. At E18, vascular density was increased by angiogenesis. The primordium of filiform papillae was observed at E17. It was the last to develop among the lingual papillae. VEGF was expressed in the cytoplasm of grown epithelial cells of filiform papillae at E19, and in blind-ended capillary sprouts formed by angiogenesis in the connective tissue cores at E20. The capillary sprouts formed loops by vascular remodeling at P1. Consequently, VEGF was expressed on the papillary epithelium and connective tissue cores of papillae during development of the papillary epithelium, and invasion by capillary sprouts into each papillae was observed thereafter. These results suggest a close relationship between expression of VEGF and angiogenesis of lingual papillae in the rat.

Anatomical study of the vertebral artery in Japanese adults.

The aim of the present study was to examine the vertebral arteries. The origins of the right and left vertebral arteries and their entrance points into the cervical transverse foramen were examined in dissections of 515 Japanese cadavers (303 males, 212 females) at Kurume University School of Medicine from 1990 to 2003. There were 515 right vertebral arteries and 514 left vertebral arteries. The right vertebral artery originated from the right subclavian artery in 514 of 515 arteries and one of the arteries arose from the bifurcation of the brachiocephalic trunk. The mean distance between the origin of the right subclavian artery and the right vertebral artery was 20.9 mm. The left vertebral artery originated from the left subclavian artery in 484 of 514 arteries and the mean distance between the origin of the left subclavian artery and the left vertebral artery was 32.1 mm. The remaining 30 arteries (5.8%) originated from the aortic arch between the left common carotid artery and the left subclavian artery and this frequency is similar to previously published data. There was no right-left difference for the entrance point of the vertebral arteries into the cervical transverse foramen and the 6th cervical vertebra (C6) was the most common entrance point. Seventy-eight percent of our cases had right and left vertebral arteries that originated in the subclavian arteries and entered the cervical transverse foramen at C6. Among the 30 left vertebral arteries that originated from the aortic arch, 20 arteries (66.7%) entered a cervical transverse foramen at a level higher than C6. This frequency was higher than that for the left vertebral artery that originated from the subclavian artery.

Intermittent pneumatic compression for prevention of pulmonary thromboembolism after gynecologic surgery.

BACKGROUND: To investigate the incidence of pulmonary embolism and risk factors for this condition after obstetric and gynecologic surgery, as well as the efficacy of intermittent pneumatic compression. METHODS: A total of 6,218 patients operated at Keio University Hospital excluding obstetric or infertility-related surgery and uterine cervical conization were evaluated retrospectively to determine the preventive effect of intermittent pneumatic compression on postoperative pulmonary embolism. RESULTS: Pulmonary embolism occurred in 42 patients (0.68%). Multivariate analysis showed that malignancy, blood transfusion, and a body mass index > or =25 kg/m2 or > or =28 kg/m2 were independent risk factors for postoperative pulmonary embolism. A significantly lower incidence of pulmonary embolism occurred in patients receiving pneumatic compression postoperatively versus those without it. Among gynecologic malignancies, endometrial cancer was a significant risk factor for pulmonary embolism. CONCLUSION: Preventive measures, including intermittent pneumatic compression, should be taken to avoid postoperative pulmonary thromboembolism in the gynecology field.

Clinicopathologic manifestations of early-onset endometrial cancer in Japanese women with a familial predisposition to cancer.

AIM: The number of patients under 40 years of age with early-onset endometrial cancer is on the rise in Japan. Preservation of fertility in younger patients is a critical issue. In order to examine the clinical and pathological characteristics of these patients, cases of early-onset endometrial cancer at a single hospital were analyzed. METHODS: Seventy-four patients were diagnosed with endometrial cancer before age 40 and included in this study after obtaining informed consent. RESULTS: The clinical characteristics included a significantly higher prevalence of complications such as nulligravidity and nulliparity (P < 0.001). Pathologically, well-differentiated endometrial carcinoma was significantly more frequent (P = 0.011). The 5-year survival rate was high (98.7%). In regards to the relationship between clinicopathological features and grade of differentiation, the prevalence of G2 and G3 carcinoma was not significantly lower (P = 0.24) in patients with obesity. Although the frequency of G2 and G3 carcinoma was significantly higher in patients with a family history of cancer (P = 0.02), their 5-year survival rate was not significantly lower (100%). CONCLUSION: This study found that these two types of early-onset endometrial cancer are clinicopathologically different. In patients with a family history of cancer, their body mass index was lower, and the frequency of G2 and G3 carcinoma was significantly higher, but their 5-year disease-free survival rate was not significantly lower.

An anomalous case of the left gastric artery, the splenic artery and hepato-mesenteric trunk independently arising from the abdominal aorta.

This report describes a rare case of an arterial anomaly in the celiaco-mesenteric region, encountered in a Japanese female cadaver for dissection at the gross anatomy laboratory of Kurume University School of Medicine in 2003. The usual celiac trunk was not identified, and the left gastric artery, the splenic artery and the hepato-mesenteric trunk independently arose from the abdominal aorta. Moreover, the hepatic artery arising from the hepato-mesenteric trunk ran behind the portal vein. The classification for this type of arterial anomaly is a Type II' of Morita's classification and Type II of Higashi and Hirai's classification, not belong to the Adachi's.

Clinical characteristics of prognostic factors in poorly differentiated (G3) endometrioid adenocarcinoma in Japan.

BACKGROUND: It has been reported that prognosis is less favorable in poorly (G3) differentiated endometrioid adenocarcinoma than in well (G1) or moderately (G2) differentiated endometrioid adenocarcinoma. The goal of this study is therefore to analyze the prognosis of G3 endometrioid adenocarcinoma and various factors that may predict a favorable prognosis. METHOD: This study included 699 Japanese cases of endometrioid adenocarcinoma at the International Federation of Gynaecology and Obstetrics (FIGO) surgical stages I-IV (including 74 G3 cases). We investigated the G1-G3 survival rates of endometrioid adenocarcinoma cases and the G2 and G3 disease-free periods. We also examined the clinicopathological characteristics of G3 endometrioid adenocarcinoma. RESULT: The prognosis was poor in stages III and IV in G3 and in G2 cases, but recurrence was observed more frequently in G3 cases than in G2 cases. Adnexal metastasis and high pre-surgery CA602 values showed significantly low P-values for survival. CONCLUSIONS: We suggest that the risk of late recurrence is higher in G3 than in G2 cases. The absence of adnexal metastasis and low pre-surgery CA19-9 values may suggest a relatively favorable prognosis in G3 endometrioid adenocarcinoma.

Association of HNPCC and endometrial cancers.

Hereditary nonpolyposis colorectal cancer (HNPCC) is among the representative familial cancers that are autosomally dominant inherited disorders. Because endometrial cancers develop at high rates in women with HNPCC, it is suggested that some endometrial cancers are familial cancers that are induced by mutations of the DNA mismatch repair (MMR) genes, as in HNPCC. To understand the clinical pathology of familial endometrial cancers that are associated with HNPCC, we surveyed the family histories of 385 patients with endometrial cancer and found that 0.5% of endometrial cancers met the new diagnostic criteria of HNPCC. From molecular and biological analyses, we found microsatellite instability in 30.8% of endometrial cancers and germline mutations of MMR genes in 8.3%. These results suggest a close relationship of MMR gene mutations to the development of endometrial cancers. For a better understanding of the clinical pathology of HNPCC-associated familial endometrial cancers, it is critical for gynecologists to perform a large multicenter study, including detailed family histories.

Two Japanese kindreds occurring endometrial cancer meeting new clinical criteria for hereditary non-polyposis colorectal cancer (HNPCC), Amsterdam Criteria II.

Hereditary non-polyposis colorectal cancer (HNPCC), also called Lynch syndrome, is an autosomal dominantly inherited disorder of cancer susceptibility. Patients with HNPCC exhibit an increased risk for HNPCC-associated extracolonic tumors such as cancer of the endometrium. HNPCC is associated with germline mutations in DNA mismatch repair (MMR) genes: hMLH1, hMSH2 and hMSH6. Here, we describe two Japanese kindreds (0.5%) who met the new clinical criteria for HNPCC, Amsterdam criteria II, from among 375 endometrial cancer patients treated at Keio University Hospital from 1990 to 2002. From these results, it was found that female HNPCC patients comprised approximately 0.5% of all endometrial cancer patients. Decreased expression of two MMR gene protein products (hMLH1 and hMSH6) was confirmed immunohistochemically in these two endometrial tumors in HNPCC kindreds. This case report provides important information on Japanese HNPCC patients occurring endometrial cancer.

Structural basis of sugar-recognizing ubiquitin ligase.

SCF(Fbs1) is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel beta-sandwich. The structure of the SBD-chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination.

Identification of germline MSH2 gene mutations in endometrial cancer not fulfilling the new clinical criteria for hereditary nonpolyposis colorectal cancer.

Endometrial cancer is the second most common malignancy in patients with hereditary nonpolyposis colorectal cancer (HNPCC). This cancer is caused by germline mutations in one of the DNA mismatch repair (MMR) genes. The present study was undertaken to analyze the relation between microsatellite instability (MSI) and germline mutations of MMR genes. We analyzed MSI in 38 cases of endometrial cancer. MSI was present in one or more (out of 5 examined) regions in 11 (29%) cases. Furthermore, alterations in MLH1 and MSH2, two culprit genes representative of HNPCC, were examined in the 11 MSI-positive patients using polymerase chain reaction-single-strand conformation polymorphism and sequencing. Germline mutations, namely, 1) a missense mutation at codon 688 (ATG-->ATA, Met-->Ile) and 2) a missense mutation at codon 390 (CTT-->TTT, Leu-->Phe) of the MSH2 gene, were found in 2 of the 11 patients (18%). Although these two cases do not fulfill the new Amsterdam criteria, they had strong family histories of colorectal and endometrial carcinoma. Our results show that genetic testing is important in cases of endometrial cancer with a history suggestive of HNPCC even if the new Amsterdam criteria are not fulfilled.