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Werner syndrome (WS) is a rare autosomal recessive progeroid syndrome characterized by the premature onset of multiple age-related disorders. The gene responsible for WS has been identified as WRN, a member of the RecQ family of helicase genes. Based on the fact that patients with WS exhibit osteoporosis and osteoarthritis, the present study was undertaken to clarify the contribution of the WRN gene to the etiology of these two common age-related disorders in normal postmenopausal women. We investigated the association of a WRN gene polymorphism, namely c.4330 T --> C leading to an amino acid substitution from Cys to Arg, with bone density and lumbar spondylosis score in unrelated Japanese postmenopausal women (n = 377). Genotypic frequencies of T/T, T/C, and C/C were 87.5%, 12.2%, and 0.3%, respectively. Bone density of the lumbar spine (L2-4) was significantly lower in women carrying the minor C allele than in non-carriers (P = 0.037). When bone density was expressed by the Z score after being adjusted by age and weight, carriers of the C allele showed lower values not only in the lumbar spine, but also in the total body (P = 0.015 and 0.042, respectively). The association study with spondylosis in postmenopausal women (n = 221) revealed that this polymorphism was not related to the severity of spondylosis expressed by the Kellgren-Lawrence score at any disk level of the lumbar spine (L2/3-L5/S1). These findings indicate that the WRN gene may be a candidate for the genetic regulation of osteoporosis, but not spondylosis, in normal Japanese postmenopausal women. [ABSTRACT FROM AUTHOR]

Mouse mutant ttw (tiptoe walking) is an excellent model for ectopic ossification. This mutant exhibits ossification in various soft tissues, which is histologically similar to human OPLL (ossification of posterior longitudinal ligament of the spine). We previously reported that ttw is caused by a nonsense mutation of the nucleotide pyrophosphatase (ENPP1) gene, and that a polymorphism of the human ENPP1 gene is associated with OPLL. These facts indicate that ENPP1 regulates ectopic ossification in viv o; however, the mechanism is unclear. ENPP1 is an ectoenzyme that generates phosphate (Pi) and pyrophosphate (PPi). PPi is a strong inhibitor of ossification. Abnormal Pi metabolism is observed in patients with OPLL, and diseases with abnormal Pi metabolism such as hypophosphatemic rickets are frequently complicated by ectopic ossification. These lines of evidence suggest Pi-PPi metabolism associated with ENPP1 may play an important role in regulation of ectopic ossification. To clarify the molecular mechanism of ectopic ossification in ttw , we examined the effect of dietary phosphate and calcium on the ttw phenotype and found a high dietary phosphate-accelerated ectopic ossification. Then we examined genes associated with the enhanced ossification in ttw on a high phosphate diet by a differential display method. We identified nine mouse genes; six genes were up-regulated by the high phosphate diet, and three were down-regulated. Six of the nine genes were novel and we cloned and characterized them. Two of the genes were highly specific to cartilage, suggesting their specific role in enchondral ossification. Our identification of the novel genes would give novel insight into the mechanism of ectopic ossification and etiology of OPLL. Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

PRG4 (proteoglycan 4)has been identified as megakaryocyte stimulating factor and articular superficial zone protein.PRG4 has characteristic motifs including somatome- din B and hemopexin domains,a chondroitin sulfate-attach- ment site and mucin-like repeats.During a screen of genes implicated in ectopic ossification,we found a novel mouse gene highly homologous to human and bovine PRG4 genes.Here, we report isolation,characterization and mapping of the gene, Prg4 together with characterization of its human orthologue. Prg4 cDNA was 3,320 bp long,encoding a 1,054 amino-acid protein.Human and mouse PRG4 genes each consisting of 12 exons spanned 18 and 16 kb,respectively.Characteristic motifs were conserved across species;however,the mucin-like repeat regions were highly diverse in length between species with a tendency that larger animals had longer repeats.Expression of human and mouse PRG4 genes was similar and found not only in cartilage,but also in liver,heart,lung,and bone.Expression of the mouse gene increased with progression of ectopic ossifi- cation.Multiple tissue-specific splicing variants lacking some of the motifs were found in both human and mouse.Although a specific role in the articular joint has previously been reported, the presence of multi-functional motifs as well as unique expression and alternative splicing patterns suggest that PRG4 functions in several distinctive biological process including reg- ulation of ossification. [ABSTRACT FROM AUTHOR]

Large-scale sequencing of selected genomic regions, coupled with in silico gene trapping, is a robust approach to identifying previously unknown genes. In this way we have found a gene (C8orf2) that is highly homologous to C. elegans C42C1.9. C8orf2 was situated on 8p11.2 between STS markers NIB1979 (proximal) and AFMA295ZD5 (distal), oriented toward the centromere. C8orf2 consisted of 16 exons spanning more than 16.5 kb of genomic DNA, and was expressed ubiquitously in human tissues. The gene encoded 339-and 152-amino acid polypeptides by alternative splicing; the larger variant con-tained a region extremely rich in charged amino acids, in par-ticular lysine and glutamic acid. C8orf2 also bore sequence homology to the human KE04p gene. Its conservation among highly divergent species suggests that C8orf2 belongs to a novel gene family. [ABSTRACT FROM AUTHOR]

Drosophila ash2 belongs to the trithorax (trx ) gene family. The ash2 product positively regulates expression of homeotic selector genes, and is implicated in early development and formation of various disc patterns in the fruit fly. Through large-scale sequencing of human genomic DNA coupled with in silico gene trapping, we identified a gene (ASH2L) on chromosome 8p11.2 whose predicted product was highly homologous to ash2 , characterized it, and identified its mouse counterpart. The human ash2 cDNA is 2368 bp long, encoding 628 amino acids. The 16-exon gene spans more than 34 kb of genomic DNA between STS markers WI-9207 (centromere) and AFMA295ZD5 (telomere) on chromosome 8, with transcription oriented telomere to centromere. The ash2 genes are highly conserved among different species, including C. elegans and yeast. The presence of a conserved bipartite nuclear localization signal and a PHD finger motif in the human ash2 gene suggests that the gene product would function as a transcriptional regulator in humans, as its homologue does in Drosophila. [ABSTRACT FROM AUTHOR]

Background: In patients with pancreaticobiliary maljunction complicated by congenital biliary dilatation, the pancreatic enzyme flows back into the bile, leading to bile duct carcinogenesis. Although the biliary tract resection and reconstruction is well documented to decrease the rate of malignancy, cancer occurrence has been reported in the residual intrahepatic or intrapancreatic bile duct, even after resection. We report a case of multiple biliary tract cancers in the liver complicated by congenital biliary dilatation, whose tumor lesions were resected en bloc without disconnecting the biliary tract by simultaneous pancreatoduodenectomy and living donor liver transplantation., Case Presentation: A 27-year-old woman presented with epigastric discomfort. Examination indicated multiple biliary tract cancers complicated by congenital biliary dilatation. Computed tomography scan revealed three papillary tumors in the right hepatic duct with increased 18 F-FDG accumulation on positron emission tomography. Contrast-enhanced ultrasound revealed another lesion in the left hepatic duct. Adenocarcinoma cells were detected using bile and choledochal brush cytology. Tumors resection by right lobectomy or trisegmentectomy of the liver and extrahepatic bile duct resection indicated a high risk of postoperative liver failure; the residual liver volumes were calculated only 277 ml or 176 ml, respectively. In addition, tumor recurrence owing to bile leakage during the surgery and carcinogenesis from the remaining bile duct were concerned. Pancreatoduodenectomy was performed without disconnecting the biliary tract, and the tumors were resected en bloc with the whole liver. The left lobe liver graft from the husband was then transplanted. After 5 years of adjuvant treatment with tegafur/gimeracil/oteracil potassium, she remained in remission eight and half years after the surgery., Conclusions: Given the mechanism and development of cancer in the congenital biliary dilatation, simultaneous pancreatoduodenectomy and liver transplantation may be considered, especially in the case of young patients., Competing Interests: Declarations. Ethics approval and consent to participate: This report has been performed in accordance with the Declaration of Helsinki, and was approved by the local ethics committee and conducted according to the guidelines of Hokkaido University. Consent for publication: Informed consent to publish has been obtained. Competing interests: The authors of this manuscript have no conflicts of interest to disclose described by the Surgical case reports., (© 2024. The Author(s).)

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome caused by germline variants in the APC gene, leading to the development of numerous colorectal polyps and significantly increases the risk of colorectal cancer. A diagnosis is typically made using colonoscopy, and genetic testing can assist in patient surveillance and carrier identification. Recent advances include the use of whole-genome array comparative genomic hybridization (a-CGH), which provides better resolution of genetic imbalances. We aimed to explore the specific features of FAP patients with whole APC gene deletions using high-resolution a-CGH and to compare patient characteristics. Two polyposis patients with whole APC deletions were identified, and the lost genetic sizes ranged from 0.3-1.1 Mb. Nervous abnormalities were a characteristic symptom in a patient with a 1.1 Mb loss. A patient with an approximately 0.3 Mb loss, which included the entire APC gene, presented a polyposis phenotype without intellectual disability. The comparison of genetic losses, with or without intellectual disability, revealed 7 genetic changes. Consequently, EPB41L4A is a candidate gene associated with the neurogenic phenotype., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Gastrointestinal stromal tumors surrounding the esophagogastric junction are often challenging to resect, with no consensus regarding the optimal surgical technique. Here in, we present a case of concurrent gastric cancer in the antrum and gastrointestinal stromal tumors adjacent to the esophagogastric junction. The patient underwent simultaneous distal gastrectomy and local resection assisted by a surgical robot, avoiding the need for total gastrectomy. The utilization of robot-assisted surgery has become an increasingly popular technique, holding promise for simplifying complex surgical procedures across diverse medical settings., (© 2024 Asia Endosurgery Task Force and Japan Society of Endoscopic Surgery and John Wiley & Sons Australia, Ltd.)

Ammonia is an extremely important storage and transport medium for renewable energy, and technology is expected to produce it on demand and onsite using renewable energy. Applying a DC (direct current) to a solid catalyst layer with semiconducting properties makes ammonia synthesis highly efficient, even at low temperatures (approximately 400 K). In this process, oxide supports with semiconducting properties play important roles as metal supports and conduction fields for electrons and protons. The influence of the degree of particle aggregation on the support properties and ammonia synthesis using an electric field was evaluated for CeO 2 , which is the best material for this purpose because of its semiconducting properties. The results showed that controlling the aggregation structure of the crystalline particles could significantly influence the surface conductivity of protons and electrons; thus, the activity could be largely controlled. The Ru-CeO 2 interaction could also be controlled by changing the crystallinity, which suppressed the aggregation of the supported Ru and significantly improved the ammonia synthesis activity using an electric field at low temperatures., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Castleman disease (CD) is a rare lymphoproliferative disease. Hyaline-vascular type unicentric CD has a good prognosis if completely resected during surgery. However, follicular dendritic cell proliferative lesions have the potential for recurrence and metastasis. A 22-year-old man was referred to our hospital with the chief complaint of nausea and vomiting. These symptoms were caused by a right mesocolonic tumor pushing the duodenum. The patient underwent laparoscopic tumorectomy and complete surgical excision. The postoperative course was uneventful, with no complications. Pathological examination confirmed that the tumor was an enlarged lymph node, typical of hyaline vascular-type CD; however, follicular dendritic cell proliferative lesions were noted. We report a rare case of hyaline-vascular-type CD with follicular dendritic cell proliferative lesions associated with malignancy, as limited case reports exist on this particular disease., Competing Interests: The authors have no conflicts of interests to declare., (Published by Oxford University Press and JSCR Publishing Ltd. © The Author(s) 2023.)

This report is the first describing a study quantitatively analysing aspects of oxide surface protonics in a dry H 2 atmosphere. Elucidating surface protonics is important for electrochemical and catalytic applications. In this study, AC impedance spectroscopy was used to investigate surface conduction properties of porous CeO 2 at low temperatures (423-573 K) and in a dry H 2 atmosphere. Results demonstrated that the conductivity increased by several orders of magnitude when H 2 was supplied. Dissociative adsorption of H 2 contributes to conduction by forming proton-electron pairs. Also, H/D isotope exchange studies confirmed protons as the dominant conduction carriers. Furthermore, H 2 adsorption equilibrium modelling based on the Langmuir mechanism was applied to explain the H 2 partial pressure dependence of conductivity. For the first time, the obtained model explains the experimentally obtained results both qualitatively and quantitatively. These findings represent new insights into surface protonics in H 2 atmosphere.

Background: Simultaneous pancreas and kidney transplantation (SPK) is a treatment option for patients with end-stage renal disease due to type 1 diabetes mellitus. We report a patient with a refractory fistula due to leakage from the duodenal stump of the pancreas graft after an SPK with bladder drainage who was successfully treated with a percutaneous direct injection of N-butyl-2-cyanoacrylate (NBCA)., Case Presentation: A 60-year-old female with a 33-year history of type 1 diabetes mellitus and a 10-year history of renal replacement therapy underwent an SPK in 2015. At the time of transplantation, an abdominal aortic aneurysm with a high risk of rupture was treated by a Y-graft replacement prior to the SPK. Bladder drainage of the pancreas graft was chosen to avoid a vessel graft infection. The patient's postoperative course was uneventful. The patient was discharged on postoperative day 93 with good-functioning pancreas and kidney grafts. One and a half years after the operation, the patient was found to have acute graft pancreatitis and a leak from the duodenal stump of the pancreas graft due to a paralytic neurogenic bladder. The insertion of an indwelling catheter into the bladder and the endoscopic-guided insertion of a catheter into the graft pancreatic duct through the duodenum/bladder anastomosis did not result in the closure of the fistula. Therefore, NBCA was injected at the site of the leak point using CT-guided technique. The fistula was completely closed immediately after the injection, with no recurrences of leaks., Conclusions: A percutaneous direct injection of NBCA is one of the treatment options to treat intractable fistulas.

We report the second case of deceased donor liver transplantation in a patient co-infected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in Japan. A 48-year-old patient with hemophilia A was infected with HIV and HCV through contaminated factor VIII concentrate in his childhood and developed cirrhosis and hepatocellular carcinoma. The patient was on the transplant list for a deceased donor liver. The patient had broad spectrum anti-HLA class I and II antibodies, which may be attributed to repeated whole blood transfusions in the past. Catastrophic coagulopathy during the surgery was predicted because of the underlying hemophilic status and severe thrombocytopenia requiring HLA-matched platelet products, which are difficult to obtain quickly. To maintain adequate platelet counts (> 5 × 10 4 /µL) while awaiting liver transplantation, a thrombopoietin receptor agonist and rituximab were administered. During surgery, factor VIII concentrate was administered according to a previously planned protocol. Adequate hemostasis was obtained, and the operation was completed without uncontrollable coagulopathy. The postoperative course was uneventful, and the patient was discharged on postoperative day 41. Detailed planning is required for surgical patients with hemophilia and HIV/HCV cirrhosis, especially for those with a diverse spectrum of anti-HLA antibodies.

Background: Post-transplant lymphoproliferative disorder (PTLD) of T cell type has been rarely reported. Accurate diagnosis of this life-threatening rare form of PTLD is important for the treatment strategy., Case Presentation: A 7-year-old boy had severe diarrhea and weight loss progressively at 7 years post-living donor liver transplantation (LDLT) for biliary atresia. Endoscopy in the gastrointestinal (GI) tract revealed multiple erosions and ulcer lesions with prominent intraepithelial lymphocytosis in the duodenum and terminal ileum. Immunohistochemical examination demonstrated that these accumulated lymphocytes mainly comprised small- to medium-sized T cells expressing CD3, CD4, CD5, CD7, and CD103, but lacking CD8, CD56, and Epstein-Barr virus-encoded small RNAs. In addition, T cell receptor β gene rearrangement was detected by polymerase chain reaction analysis. Comprehensively, the lesions were best interpreted as post-transplant indolent T cell lymphoproliferative disorder (LPD) of the intestine. Clinical remission was achieved by reducing the immunosuppressant., Conclusion: A rarely reported indolent type of T cell LPD in post-LDLT was diagnosed by direct inspection and histological investigation. Although the histological classification and therapeutic strategy for post-transplant indolent T cell LPD have not been established, reducing immunosuppression allowed complete remission in our case. To prevent the incidence of PTLD and de novo malignancy, developing a methodology to set a proper dose of immunosuppressant is required.

Therapeutic drug monitoring for voriconazole, an antifungal agent, is essential for maximizing efficacy and preventing toxicity. The aim of this study was to elucidate the optimal maintenance dose of voriconazole in patients with severe liver cirrhosis (Child-Pugh class C) by reviewing the plasma trough concentrations obtained by therapeutic drug monitoring and daily doses of voriconazole. We retrospectively evaluated 6 patients with Child-Pugh class C cirrhosis who received oral voriconazole treatment and were liver transplant recipients or were awaiting liver transplantation. We compared their voriconazole trough concentrations and daily maintenance doses to those of patients who did not have liver cirrhosis (n=56). We found that plasma voriconazole trough concentrations in all patients with Child-Pugh class C were almost within therapeutic range, and the median plasma trough concentration at steady state was not significantly different from that of patients who did not have liver cirrhosis. In addition, the median daily maintenance dose of voriconazole was significantly lower (2.13 mg/kg/d) than that of the control patients (6.27 mg/kg/d), suggesting that trough voriconazole concentrations are elevated in Child-Pugh class C patients. Thus, we conclude that oral voriconazole maintenance doses in patients with Child-Pugh class C should be reduced to approximately one-third that of patients with normal liver function, with the follow-up dose adjusted by therapeutic drug monitoring.

Background: Hepaticojejunostomy may be used for biliary reconstruction in certain cases of liver transplantation. In this occasion, Roux-en-Y biliary reconstruction is predominantly performed. Petersen's hernia is an internal hernia that can occur after Roux-en-Y reconstruction, and it may lead to extensive ischemic changes affecting incarcerated portions of the small bowel or Roux limb resulting in severe complications with a poor prognosis., Case Presentation: The present case was a 44-year-old male who underwent living donor liver transplantation (LDLT) for familial amyloid polyneuropathy and in whom biliary reconstruction was performed with Roux-en-Y hepaticojejunostomy. Two years after liver transplantation, symptomatic bowel strangulation was diagnosed by CT examination and emergent surgery was performed accordingly. On exploration, an ischemic limb associated with Petersen's hernia was observed. Although repositioning of the incarcerated bowel loop gradually improved the color of the limb, the limb failed to completely recover to a normal color. To allow accurate evaluation for the viability of the limb, we decided to perform a second-look operation after 48 h. On re-exploration, the surface of the limb remained a dark color; however, intraoperative endoscopic findings revealed only partial necrosis of the mucosa. Next, we resected the portion of ischemic damaged limb only following side-to-side jejunojejunostomy. Consequently, redoing of biliary reconstruction could be avoided and the original hepaticojejunostomy site was preserved. Although the stricture of the remnant Roux limb occurred 1 month thereafter, it was successfully managed by balloon dilation via percutaneous transhepatic biliary drainage route., Conclusions: The occurrence of Petersen's hernia should always be considered in cases of liver transplantation with Roux-en-Y biliary reconstruction. On the basis of an accurate assessment of the extent of jejunal limb injury, reanastomosis of the hepaticojejunostomy, a potentially high-risk surgical procedure, can be avoided in emergent situations.

Objective and Design: To examine the effect of 3-[(dodecylthiocarbonyl)-methyl]-glutarimide (DTCM-G), a novel anti-inflammatory agent that inhibits lipopolysaccharide (LPS) activation of RAW264.7 macrophages, on murine models of colitis and RAW264.7 cells., Materials and Methods: Colitis was induced by rectally infusing trinitrobenzenesulfonic acid (TNBS) (1.5 mg in 50% ethanol) in BALB/c mice or orally administering 3% dextran sulfate sodium (DSS) for 5 days in C57BL/6 mice. The severity of colitis was assessed after intraperitoneally injecting DTCM-G (40 mg/kg). The anti-inflammatory properties of DTCM-G and its mechanisms were investigated in LPS-stimulated RAW264.7 cells., Results: DTCM-G significantly ameliorated TNBS-induced colitis, according to the body weight loss, disease activity index, colonic obstruction, macroscopic colonic inflammation score, mucosal myeloperoxidase activity, and histopathology. Immunohistochemistry and isolated lamina propria mononuclear cells showed significantly reduced colonic F4/80(+) and CD11b(+) macrophage infiltration. DTCM-G significantly suppressed tumor necrosis factor (TNF)-α and interleukin (IL)-6 messenger RNA expression in the colon and attenuated DSS-induced colitis, according to the disease activity index and histopathology. In RAW264.7 cells, DTCM-G suppressed LPS-induced TNF-α/IL-6 production and enhanced glycogen synthase kinase-3β phosphorylation., Conclusions: DTCM-G attenuated murine experimental colitis by inhibiting macrophage infiltration and inflammatory cytokine expression. Thus, DTCM-G may be a promising treatment for inflammatory bowel disease.

Background: Pancreatic islet transplantation (PITx) is an attractive treatment option for restoring appropriate glucose homeostasis in type 1 diabetes patients. Although islet grafts can successfully engraft after PITx, large numbers of islet grafts are required mainly because immune reactions, including inflammation, destroy islet grafts. In these processes, nuclear factor (NF)-κB plays a central role. We hypothesized that the inhibition of NF-κB activation would ameliorate inflammatory responses after PITx and aid successful engraftment., Methods: To test this hypothesis, a newly developed NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), was used on a syngeneic mouse PITx model. One hundred seventy-five islets from C57BL/6 (B6) mice were transplanted into streptozotocin-induced diabetic B6 mice. The recipient mice were administered DHMEQ for 1, 2, or 3 days after PITx. The underlying mechanisms of DHMEQ on islet graft protection were investigated in an in vitro coculture model of pancreatic islets and macrophages., Results: With a vehicle treatment, only 11.1% of the islet-recipients achieved normoglycemia after PITx. In sharp contrast, DHMEQ treatment markedly improved the normoglycemic rate, which was associated with the suppression of serum high mobility group complex-1 (HMGB1) and proinflammatory cytokines, including tumor necrosis factor-α, monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, interleukin-1β, and interleukin-6, after PITx. In a murine macrophage-like cell line, DHMEQ inhibited HMGB1-driven activation and proinflammatory cytokine secretion and further prevented death isolated islets after coculture with these activated macrophages., Conclusions: Inhibition of NF-κB activation by DHMEQ after PITx reduces the HMGB1-triggered proinflammatory responses and results in engraftment of transplanted islets even with fewer islet grafts.

Background: Long-term graft deterioration remains a major obstacle in the success of pancreatic islet transplantation (PITx). Antigen-independent inflammatory and innate immune responses strengthen subsequent antigen-dependent immunity; further, activation of nuclear factor (NF)-κB plays a key role during these responses. In this study, we tested our hypothesis that, by the inhibition of NF-κB activation, the suppression of these early responses after PITx could facilitate graft acceptance., Methods: Full major histocompatibility complex (MHC)-mismatched BALB/c (H-2) mice islets were transplanted into streptozotocin-induced diabetic C57BL/6 (B6: H-2) mice. The NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) was administered for either 3 or 14 days after PITx. To some PITx recipients, tacrolimus was also administered. Islet allograft survival, alloimmune responses, and in vitro effects of DHMEQ on dendritic cells (DCs) were assessed., Results: With a vehicle treatment, 600 islet allografts were promptly rejected after PITx. In contrast, 3-day treatment with DHMEQ, followed by 2-week treatment with tacrolimus, allowed permanent acceptance of islet allografts. The endogenous danger-signaling molecule high mobility group complex 1 (HMGB1) was elevated in sera shortly after PITx, whereas DHMEQ administration abolished this elevation. DHMEQ suppressed HMGB1-driven cellular activation and proinflammatory cytokine secretion in mouse bone marrow-derived DCs and significantly reduced the capacity of DCs to prime allogeneic T-cell proliferation in vitro. Finally, the DHMEQ plus tacrolimus regimen reverted the diabetic state with only 300 islet allografts., Conclusions: Inhibition of NF-κB activation by DHMEQ shortly after PITx suppresses HMGB1, which activates DCs and strengthens the magnitude of alloimmune responses; this permits long-term islet allograft acceptance, even in case of fewer islet allografts.

Background/purpose: In order to perform a preclinical trial of pancreatic islet transplantation (PIT) in nonhuman primates, insulin-dependent diabetes mellitus (IDDM) must be induced. Methods for IDDM induction are administration of streptozotocin (STZ) or total pancreatectomy (TP). While STZ-induced IDDM is not always reliable, TP is appropriate for IDDM induction. However, TP is very difficult because of the complex surgical procedure required, necessary confirmation of IDDM, and difficulty in postoperative management. In this study, we tried to establish a reliable IDDM model for PIT in cynomolgus monkeys., Methods: TP was performed in 5 male cynomolgus monkeys (Macaca fascicularis). This was followed by scheduled measurements of blood glucose, C-peptide levels, insulin levels, oral intake, and insulin requirements. IDDM induction was confirmed by serum C-peptide levels and intravenous glucose tolerance test (IVGTT). Allogeneic PIT was performed 14 days later with immunosuppressive therapy., Results: TP successfully induced IDDM in all animals, confirmed by reduction of fasting serum C-peptide levels. Negative responses of serum C-peptide levels and insulin in IVGTT provided further confirmation of IDDM induction. No mortality or morbidity was encountered in any of the animals., Conclusions: TP successfully induced IDDM for PIT in cynomolgus monkeys.

Endochondral ossification is an essential process not only for physiological skeletal development and growth, but also for pathological disorders such as osteophyte formation in osteoarthritic joints and ectopic ossification with ageing. We recently identified a novel cartilage-specific molecule carminerin. Although the carminerin-deficient mice developed and grew normally, pathological endochondral ossification were suppressed. We conclude that carminerin contributes to chondrocyte calcification during pathological endochondral ossification without affecting physiological skeletal conditions, suggesting that this molecule could be a therapeutic target for these disorders.

Contribution of genetic backgrounds to the etiology of lumbar spondylosis has been suggested by epidemiological studies. This study was designed to determine the association of restriction fragment length polymorphisms (RFLPs) of estrogen receptor (ER), vitamin D receptor (VDR), parathyroid hormone (PTH) and interleukin-1beta (IL-1beta) genes with the radiological severity of lumbar spondylosis at the disk level from L1/2 to L5/S1 in Japanese post-menopausal women. ER and VDR RFLP haplotypes were associated with the severity of spondylosis in the upper levels (L1/2 and L2/3) more than in the lower levels. Association of ER genotype was more pronounced in the group younger than average than in the older group, while that of VDR genotype was more significant in the older group. Neither PTH nor IL1-beta RFLP was associated with the severity at any levels in either stratified group. We thus conclude that ER and VDR genes may contribute to lumbar spondylosis in a distinct manner: estrogen sensitivity influences the severity in the early phase after menopause while vitamin D plays an important role at older ages when the contribution of estrogen loss is weaker.

Research to date has identified several genes that are implicated in the etiology of ossification of the posterior longitudinal ligament of the spine (OPLL); however, their pathogenetic relevance remains obscure. The aim of this study is to identify susceptibility genes for OPLL through a large-scale case-control association study and to re-examine previously reported associations. A total of 109 single nucleotide polymorphisms (SNPs) in 35 candidate genes were genotyped for 711 sporadic OPLL patients and 896 controls. The differences in allelic and genotypic distribution between patients and controls were assessed using the chi (2) test with Bonferroni's correction. We also analyzed the association by separating patients into subgroups according to sex, age and the number of ossified vertebrae. The nominal P values fell below 0.05 for five SNPs in three genes. An intronic SNP in the TGF3 gene (P=0.00040) showed the most significant association. Previously reported associations of COL11A2, NPPS and TGFB1 with OPLL could not be reproduced. Further, no significant associations were detected in stratified analyses based on sex, age or the number of ossified vertebrae. TGFB3 warrants further investigation because it is located within a genomic region that has been positively linked with OPLL.

Endochondral ossification is an essential process not only for physiological skeletal development and growth, but also for pathological disorders. We recently identified a novel cartilage-specific molecule, carminerin (also known as cystatin 10 and encoded by Cst10), which is upregulated in synchrony with cartilage maturation and stimulates the later differentiation of cultured chondrocytes. Although carminerin-deficient (Cst10-/-) mice developed and grew normally, they had a microscopic decrease in the calcification of hypertrophic chondrocytes at the growth plate. When we created experimental models of pathological endochondral ossification, we observed suppression of chondrocyte calcification during formation of osteoarthritic osteophytes, age-related ectopic ossification and healing of bone fractures in Cst10-/- mice. Cultured Cst10-/- chondrocytes showed a reduction in calcification with activation of an SRY site in the promoter of the gene encoding nucleotide pyrophosphatase phosphodiesterase 1 (NPP1, encoded by Enpp1). Functional NPP1 is required for carminerin deficiency to suppress the pathological endochondral ossifications listed above. Carminerin is the first cartilage-specific protein that contributes to chondrocyte calcification during endochondral ossification under physiological and pathological conditions through the transcriptional inhibition of NPP1.

This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes.

Study Design: A case-control association study and a stratified study investigating the genetic etiology for ossification of the posterior longitudinal ligament of the spine., Objective: To determine the association of restriction fragment length polymorphisms of estrogen receptor, vitamin D receptor, parathyroid hormone, and interleukin-1alpha and -1beta with susceptibility to ossification of the posterior longitudinal ligament of the spine and its severity., Summary of Background Data: Contribution of genetic backgrounds to the etiology for ossification of the posterior longitudinal ligament of the spine has been suggested by epidemiologic studies., Methods: Genomic deoxyribonucleic acid samples obtained from 120 patients (77 men and 43 women) with ossification of the posterior longitudinal ligament of the spine and 306 control subjects without the disorder (166 men and 140 women) were amplified by polymerase chain reaction, and polymorphism genotypes were determined by restriction endonuclease digestion. The distribution of genotypes was compared between patients with the disorder and control subjects. In addition, the severity of ossification was determined by the number of ossified vertebrae in patients with the disorder, and associations of the severity with age, gender, and genotypes were examined., Results: Estrogen receptor (P = 0.007) and interleukin-1beta (P = 0.001) polymorphisms exhibited different distributions between patients with ossification of the posterior longitudinal ligament of the spine and control subjects in women, but not in men. In patients with the disorder, the severity of ossification was negatively correlated with age in women (P = 0.013), but not in men. Estrogen receptor polymorphism was associated with the severity only in women (P = 0.001)., Conclusions: The contribution of genetic backgrounds is likely to be stronger in women than in men with ossification of the posterior longitudinal ligament of the spine. Estrogen receptor polymorphism was associated with both initiation and promotion of the disorder, but interleukin-1beta polymorphism was associated only with its initiation in women.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a disease that causes paralysis by compressing the spinal cord. Based on the fact that the nucleotide pyrophosphatase (Npps) gene is responsible for ectopic ossification in ttw, an OPLL model mouse, the possibility was explored whether the human NPPS gene is associated with susceptibility to and severity of OPLL. First, we screened for single-nucleotide polymorphisms (SNPs) in the human NPPS locus using selected 25 OPLL patients with young onset (< 35 years old) or severe ossification (> 10 ossified vertebrae), and identified three novel SNPs in the locus. A case-control association study between 180 OPLL patients and 265 non-OPLL controls showed that one of these SNPs, IVS15-14T --> C substitution, was more frequently observed in OPLL patients (p = 0.022), especially in those with severe ossification (p < 0.0001) and young onset (p = 0.002), than in controls. A stratified study with the number of ossified vertebrae in OPLL patients revealed that IVS15-14T --> C substitution (p = 0.013) as well as young onset (p = 0.046) and female sex (p = 0.006) were associated with severe ossification. We conclude that the IVS15-14T --> C substitution in the human NPPS gene is associated not only with susceptibility to, but also with severity of OPLL.

The Trp-Asp (WD) motif has been shown to exist in a number of proteins. Genes containing repeats of the WD motif compose a large gene family associated with a variety of cellular functions and can be divided into a number of functional subfamilies. By means of the differential display method using ttw, a mouse model for the early stage of ectopic ossification, we have identified a novel mouse gene, Wdr8 (WD repeat domain 8), which contains two WD repeats, together with its human orthologue. The human and mouse WDR8 genes encode 460 and 462 amino acids, respectively, with 89% identity, and are expressed in almost all tissues, including bone and cartilage, and in bone-forming cells, including osteoblasts and chondrocytes. Wdr8 expression in cartilage was differentially displayed by stimuli for ectopic ossification in ttw and was observed strongly only at a transition period from hypertrophic to mineralizing stages in ATDC5, a chondrogenic cell line that exhibits endochondral ossification, suggesting a potential role for Wdr8 in the process of ossification. The WDR8 protein is highly conserved among a variety of species, but is distinctly different from other WD-repeat proteins, indicating that it represents a novel subfamily of the WD-repeat gene family., (Copyright 2001 Academic Press.)

The crystal structure of cytochrome c(6) from the red alga Porphyra yezoensis has been determined at 1.57 A resolution. The crystal is tetragonal and belongs to space group P4(3)2(1)2, with unit-cell parameters a = b = 49.26 (3), c = 83.45 (4) A and one molecule per asymmetric unit. The structure was solved by the molecular-replacement method and refined with X-PLOR to an R factor of 19.9% and a free R factor of 25.4%. The overall structure of cytochrome c(6) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys14 and Cys17, and the iron has an octahedral coordination with His18 and Met58 as the axial ligands. The sequence and the structure of the eukaryotic red algal cytochrome c(6) are very similar to those of a prokaryotic cyanobacterial cytochrome c(6) rather than those of eukaryotic green algal c(6) cytochromes.

In the double-door laminoplasty procedure used for patients with cervical myelopathy, techniques to obtain a wider spinal canal have been tested. We hypothesized that making the lateral gutters as lateral as possible may be effective for obtaining a wider spinal canal. In this study we aimed to test our hypothesis by using a new measurement method to assess the postoperative cross-sectional area of the spinal canal. Fifty-six vertebrae were operated on in 11 patients, using the STSS spacer (Asahi Optical, Tokyo, Japan). On computed tomography (CT) scans, the most posterior edge of a space-occupying lesion was defined as point A and a horizontal line through point A was drawn parallel to the posterior wall of the vertebral body (line X). Two oblique lines were drawn along the medial margin of the bilateral split laminae (lines R and L). The triangular area enclosed by lines X, R, and L was defined as the substantial area of the spinal canal (SASC). A horizontal line through the median points of the bilateral facet joint in the anteroposterior direction was defined as line B. According to the relative position of line X to line B, two types of vertebra were observed: In type 1, line X was located ventral to line B; in type 2, line X was located dorsal to line B. The mean data values for type 1 vertebra were: SASC, 133.1 mm2; ATD, 22.9 mm; angle R, 61.3 degrees; and angle L, 61.8 degrees. The mean data values for type 2 vertebra were: SASC, 99.8 mm2; ATD, 20.1 mm; angle R, 58.6 degrees; and angle L, 57.5 degrees. There were significant differences between types 1 and 2 vertebrae in values both for SASC and ATD (P < 0.01). There were no significant differences between types 1 and 2 vertebrae regarding angles R and L. The difference in SASC in the two types appeared to arise from the difference in ATD. In other words, the degree of enlargement of the spinal canal was determined not by the inclination of the bilateral split laminae, but by the transverse diameter. This result supports the validity of our hypothesis. In a double-door laminoplasty, to obtain a wider spinal canal, the lateral gutters must be made as lateral as possible (i.e., at the medial border of the facet joints, especially in type 2 vertebra). On the preoperative CT scan, the relative positions of the large-volume lesion and the facet joint must be noted.