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Malignant peritoneal mesothelioma (MPM) is a rare malignant tumor with peritoneal thickening. Tuberculous peritonitis also shows peritoneal thickening, so differentiating between the two is important but difficult if latent tuberculosis infection (LTBI) is present. We herein report a patient with MPM and LTBI. A 79-year-old man was diagnosed with peritoneal thickening on computed tomography. Interferon gamma release assay (IGRA) results were positive, suggesting tuberculous peritonitis. He underwent a laparoscopic omental biopsy and was diagnosed with MPM, which can occur together with LTBI. If peritoneal thickening is observed, an IGRA should be performed early, and the possibility of LTBI should be considered.

A 52-year-old man was found to have an increase in carbohydrate antigen (CA)19-9. He underwent endoscopic ultrasound/fine needle aspiration (EUS-FNA) of the soft tissue shadow near the pancreas and was diagnosed with adenocarcinoma. The cancer was judged to be of unknown primary. Cancers of unknown primary make up 3-5% of malignant tumors, and it is difficult to determine the treatment policy. FOLFIRINOX’s efficacy in cancer of unknown primary is unclear. In this case, he received chemotherapy with FOLFIRINOX. After five cycles of treatment, the best effect was stable disease (SD). However, he died about 6 months after the onset of symptoms. Pathological anatomy after death revealed that the primary lesion was in the pancreas. FOLFIRINOX may be effective for cancers of unknown primary. Pathological anatomy is important to confirm the validity of treatment. It is important to consider giving chemotherapy if there is a presumed primary tumor even if the cancer is of unknown primary. J Med Cases. 2021;12(11):442-445 doi: https://doi.org/10.14740/jmc3812

Objectives There are patients who do not undergo colonoscopy even if the fecal immunochemistry test (FIT) results are positive and even with repeated positive test results the following year. We aimed to investigate colorectal cancer (CRC) risk in examinees with positive FIT results in our annual screening program. Methods We analyzed patients who underwent initial colonoscopy from April 2010 to March 2017 because of positive FIT results using an endoscopy database in our hospital. We investigated the difference in the risk of advanced colorectal neoplasia as a surrogate marker of CRC between those who had an initial positive test and those who had repeated positive tests. Results A total of 748 patients were included in this analysis. The advanced neoplasia detection rates were 7.6% (50/656) and 18.5% (17/92) for the initial and repeated positive test groups, respectively. Subgroup analysis of those with repeated positive tests revealed that the detection rates in examinees with positive tests 1-2 and >2 years ago were 16.7% (6/36) and 19.6% (11/56), respectively. The odds ratios for advanced neoplasia detection in patients with positive tests 1-2 and >2 years ago compared with those in the initial positive test group were 2.72 (95% confidence interval [CI], 1.04-7.10) and 3.09 (95% CI, 1.47-6.48), respectively. Conclusions The risk of CRC appears more than doubled in patients with a repeated positive FIT result. Prompt colonoscopy is recommended for FIT-positive cases.

We report the case of diffuse large B-cell lymphoma (DLBCL) with a fistula from the ileum to the cecum. A 57-year-old male came to the hospital complaining of abdominal pain. He underwent an abdominal computed tomography with contrast, which showed full-thickness wall thickening at the ileocecal region. He underwent a lower gastrointestinal endoscopy. No tumor was found at the ileocecal valve, and macroscopic findings were normal. The scope was advanced to the cecum, an additional outpouching was found. The outpouching appeared to be an ileocecal fistula. The diagnosis was DLBCL.

We experienced a case of gastric cancer with multiple liver metastases characterized by frequent hypoglycemic attacks. Hypoglycemia was observed on admission. We suspected that the cause of this hypoglycemia was non-islet cell tumor hypoglycemia (NICTH). Staining of the tissue with an insulin-like growth factor (IGF)-II antibody revealed that IGF-II was present in the tumor cells. This finding suggested that the tumor was producing IGF-II, which leads to NICTH. After starting parenteral nutrition, the patient emerged from the hypoglycemic coma. He remained out of the coma until he died of liver failure.

An 81-year-old man was diagnosed with Goodpasture syndrome (GS) because he met the criteria of positive anti-GBM antibodies, rapid progressive glomerulonephritis and pulmonary hemorrhage. After starting plasmapheresis and steroid pulse therapy, he experienced tarry stool and contrast-enhanced CT revealed an aneurysmal finding in the jejunum. Paroral enteroscopy showed a jejunal Dieulafoy's lesion (DL) with gush-out hemorrhage. Hemostasis was successfully achieved by hemoclipping, and he then experienced no re-bleeding events. GS can present as a jejunal DL, and contrast-enhanced CT is useful for investigating the etiology and site of small intestinal bleeding, which can lead to smooth, effective endoscopic hemostasis.

Magnetic compression anastomosis (MCA) was developed as a low-invasive treatment for gastro-enteric or entero-enteric obstruction. A 72-year-old man underwent subtotal gastrectomy with Billroth II reconstruction for early gastric cancer. After the operation, he suffered from repeated aspiration pneumonia due to anastomotic obstruction caused by jejunal kinking at the efferent loop of anastomosis. We therefore performed jejunojejunostomy via the MCA technique, as his situation was not improved despite conservative therapy and he had a high reoperative risk. We prepared two flat plate-shaped neodymium magnets (15 × 3 mm) each with a small hole, and a nylon thread was passed through each hole. Each magnet was then delivered endoscopically to the anal side of the jejunal kinking, subsequently to the anastomosis, using biopsy forceps. The two magnets immediately became attracted towards each other transmurally. Oozing hemorrhage with clot at the mated magnets was observed 10 days after starting the compression. After retrieving the magnets, we confirmed the completion of jejunojejunostomy and then successfully achieved hemostasis of the anastomotic hemorrhage using argon plasma coagulation. The widely patent anastomosis was confirmed endoscopically 1 month after canalization; and he has been asymptomatic and able to eat a normal diet ever since. Endoscopic MCA is an effective, low-invasive treatment for anastomotic obstruction after subtotal gastrectomy. A standardized, safer procedure should be established for general use in the clinical setting.

Background The usefulness of prophylactic biliary stenting for patients with common bile duct stones (CBDS) and gallstones (GS) to prevent recurrent biliary events after endoscopic sphincterotomy (EST) and CBDS extraction before elective cholecystectomy remains controversial. The aim of this study was to evaluate the risk of recurrent CBDS around the perioperative period and clarify its risk factors. Methods The clinical data of all patients who received prophylactic biliary stenting after EST for CBDS and later underwent cholecystectomy for GS followed by stent extraction in our institution were retrospectively reviewed. The numbers of residual CBDS at the end first and second endoscopic retrograde cholangiography (ERC) studies were compared. Univariate and multivariate analyses were performed using a logistic regression model to determine risk factors for recurrent CBDS in the perioperative period. Results Forty-two consecutive patients received prophylactic biliary stenting and subsequent cholecystectomy for GS. Three of these patients were excluded from this study because the number of residual stones was not confirmed. The median maximum CBDS diameter at second ERC was 0 mm (range, 0 - 10 mm); six patients had multiple CBDS (≥ 5). The number of CBDS at second ERC was increased in comparison to that at the first ERC in 15 patients (38.4%), and was unchanged or decreased in 24 patients. The median minimum cystic duct diameter was 4 mm (range, 1 - 8 mm). The median interval between first ERC and operation was 26 days (range, 2 - 131 days). The median interval between operation and second ERC was 41 days (range, 26 - 96 days). Laparoscopic cholecystectomy (LC) was performed in 38 patients, one of whom was converted from LC to open cholecystectomy. Postoperative complications (transient bacteremia) occurred in one patient. The cystic duct diameter was an independent risk factor for an increased number of CBDS at second ERC in the multivariate analysis (odds ratio 0.611 (95% confidence interval (0.398 - 0.939)), P = 0.03). Conclusion Recurrent CBDS around the perioperative period of cholecystectomy is not a rare complication after EST and the removal of CBDS with concomitant GS. Prophylactic biliary stenting is considered useful for preventing CBDS-associated complications, especially for patients in whom the cystic duct diameter is larger (≥ 5 mm).

A woman in her 70s with systemic sclerosis experienced dyspnea, and consequently, she was diagnosed with an esophago-pleural fistula, which was caused by a perforated esophageal ulcer. We administered conservative treatments including continuous pleural drainage and total parenteral nutrition. The fistula was closed but recurred, at which point we attempted to close the fistula by filling and shielding using polyglycolic acid (PGA) sheets and fibrin glue (FG). We were able to safely and smoothly fill and shield the fistula using the PGA sheets with a guidewire. We show that endoscopic closure of an esophago-pleural fistula using this technique is an effective, low-invasive treatment for gastrointestinal perforation and refractory fistulas.

A 69-year-old man was referred to our department with acute hepatitis. He had been newly treated with benidipine hydrochloride for two months. His blood test results were as follows: aspartate aminotransferase, 1,614 IU/L; alanine aminotransferase, 1,091 IU/L and anti-smooth muscle antibody, ×80. Needle liver biopsy specimen showed interface hepatitis with mainly lymphocytic infiltration and bridging fibrosis in the periportal area. Immunohistochemistry revealed lymphocytic infiltration positive for IgG4. We diagnosed him with IgG4-related AIH with an etiology that was suspected of being drug-induced. Oral prednisolone was started and then tapered after achieving biochemical remission. Hepatitis recurred after the cessation of steroids; however, remission was achieved with ursodeoxycholic acid.

Background and aim Because the risk of colorectal cancer has not been well examined in fecal immunochemistry test (FIT)-positive patients who previously underwent colonoscopy, this study aimed to investigate this topic. Methods This was a single-center, observational study of prospectively collected data in Japan. FIT-positive, average-risk patients who underwent colonoscopy were divided into groups as follows: those who never underwent colonoscopy in the past (no colonoscopy group), those with a history of colonoscopy between 6 months and 5 years (0.5- to 5-year colonoscopy group), and those with a history of colonoscopy more than 5 years ago (> 5-year colonoscopy group). We investigated the prevalence of advanced neoplasia and invasive cancer among these groups using multiple logistic regression analysis. Results Detection rates of advanced neoplasia in the no colonoscopy group, 0.5- to 5-year colonoscopy group, and > 5-year colonoscopy group were 14.8% (240/1626), 3.9% (13/330), and 6.9% (17/248), respectively. Detection rates of invasive cancer in each aforementioned group were 5.7% (92/1,626), 0.3% (1/330), and 1.2% (3/248), respectively. Odds ratios of advanced neoplasia in the 0.5- to 5-year colonoscopy group and > 5-year colonoscopy were 0.23 (95% confidence interval [CI]: 0.13-0.42) and 0.40 (95% CI: 0.24-0.68), respectively, in multivariate analysis. The odds ratios of invasive cancer in each aforementioned group were 0.05 (95% CI: 0.01-0.37) and 0.19 (95% CI: 0.06-0.61), respectively. Conclusion Re-screening with the FIT should not be recommended for at least 5 years for average-risk patients after colonoscopy without high-risk neoplasms, because the risks of colorectal cancer are low in such patients.

Magnetic compression anastomosis (MCA) has been developed as a non-surgical alternative treatment for biliary obstruction without serious complications. A 70-year-old woman who had undergone pancreaticoduodenectomy with modified Child reconstruction for pancreatic head cancer suffered from obstructed choledochojejunostomy with no recurrent findings 4 months after the operation. Cholangiography using the percutaneous transhepatic cholangiographic drainage (PTCD) and fluoroscopy revealed complete obstruction of the upper common bile duct, and the length of the obstruction was 7 mm. Intraductal ultrasonography (IDUS) showed fibrous heterogenous hyperechoic appearance without fluid collection, vessels or foreign bodies at the site of the obstruction. We performed choledochojejunostomy using the MCA technique. One magnet was inserted into the obstruction of the hepatic side through the PTCD fistula. Another was delivered endoscopically to the obstruction of the jejunal side. The two magnets were immediately attracted towards each other transmurally, and reanastomosis was confirmed 7 days after starting the compression. The magnets were easily retrieved endoscopically. A 16-Fr indwelling drainage tube was placed in the jejunum through the PTCD. The internal tube is still in place 6 months after reanastomosis, and no MCA-related complications have been observed. In conclusion, MCA is a safe, effective, low-invasive treatment for biliary obstruction, and IDUS is useful for the pretreatment assessment of feasibility and safety.

A 58-year-old male receiving two types of antithrombotic medication developed acute obstructive suppressive cholangitis due to choledocholithiasis. During the first endoscopic retrograde cholangiopancreatography (ERCP) procedure, we performed biliary plastic stenting. Seven days after this procedure and with continued antithrombotic treatment, we performed ERCP with endoscopic sphincterotomy and stone extraction. Twelve hours after this procedure, the patient suffered transient unconsciousness and progression of anemia. Sixty hours after the procedure, he experienced right hypochondralgia and hiccups. Ultrasonography and computed tomography revealed a subcapsular hepatic hematoma. Bleeding was successfully arrested with selective arterial embolization. We suspected that the cause of these problems was vessel injury from the rigid portion of the guidewire during the ERCP procedure.

Because the risk of colorectal cancer has not been well examined in fecal immunochemistry test (FIT)-positive patients who previously underwent colonoscopy, this study aimed to investigate this topic.This was a single-center, observational study of prospectively collected data in Japan. FIT-positive, average-risk patients who underwent colonoscopy were divided into groups as follows: those who never underwent colonoscopy in the past (no colonoscopy group), those with a history of colonoscopy between 6 months and 5 years (0.5- to 5-year colonoscopy group), and those with a history of colonoscopy more than 5 years ago ( 5-year colonoscopy group). We investigated the prevalence of advanced neoplasia and invasive cancer among these groups using multiple logistic regression analysis.Detection rates of advanced neoplasia in the no colonoscopy group, 0.5- to 5-year colonoscopy group, and 5-year colonoscopy group were 14.8% (240/1626), 3.9% (13/330), and 6.9% (17/248), respectively. Detection rates of invasive cancer in each aforementioned group were 5.7% (92/1,626), 0.3% (1/330), and 1.2% (3/248), respectively. Odds ratios of advanced neoplasia in the 0.5- to 5-year colonoscopy group and 5-year colonoscopy were 0.23 (95% confidence interval [CI]: 0.13-0.42) and 0.40 (95% CI: 0.24-0.68), respectively, in multivariate analysis. The odds ratios of invasive cancer in each aforementioned group were 0.05 (95% CI: 0.01-0.37) and 0.19 (95% CI: 0.06-0.61), respectively.Re-screening with the FIT should not be recommended for at least 5 years for average-risk patients after colonoscopy without high-risk neoplasms, because the risks of colorectal cancer are low in such patients.

We investigated the usefulness of screening for colorectal cancer (CRC) using immunological fecal occult blood test (FOBT) in 472 scheduled inpatients (median age, 68.6 years) who underwent screening for CRC via FOBT (single stool sample) at our hospital. The recall rate for further examination was 26.6% (126/472), and the rate of patients who underwent further examination was only 34.9% (44/126). The overall colorectal neoplasm detection rate, overall CRC detection rate, and positive predictive value for CRC in inpatients were 5.5% (26/472), 1.4% (7/472), and 5.5% (7/126), respectively, which were higher than those of population-based screening for CRC. Screening for CRC using FOBT in inpatients is a non-invasive and efficient method to detect latent CRC.

Hypertension and oxidant stress predispose to the onset and progression of arteriosclerotic diseases. In this study, the components of two kinds of porcine liver hydrolysates (LH-I and LH-II) were analyzed, and the antioxidant effects and angiotensin converting enzyme (ACE)-inhibiting effects of LH-I and LH-II were examined in vitro. Furthermore, the effects of LH-I and LH-II on the blood pressure were examined in spontaneously hypertensive rats (SHR). The results showed that peptides and amino acids accounted for 70% or more of the constituents of both LH-I and LH-II. The results of gel filtration HPLC showed that most of the nitrogen-containing components were peptides or amino acids with molecular weights of 6000 or less. The DPPH radical scavenging activities of LH-I and LH-II were 55.6 and 38.1 μM Trolox Equivalent/g, respectively. The IC(50) values for the ACE-inhibiting activity of LH-I and LH-II were 0.18 and 0.31 mg/mL, respectively. Oral administration of 1 g/rat of LH-I or LH-II to SHR resulted in significant lowering of the blood pressure. These findings indicate that both LH-I and LH-II have antioxidant activity and ACE-inhibiting activity. Moreover, both exerted a blood pressure-lowering effect in SHR. The antioxidant activity and ACE-inhibiting activity of LH-I and LH-II are presumed to be based on the actions of the component peptides.

Adjuvant arthritis was induced in rats in the growth stage (aged 6 weeks) and those in the mature stage (aged 4 months), and changes in the systemic bone turnover and the effects of methotrexate (MTX, CAS 133073-73-l) were compared. After induction of adjuvant arthritis, the paw edema ratio and the urinary deoxypyridinoline (u-Dpy) level increased in both age groups. No marked changes were observed in the serum osteocalcin (s-OC) level in either group. In the 6-week-old rats, arthritis completely inhibited the bone mass, and strength of the femur and lumbar vertebral body. The 4-month-old rats showed more marked changes than the 6-week-old rats in the bone mass and strength of the lumbar, vertebral body. MTX administration (0.05, 0.1 and 0.2 mg/kg/day) resulted in significant dose-dependent inhibition of arthritis-induced changes, and the effects of MTX were similar between the two age groups. MTX was useful at each age. These results suggest that 4-month-old rats with arthritis are more appropriate as a mode1 for evaluation of drugs for bone metabolic turnover in human chronic rheumatoid arthritis.

In clinical trials, acotiamide hydrochloride (acotiamide: Z-338) has been reported to be useful in the treatment of functional dyspepsia. Here, we investigated the effects of acotiamide on gastric contraction and emptying activities in rats in comparison with itopride hydrochloride (itopride) and mosapride citrate (mosapride). We also examined in vitro the compound's inhibitory effect on acetylcholinesterase (AChE) activity derived from rat stomach. In in vivo studies, acotiamide (30 and 100mg/kg s.c.) and itopride (100mg/kg s.c.) markedly enhanced normal gastric antral motility in rats. In gastric motility dysfunction models, acotiamide (100mg/kg s.c.) and itopride (100mg/kg s.c.) improved both gastric antral hypomotility and the delayed gastric emptying induced by clonidine, an α(2)-adrenoceptor agonist. In contrast, mosapride (10mg/kg s.c.) had no effect on these models. Like the AChE inhibitors itopride (30 mg/kg s.c.) and neostigmine (10 μg/kg s.c.), acotiamide (10mg/kg s.c.) also clearly enhanced gastric body contractions induced by electrical stimulation of the vagus, which were abolished by atropine and hexamethonium, whereas mosapride (3 and 10mg/kg s.c.) did not. In in vitro studies, acotiamide concentration-dependently inhibited rat stomach-derived AChE activity (IC(50)=2.3 μmol/l). In addition, stomach tissue concentrations of acotiamide after administration at 10mg/kg s.c. were sufficient to produce inhibition of AChE activity in rat stomach. These results suggest that acotiamide stimulates gastric motility and improves gastric motility dysfunction in rats by inhibiting AChE activity, and may suggest a role for acotiamide in improving gastric motility dysfunction in patients with functional dyspepsia.

Fully modified 4'-thioDNA, an oligonucleotide only comprising 2'-deoxy-4'-thionucleosides, exhibited resistance to an endonuclease, in addition to preferable hybridization with RNA. Therefore, 4'-thioDNA is promising for application as a functional oligonucleotide. Fully modified 4'-thioDNA was found to behave like an RNA molecule, but no details of its structure beyond the results of circular dichroism analysis are available. Here, we have determined the structure of fully modified 4'-thioDNA with the sequence of d(CGCGAATTCGCG) by NMR. Most sugars take on the C3'-endo conformation. The major groove is narrow and deep, while the minor groove is wide and shallow. Thus, fully modified 4'-thioDNA takes on the A-form characteristic of RNA, both locally and globally. The only structure reported for 4'-thioDNA showed that partially modified 4'-thioDNA that contained some 2'-deoxy-4'-thionucleosides took on the B-form in the crystalline form. We have determined the structure of 4'-thioDNA in solution for the first time, and demonstrated unexpected differences between the two structures. The origin of the formation of the A-form is discussed. The remarkable biochemical properties reported for fully modified 4'-thioDNA, including nuclease-resistance, are rationalized in the light of the elucidated structure.

The synthesis and properties of fully modified 4'-thioDNAs, oligonucleotides consisting of 2'-deoxy-4'-thionucleosides, were examined. In addition to the known literature properties (preferable hybridization with RNA and resistance to endonuclease hydrolysis), we also observed higher resistance of 4'-thioDNA to 3'-exonuclease cleavage. Furthermore, we found that fully modified 4'-thioDNAs behaved like RNA molecules in their hybridization properties and structural aspect, at least in the case of the 4'-thioDNA duplex. This observation was confirmed by experiments using groove binders, in which a 4'-thioDNA duplex interacts with an RNA major groove binder, lividomycin A, but not with DNA groove binders, to give an increase in its thermal stability. Since a 4'-thioDNA duplex competitively inhibited the hydrolysis of an RNA duplex by RNase V1, it was not only the physical properties but also this biological data suggested that a 4'-thioDNA duplex has an RNA-like structure.

The hepatoprotective effect of ZNC-2381 (1-(4-aminophenyl) methyl-3-(3-nitrophenyl)-1,3-dihydroimidazo[4,5-b]pyridine-2-one), a novel 2-one dihydroimidazopyridine derivative, has been evaluated in several experimental models of hepatic injury. In mice, oral ZNC-2381, administered at doses of 3, 10 or 30 mg kg−1, 1 h before induction of hepatic injury with concanavalin A, dose-dependently inhibited increases in serum alanine aminotransferase (ALT) activity. Apoptosis of liver cells, as indicated by DNA fragmentation (nucleosome assay) and DNA-ladder formation (electrophoresis), was also inhibited dose-dependently. ZNC-2381 dose-dependently inhibited concanavalin A-induced increases in serum tumour necrosis factor (TNF)-α levels, and TNF-α mRNA expression in the liver. Oral ZNC-2381 also dose-dependently inhibited increases in serum ALT activity in mice with hepatic injury induced by Propionibacterium acnes and a bacterial lipopolysaccharide (LPS) or d-galactosamine-LPS, and in rats with d-galactosamine-induced hepatic injury. These results indicate that oral ZNC-2381 inhibits cytokine (TNF-α) production and cytokine-related hepatocellular apoptosis, and might thus prevent different types of hepatic injury.

Urinary pyridinoline (U-Py) is a useful marker of bone and cartilage loss in rheumatoid arthritis (RA) . The serum pyridinoline (S-Py) concentration instead of U-Py was measured both in rats with adjuvant-induced (AA) and mice with collagen-induced (CIA) arthritis by high perfomance liquid chromatography (HPLC) . On the seventh day after adjuvant innoculation, the AA rats showed a significant increase in S-Py levels. The level of S-Py was gradually increased with the development of AA. Moreover, in CIA mouse, another arthritic model, on sixty days after innoculation of the type II collagen the S-Py level was also significantly higher than that in the normal mice.These findings indicate that the measurement of S-Py instead of U-Py may be useful as marker of bone and cartilage destruction in patients with rheumatoid arthritis.

Effects of anti-rheumatic drugs, gold sodium thiomalate (GST), methotrexate (MTX), D-penicillamine (D-P) and bucillamine (BU), on rhIL-1β-induced nitric oxide (NO) production in rat synovial fibroblast-like cells (SFC) cultured in vitro were investigated. The SFC (passage 4) were cultured for 48hrs in Dulbecco's modified Eagle's medium containing an NO inducer, LPS or rhIL-1β. NO production by SFC was assessed by using Griess reagent. RhIL-1β but not LPS, induced a significant increase in NO production in SFC. The rhIL-1β-induced NO production was inhibited by the presence of GST in a dose dependent manner, whereas MTX (100 ng/ml) had no effect. In contrast, both D-P (10 μg/ml) and BU (3 μg/ml) significantly stimulated rhIL-1β-induced NO production in SFC. This effect of SH-containing antirheumatic drugs was completely abolished by the presence of NO inhibitors, aminoguanidine (AG : 1 mM) and cycloheximide (CH : 2 μM) . AG but not CH, added at a later stage of culture (24-48hrs) abolished rhiL 1β induced NO production, indicating that these SH-compounds stimulate NO synthase activity in SFC.The present study suggests that SH-anti-rheumatic drugs stimulate rhIL-1β-induced NO production in SFC, and that the increase in NO may partly inhibit the bone destruction in rheumatoid arthritis.

2'-Deoxy-4'-thioribonucleic acid (4'-thioDNA) having a sulfur atom instead of an oxygen atom in the furanose ring has a nuclease resistance and hybridization ability higher than that of natural DNA. Despite its great potential for various biological applications, a long 4'-thioDNA having all four kinds of 2'-deoxy-4'-thionucleosides has not been reported. In this study, we describe systematic analysis of the incorporation of 2'-deoxy-4'-thionucleoside 5'-triphosphates (dSNTPs) using various DNA polymerases. We found that family B DNA polymerases, which do not have 3'→5' exonuclease activity, could efficiently incorporate dSNTPs via single nucleotide insertion and primer extension. Moreover, 104-mer PCR product was obtained even under the conditions in the presence of all four kinds of dSNTPs when KOD Dash DNA polymerase was used. The resulting PCR product was converted into a natural dsDNA by using PCR with dNTPs, and sequencing of the natural dsDNA revealed that the PCR cycle successfully proceeded without losing the sequence information of the template. To the best of our knowledge, this is the first example of accurate PCR amplification of highly modified DNA in the presence of only unnatural dNTPs.

Human monocytic leukemia THP-1 cells were induced to differentiate into macrophage-like cells by treatment with cardiotonic steroid bufalin, which was previously shown to interact with the Na+, K+-ATPase with similar kinetics to ouabain, a specific inhibitor of the enzyme. This induction of differentiation was characterized by loss of proliferation, cell adherence, increased ability to reduce Nitro Blue tetrazolium (NBT), and increased expression of interleukin 1 beta (IL-1 beta). During this process, bufalin downregulated c-myb and c-myc expressions and induced c-fos and Egr-1 transcripts. Ouabain also caused similar changes in proto- oncogene expression and induced phenotypic markers of differentiated cells at concentrations comparable to bufalin. The 12-O-tetradecanoyl phorbol-13-acetate resistant THP-1 cell variant, which was unresponsive to this agent as to growth inhibition and proto-oncogene expression, responded to bufalin. The finding that protein kinase inhibitor H7 failed to bufalin-mediated c-fos induction further supports the theory that the signal transduction machinery caused by bufalin is separable from the phorbol ester. The cytotoxic effect of high doses of bufalin apparently disappeared in the medium where Na+ was replaced with choline ions. Furthermore, bufalin failed to induce c-fos expression and to downregulate c-myb transcripts in the low-Na+ medium. These findings indicate that an increased intracellular Na+ concentration resulting from the Na+, K(+)-ATPase inhibition possibly triggers the change in proto-oncogene expression evoked by bufalin.

Nitric oxide (NO) produced by the inducible NO synthase (iNOS) in the some tissue is associated with acute and chronic inflammatory process. This study is to examine that the effect of methotrexate (MTX) on the NO production from rat peritoneal macrophages by the iNOS. Macrophages from normal rats were cultured in the presence of drug followed by activation with lipopolysaccharide (LPS) . After 24 hrs, NO-2 as a metabolite of NO was measured by Griess reaction. MTX, bucillamine (BU), and indomethacin (IM) did not influence on the NO-2 accumulation with LPS. Aminoguanidine (AG), a preferential inhibitor of iNOS, and dexamethasone (DEXA) abolished NO-2 accumulation with LPS in concentration dependent manner.On the other hand, adjuvant-induced arthritis (AA) was induced in rats by the injection of Mycobacterium tuberculosis into the right hind foot pad. Drugs were administered daily by the oral route from day 1 to day 21. On day 22, macrophages from AA rats had an enhanced capacity to produce NO in vitro. MTX (0.1 mg/kg), DEXA (0.5 mg/kg) and IM (1 mg/kg) reduced the paw swelling and inhibited the increased NO production by macrophages. AG (100 mg/kg) only partly inhibited the increased NO production, while BU (10 mg/kg) did not influence the paw swelling and the increased NO production.Our data revealed that oral administration of MTX to AA rats significantly inhibited ex vivo LPS-induced NO production from peritoneal macrophages in accordance with its in vivo prophylactic effect of arthritis, suggesting that NO blockade might be one of the important mode of action for MTX in animal models of arthritis.

Insulin resistance associated with visceral fat obesity has been suggested to be the pathological basis of metabolic syndrome. Many studies have demonstrated increased oxidant stress in diabetic patients and animal models of diabetes mellitus. In this study, the effect of liver hydrolysate administration on the blood glucose was examined in SHR/NDmcr-cp (SHR-cp) rats that show spontaneously occurring metabolic syndrome-like abnormalities. The SHR-cp rats were fed diets containing 5% liver hydrolysate for 12 weeks, and the fasting blood glucose and HbA1c were determined every 3 weeks. After administration of the liver hydrolysate-containing feed for 12 weeks, an oral glucose tolerance test was conducted and the plasma angiotensin II (AngII) concentrations were determined. The liver hydrolysate administration had no effect on the blood insulin levels in the oral glucose tolerance test, but significantly inhibited the d-glucose-induced increases of the blood glucose levels. Furthermore, the liver hydrolysate had almost no effect on the fasting blood glucose level, but tended to inhibit the increase of HbA1c. The plasma AngII concentration after the 12-week administration of liver hydrolysate remained significantly lower than that in the control group. These results indicate that a component of liver hydrolysate inhibits d-glucose-induced increase of the blood glucose level, and may improve insulin resistance. The angiotensin converting enzyme (ACE)-inhibiting effect and antioxidant effect of liver hydrolysate may be involved in this effect.

We describe the design and proof of concept of a pair of chemical probes for investigating DNA-protein interactions-specifically, the incorporation of 7-bromo-7-deazaadenine and 3-bromo-3-deazaadenine 2'-deoxynucleosides (Br(7)C(7)dA and Br(3)C(3)dA) into oligodeoxynucleotides (ODNs)-and their utility. Whereas the bromo substituent of the Br(7)C(7)dA unit in an ODN duplex acts sterically to inhibit binding with NF-kappaB, which interacts with the duplex in its major groove, the bromo substituent of the Br(3)C(3)dA unit acts sterically to inhibit binding with RNase H, which interacts with the duplex in its minor groove. In addition, the utilization of ODNs containing 7-deazaadenine and 3-deazaadenine 2'-deoxynucleosides (C(7)dA and C(3)dA), together with the pair of chemical probes, afforded valuable information on the requirement for nitrogen atoms located in either the major or minor grooves. Accordingly, we were able to show the utility of ODNs containing Br(7)C(7)dA, Br(3)C(3)dA, C(7)dA, and C(3)dA for the investigation of DNA-protein interactions.

4'-thio DNA consisting of 2'-deoxy-4'-thionucleosides exhibits resistance to both endonuclease and 3'-exonuclease cleavages. Interestingly, we found that 4'-thioDNA duplex behaved like RNA molecules in hybridization properties and structural aspects. Here, we have determined the structure of 4'-thioDNA duplex in solution by NMR. Most residues take on C3'-endosugar puckering, which is characteristic to A-form. The major groove of 4'-thioDNA duplex is narrow and deep, while the minor groove is wide and shallow. These features are also characteristic to A-form. Thus, although DNA duplex usually takes on B-form in solution, 4'-thioDNA takes on A-form, like RNA molecules. A-form-like groove features of 4'-thioDNA can account for the fact that 4'-thioDNA duplex interacts with an RNA major groove binder, but not with DNA groove binder. Interaction of 4'-thioDNA with RNase V1 and resistance to endonuclease may also be accounted for in the same context.

Isodeoxynucleic acid (INA) is one of the most attractive sugar modified nucleic acids for therapeutic use. We report here the enzymatic synthesis and properties of INA. All four isodeoxynucleoside triphosphates (INTPs) were synthesized for use in a single nucleotide insertion reaction. K(m) and V(max) values showed the almost same order as natural dNTPs. Primer extension reactions were also carried out to investigate the function of INTPs as substrates for several DNA polymerases. INTPs were incorporated into N + 2 position. One INTP and three dNTPs were able to generate a full-length product (27 mer). These results suggested that template-primer duplex containing one isodeoxynucleoside could be recognized by DNA polymerases. However, duplexs containing more than two isodeoxynucleosides could not be recognized by DNA polymerases because of conformational changes by isodeoxynucleosides.

We describe the synthesis of 1,8-naphthyridine C-nucleosides, Na-N(O) and Na-O(N), and full details of hybridization properties of the duplexes containing naphthyridine:imidazopyridopyrimidine pairs. The desired compounds were obtained from Na-O(N) derivative 1 via conversions of the substituents of 2- and 7-positions, respectively. The 17mer duplex containing one Na-O(O):Im-N(N) pair in the center was stabilized by +11.4 degrees C relative to that containing a T:A pair, and this value was highest among the naphthyridine:imidazopyridopyrimidine pairs.

We report herein a practical synthesis of 4'-thiothymidine (15) and appropriately protected 2'-deoxy-4'-thiocytidine (16), -thioadenosine (27), and -thioguanosine (29) derivatives, substrates for the synthesis of 4'-thioDNA, from the corresponding 4'-thioribonucleosides. 2'-deoxy-4'-thiopyrimidine nucleosides were synthesized using a radical reaction of the corresponding 2'-alpha-bromo derivatives, which were prepared via 2,2'-O-anhydro derivatives. 2'-deoxy-4'-thiopurine nucleosides were synthesized using the same radical reaction of the corresponding 2'-beta-bromo derivatives.

The functions of leukocyte-derived chemotaxin 2 (LECT2), a novel liver-specific protein, are not well defined, especially after hepatic injury. The changes in expression of LECT2 mRNA were investigated after concanavalin A (Con A)-induced hepatic injury in mice. Serum glutamate pyruvate transaminase (s-GPT) activity and the percentage of liver DNA fragmentation, an indicator of hepatic apoptosis, increased 8 h after intravenous administration of Con A (13 mg/kg). Expression of LECT2 mRNA was reduced from 8-24 h after injection of Con A, but was detected again 48 h after recovery from hepatic injury. Expression of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma mRNA was observed in liver 2 h after Con A injection. Z-Val-Ala-Asp(OMe)-CH2F (Z-VAD-FMK), a caspase inhibitor, was administered to mice to investigate whether LECT2 was involved in apoptosis of liver cells after Con A injection. Z-VAD-FMK inhibited s-GPT activity and DNA fragmentation in the liver 8 h after Con A-induced hepatic injury, but did not prevent the reduction of LECT2 mRNA, or induction of TNF-alpha and IFN-gamma mRNA expression. When the relation between expression of LECT2, TNF-alpha and IFN-gamma mRNAs was examined 8 h after Con A injection in wild-type or immunodeficient (nu-/nu-) mice, the increase in TNF-alpha and IFN-gamma mRNA expression was found to be closely related to a reduction in LECT2 mRNA expression. These results suggest that the reduction in expression of LECT2 mRNA is not directly involved in apoptosis and may be inversely related to the expression of TNF-alpha and/or IFN-gamma mRNA in the injured liver.

We examined the effects of ZNC-2381 (1-(4-aminophenyl)methyl-3-(3-nitrophenyl)-1,3-dihydroimidazo[4,5-b] pyridine-2-one), a new oral hepatoprotective agent, on hepatocellular caspase-3 activity and apoptosis induced by anti-mouse Fas antibody (anti-Fas ab) in mice. Oral ZNC-2381, administered at doses of 10, 30 and 100 mg/kg 1 h before inducing hepatic injury with anti-Fas ab, dose-dependently inhibited the increase in serum alanine aminotransferase (s-ALT) activity 8 h after injection of anti-Fas ab. Increases in DNA fragmentation (nucleosome assay) and caspase-3 activity in the liver 2 h after injection of anti-Fas ab were also inhibited by ZNC-2381 in a dose-dependent manner. As shown by histopathological examination, ZNC-2381 dose-dependently inhibited the appearance of TUNEL-positive apoptotic cells in the liver. Moreover, in studies in vitro, ZNC-2381 (1– 100 µmol/l) concentration-dependently inhibited increases in DNA fragmentation and caspase-3 activity caused by anti-Fas ab in isolated mouse hepatocytes. N- Acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-cho), a caspase-3-specific inhibitor, inhibited hepatocellular apoptosis caused by anti-Fas ab both in vivo and in vitro, as well as the increase in s-ALT activity in vivo. These results demonstrate that orally administered ZNC-2381 inhibits hepatocellular apoptosis induced by anti-Fas ab and presents the progression of hepatic injury. We propose that the mechanism of action of ZNC-2381 may involve blockade of the signal transduction pathway (caspase-3) of apoptosis mediated by anti-Fas ab.

We investigated whether tumor necrosis factor (TNF) and caspase-3 activity are involved in the induction of hepatocellular apoptosis in D-galactosamine (D-GalN)-induced hepatotoxicity in mice. Acute hepatotoxicity was induced by the intraperitoneal injection of D-GalN into female BALB/c mice. D-GalN (0.75-3.0 g/kg) increased the serum glutamate pyruvate transaminase (s-GPT) activity and the percentage of liver DNA fragmentation, an indicator of hepatotoxicity, after 48 h, in a dose-dependent manner. Furthermore, after D-GalN (3.0 g/kg) administration, increased liver DNA fragmentation was detected biochemically at 24 h, then increased s-GPT activity accompanied by increased liver DNA fragmentation was observed after 48 h. The serum TNF (s-TNF) level and the TNF mRNA expression in the liver after D-GalN (3.0 g/kg, i.p.) administration were examined by an ELISA kit and reverse transcription polymerase chain reaction (RT-PCR), respectively, to investigate the relation between the s-GPT activity and liver DNA fragmentation. The s-TNF level and TNF mRNA expression in the liver after D-GalN (3.0 g/kg) administration were detected earlier than liver DNA fragmentation, then increased with time. However, there was almost no association of caspase-3 activity with the increase in liver DNA fragmentation. Increases in the s-TNF level, TNF mRNA expression and the percentage of DNA fragmentation in the liver and s-GPT activity were inhibited by dexamethasone (Dex; 0.4-2.5 mg/kg, i.p.) in a dose-dependent manner. Based on these findings, it was considered that the intracellular apoptosis signal in D-GalN-induced hepatotoxicity in mice did not depend on caspase-3 activity, and that other signals mediated by TNF may be involved.

Depletion of the proteoglycan content of articular cartilage was induced by injecting bradykinin (30-300 mumol/l, 50 microliters/knee) into the left knee articular cavities of rats 3 times a day for 2 days. The degree of the reduction in the intensity of histopathological safranin O staining was used as an index of proteoglycan depletion. Bradykinin reduced the cartilage proteoglycan contents of the knee joints of non-injected limbs in a dose-dependent manner and at 300 mumol/l markedly reduced these contents, but evoked no inflammatory changes. The extent of the reduction of the cartilage proteoglycan contents induced by bradykinin injection depended on the dose and injection frequency. Chondroitin sulfate-C (CAS 25322-46-7, Chondroitin ZS Tab) (30-1,000 mg/kg/day) administered orally to rats for 14 days inhibited the bradykinin-induced proteoglycan depletion of the articular cartilage in a dose-dependent manner. These results suggest that a reduction of the proteoglycan content of cartilage, like that associated with osteoarthritis, was induced by injecting bradykinin into the knee articular cavities of rats and chondroitin sulfate-C protected against this effect.

Herein we describe the synthesis of 4‘-thio-dTTP (1) and 4‘-thio-dCTP (2) and their susceptibility for PCR amplification. Double stranded 4‘-thioDNAs were amplified sufficiently by KOD dash DNA polymerase under appropriate conditions. Through this PCR study, it was shown that not only DNA-directed 4‘-thioDNA synthesis but also 4‘-thioDNA-directed 4‘-thioDNA synthesis occurred in the presence of 1 and/or 2. We then tested the ability of the resulting 4‘-thioDNAs to act as templates for transcription. In this experiment, the 4‘-thioDNAs as templates afforded RNA in 38−49% yield relative to the case of the natural DNA template. We further investigated whether transcription occurred in mammalian cells. As a result, not only natural DNA but also 4‘-thioDNAs were transcribed to afford a shRNA which showed gene-silencing effects via RNAi machinery.

In this presentation, we discuss PCR amplification of 4'-thioDNA using 2'-deoxy-4'-thionucleoside triphosphate (sdNTPs). The amplified 4'-thioDNA acted as templates for not only in vitro transcription by T7 RNA polymerase, but also transcription in cells by RNA polymerase III. Accordingly, we succeeded to inhibit gene expression in cells by transfection of 4'-thioDNA that expresses a short-hairpin RNA (shRNA).