Nicotinamide Improves Delayed Tooth Eruption in Runx2+/- Mice.

Patients with cleidocranial dysplasia (CCD) caused by mutations in RUNX2 have severe dental anomalies, including delayed or absent eruption of permanent teeth. This requires painful and expensive surgical/orthodontic intervention because of the absence of medicine for this condition. Here, we demonstrate that nicotinamide, a vitamin B3 and class III histone deacetylase inhibitor, significantly improves delayed tooth eruption in Runx2+/- mice, a well-known CCD animal model, through the restoration of decreased osteoclastogenesis. We also found that Csf1 mRNA and protein levels were significantly reduced in Runx2+/- osteoblasts as compared with wild type whereas RANKL and OPG levels had no significant difference between wild type and Runx2+/- osteoblasts. The nicotinamide-induced restoration of osteoclastogenesis of bone marrow-derived macrophages in Runx2+/- mice was due to the increased expression of RUNX2 and CSF1 and increased RANKL/OPG ratio. RUNX2 directly regulated Csf1 mRNA expression via binding to the promoter region of the Csf1 gene. In addition, nicotinamide enhanced the RUNX2 protein level and transacting activity posttranslationally with Sirt2 inhibition. Taken together, our study shows the potential and underlying molecular mechanism of nicotinamide for the treatment of delayed tooth eruption by using the Runx2+/- murine model, suggesting nicotinamide as a candidate therapeutic drug for dental abnormalities in patients with CCD.

PIN1 is a new therapeutic target of craniosynostosis.

Gain-of-function mutations in fibroblast growth factor receptors (FGFRs) cause congenital skeletal anomalies, including craniosynostosis (CS), which is characterized by the premature closure of craniofacial sutures. Apert syndrome (AS) is one of the severest forms of CS, and the only treatment is surgical expansion of prematurely fused sutures in infants. Previously, we demonstrated that the prolyl isomerase peptidyl-prolyl cis-trans isomerase interacting 1 (PIN1) plays a critical role in mediating FGFR signaling and that Pin1+/- mice exhibit delayed closure of cranial sutures. In this study, using both genetic and pharmacological approaches, we tested whether PIN1 modulation could be used as a therapeutic regimen against AS. In the genetic approach, we crossbred Fgfr2S252W/+, a mouse model of AS, and Pin1+/- mice. Downregulation of Pin1 gene dosage attenuated premature cranial suture closure and other phenotypes of AS in Fgfr2S252W/+ mutant mice. In the pharmacological approach, we intraperitoneally administered juglone, a PIN1 enzyme inhibitor, to pregnant Fgfr2S252W/+ mutant mice and found that this treatment successfully interrupted fetal development of AS phenotypes. Primary cultured osteoblasts from Fgfr2S252W/+ mutant mice expressed high levels of FGFR2 downstream target genes, but this phenotype was attenuated by PIN1 inhibition. Post-translational stabilization and activation of Runt-related transcription factor 2 (RUNX2) in Fgfr2S252W/+ osteoblasts were also attenuated by PIN1 inhibition. Based on these observations, we conclude that PIN1 enzyme activity is important for FGFR2-induced RUNX2 activation and craniofacial suture morphogenesis. Moreover, these findings highlight that juglone or other PIN1 inhibitors represent viable alternatives to surgical intervention for treatment of CS and other hyperostotic diseases.

Single-Stage Coil Embolization of Multiple Intracranial Aneurysms: Technical Feasibility and Clinical Outcomes.

PURPOSE: Not infrequently, intracranial aneurysms may be multifocal. However, strategies conferring open surgical access to opposite sides of the brain are limited. Given the recent advances in protection devices and coiling technique, a study of single-stage coil embolization in patients with multiple intracranial aneurysms was undertaken, assessing procedural safety and efficacy. METHODS: Data prospectively accrued between January 2010 and September 2013 were systematically reviewed, assessing clinical and morphologic outcomes of single-stage coil embolization in 172 patients with multiple aneurysms (≥ 2 aneurysms each; total, 371 aneurysms). RESULTS: Internal carotid artery (n = 132) was the most common site, with progressively fewer aneurysms found elsewhere (middle cerebral artery, 103; anterior communicating artery, 41; posterior communicating artery, 38). In 26 patients, one-stage embolization of three or more aneurysms took place (25 patients with three each; 1 patient with four). Stents were applied in 109 aneurysms, and in 33 lesions, balloons were used. Occlusion was achieved in 326 aneurysms (87.9 %) through coil embolization, and attempted coiling rarely failed (3 of 371, 0.8 %). Mean procedural time was 111.5 ± 37.8 min. Although procedure-related adverse events included three instances of treatment failure, asymptomatic thrombi in four patients, and aneurysmal leakage in one patient, procedural morbidity was low (1 of 172, 0.6 %), and no procedure-related deaths occurred. Postembolization follow-up of 303 aneurysms at > 6 months (mean, 15.4 ± 9.8 months) showed complete occlusion in the vast majority (275 of 303, 90.8 %), with comparatively fewer instances of minor (19 of 303, 6.3 %) and major (9 of 303, 3.0 %) recanalization. Four patients experienced delayed cerebral infarction, but only one suffered permanent neurologic deficit (Glasgow outcome scale 4). CONCLUSION: Single-stage coil embolization of multiple unruptured intracranial aneurysms is technically feasible. The time required for such procedures and the rate of complications observed seem acceptable.

Primary and revision efficacy of cross-wired metallic stents for endoscopic bilateral stent-in-stent placement in malignant hilar biliary strictures.

BACKGROUND AND STUDY AIMS: Endoscopic bilateral drainage for inoperable malignant hilar biliary strictures (HBS) using metal stents is considered to be technically difficult. Furthermore, endoscopic revision of bilateral stenting after occlusion can be challenging. This study was performed to evaluate the long-term efficacy of endoscopic bilateral stent-in-stent placement of cross-wired metallic stents in high-grade malignant HBS and planned endoscopic bilateral revision. PATIENTS AND METHODS: A total of 84 patients with inoperable high-grade malignant HBS were enrolled from three academic tertiary referral centers. Two cross-wired metal stents were inserted using a bilateral stent-in-stent placement method. Bilateral endoscopic revision was also performed during follow-up using either identical metal stents or plastic stents. The main outcome measurements were technical and functional success, complications, stent patency, and endoscopic revision efficacy. RESULTS: The technical and clinical success rates of endoscopic bilateral stent-in-stent placement of cross-wired metallic stents were 95.2% (80/84) and 92.9% (78/84), respectively. Median patency (range) and survival were 238 days (10-429) and 256 days (10-1130), respectively. Obstruction of primary bilateral stents occurred in 30.8% (24/78) of patients with functionally successful stent placement. The technical and clinical success rates of planned bilateral endoscopic revision for occluded stents were 83.3% (20/24) and 79.2% (19/24), respectively. For revision, bilateral metallic stents were placed in 11 patients (55.0%); the remaining patients received plastic stents. CONCLUSIONS: Palliative endoscopic bilateral stent-in-stent placement of cross-wired metallic stents was effective in patients with inoperable HBS. Revision endoscopic bilateral stenting may be feasible and successful in cases where the primary deployed metal stents are occluded.

Direct peroral cholangioscopy using an ultraslim upper endoscope for management of residual stones after mechanical lithotripsy for retained common bile duct stones.

BACKGROUND AND STUDY AIMS: The incidence of residual stones after mechanical lithotripsy for retained common bile duct (CBD) stones is relatively high. Peroral cholangioscopy using a mother-baby system may be useful for confirming complete extraction of stones, but has several limitations regarding routine use. We evaluated the role of direct peroral cholangioscopy (DPOC) using an ultraslim upper endoscope for the evaluation and removal of residual CBD stones after mechanical lithotripsy. PATIENTS AND METHODS: From August 2006 to November 2010, 48 patients who had undergone mechanical lithotripsy for retained CBD stones with no evidence of filling defects in balloon cholangiography were recruited. The bile duct was inspected by DPOC after balloon cholangiography. Detected residual CBD stones were directly retrieved with a basket or balloon catheter under DPOC. The incidence of residual stones detected by DPOC, and the success rate of residual stone retrieval under DPOC were investigated. RESULTS: DPOC was successfully performed in 46 of the 48 patients (95.8%). Of these, 13 patients (28.3%) had residual CBD stones (mean number 1.4, range 1-3; mean diameter 4.5 mm, range 2.3-9.6). The residual stones were removed directly under DPOC in 11 of these patients (84.6%). There were no complications associated with DPOC or stone removal. CONCLUSION: DPOC using an ultraslim upper endoscope is a useful endoscopic procedure for the evaluation and extraction of residual stones after mechanical lithotripsy for retained CBD stones.

Reduction of N-tropic mutant porcine endogenous retrovirus infectivity by human tripartite motif-containing 5-isoform alpha.

In cases of retroviral infection, the host cell deploys antiviral proteins as a type of innate immunity. Tripartite motif-containing 5-isoform alpha (TRIM5α) is a potent antiviral protein. TRIM5α has been reported to restrict human immunodeficiency virus (HIV) 1 infection in rhesus monkey cells by targeting the incoming viral capsid at the postentry or preintegration stage of the viral life cycle. As a consequence, virus replication and reverse transcription are interrupted. TRIM5α of human origin has also been shown to inhibit N-tropic murine leukemia virus infection. To investigate the inhibitory effect of TRIM5α on porcine endogenous retrovirus (PERV) infection in humans, we constructed a 293T cell line stably expressing human TRIM5α (293T-huTRIM5α) and tested the infectivity of vesicular stomatitis virus glycoprotein envelope pseudotyped viruses (wild-type PERV [wt-PERV], N-tropic mutant PERV, N-tropic murine leukemia virus, and MoMLV). Infectivity of N-tropic mutant PERV was reduced by 43.3% in 293T-huTRIM5α cells, a decrease in efficiency that was more than 3-fold greater than that of wt-PERV in 293T-huTRIM5α cells. Human TRIM5α exhibited inhibitory activity against N-tropic MLV and N-tropic mutant PERV, but showed no antiviral activity against Moloney murine leukemia virus or wt-PERV.

Prospective study of the effectiveness of percutaneous transhepatic photodynamic therapy for advanced bile duct cancer and the role of intraductal ultrasonography in response assessment.

BACKGROUND AND STUDY AIMS: We evaluated the therapeutic effects of percutaneous transhepatic photodynamic therapy (PDT) in patients with advanced bile duct cancer. The utility of intraductal ultrasonography (IDUS) for the assessment of responses and for regular follow up after PDT was also examined. METHODS: Percutaneous transhepatic biliary drainage (PTBD) was initiated before PDT. Following dilation and maturation of the PTBD tract, percutaneous PDT was performed. Intraluminal photoactivation was carried out using percutaneous cholangioscopy 2 days after intravenous application of a hematoporphyrin derivative. All patients were additionally provided with percutaneous bile duct drainage catheters after PDT. IDUS was conducted monthly to measure the thickness of the tumor mass before and after PDT. RESULTS: 24 patients with advanced cholangiocarcinomas (Bismuth IIIa, n = 4; IIIb, n = 10; IV, n = 10) were treated with PDT. At 3 months after PDT, the mean thickness of the tumor mass had decreased from 8.7 +/- 3.7 mm to 5.8 +/- 2.0 mm (P < 0.01). At 4 months after PDT, the thickness of the mass had increased to 7.0 +/- 3.7 mm. Quality of life indices improved dramatically and remained stable 1 month after PDT; the Karnofsky index increased from 39.1 +/- 11.36 to 58.2 +/- 22.72 points (P = 0.003). The 30-day mortality rate was 0 %, and the median survival time was 558 +/- 178.8 days (current range 62 - 810 days). CONCLUSIONS: PDT using percutaneous cholangioscopy is safe and effective for advanced hilar cholangiocarcinoma, and seems to prolong survival. IDUS is useful for evaluating changes in the thickness of the tumor mass after PDT.

Invasive intraductal papillary mucinous tumor of the pancreas with simultaneous invasion of the stomach and duodenum.

An 81-year-old woman was admitted with epigastric pain and weight loss. She had been diagnosed with an intraductal papillary mucosal tumor (IPMT) 7 years previously, but had refused surgery for religious reasons. Esophagogastroduodenoscopy revealed a nodular, elevated lesion that was discharging mucin into the duodenal bulb and posterior wall of the upper body of the stomach. Endoscopic ultrasonography, abdominal computed tomography, and endoscopic retrograde cholangiography were carried out, and a highly invasive IPMT with simultaneous invasion of the stomach and duodenum was diagnosed.

Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both L-ornithine and L-lysine.

The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native M(r) of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with K(m) values of 562 microM and 1592 microM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by alpha-difluoromethylornithine (K(i) 1.15 microM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys(95)) and cysteine (Cys(96), Cys(338) and Cys(377)) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys(96), each mutation caused a substantial loss in enzyme activity. Most notably, Lys(95) increased the K(m) for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the k(cat) for ODC and lysine decarboxylase (LDC) activity respectively. The Cys(377)-->Ala mutant possessed a k(cat) that was lowered by 23-fold, and the K(m) value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC activities occur in the same catalytic site.

Isolation and properties of arginase from a shade plant, ginseng (Panax ginseng C.A. Meyer) roots.

Arginase (EC 3.5.3.1) was purified to homogeneity from root tissues of three-year-old ginseng (Panax ginseng C.A. Meyer), shade plant, and was found to be an extraordinarily large molecule relatively stable to heat. The enzyme was decameric having a molecular mass of 352,000 Da, with an optimal temperature and pH of 60 degrees C and 9.5, respectively. Analogues of arginine could not replace it as substrate, and a cysteine residue is at or near the active site. Maximum activity was obtained with Mn(2+) and Co(2+) also activated the proteins, whereas, both agmatine and 5'-deoxy-methylthioadenosine were inhibitors. Specific activities of the enzyme in sliced ginseng roots were increased by plant hormones such as GA(3), IAA, kinetin and putrescine, whereas the activities of the purified enzyme were unaffected by putrescine. Increases in arginase activities by these plant hormones could affect metabolism of polyamine intracellularly.

Fixation of a modified covered esophageal stent: its clinical usefulness for preventing stent migration.

BACKGROUND AND STUDY AIMS: Membrane-covered self-expandable metal stents are effective in preventing tumor ingrowth and stent obstruction in patients with inoperable esophageal cancer, but migration of stents continues to be a major problem. We therefore constructed a modified covered self-expandable esophageal metal stent capable of being fixed using a silk thread. The stent was studied prospectively to define its palliative characteristics and whether it is effective in preventing migration. PATIENTS AND METHODS: Modified covered self-expandable metal stents were placed in 17 patients with malignant gastric cardiac cancer involving the esophagogastric junction, 41 patients with esophageal cancer, and three patients with tracheoesophageal fistulas. Clinical and radiographic follow-up examinations were carried out at regular intervals. RESULTS: Placement of the stent was successful in all patients, with good symptomatic relief and no serious stent-related complications such as esophageal perforation or hemorrhage. Acute stent placement problems, such as incomplete expansion or acute angulation of the stent, were noted in four patients. However, during a mean follow-up period of 7.5 months (range 1 to 17 months), there was no stent migration. CONCLUSIONS: Modified covered self-expandable esophageal metal stents of this type would be very effective in preventing stent migration, especially in patients with malignant gastric cardiac cancer extending to the lower esophagus, those with short-segment esophageal cancer, and those with tracheoesophageal fistulas.

The role of percutaneous transhepatic papillary balloon dilation in percutaneous choledochoscopic lithotomy.

BACKGROUND: When choledochoscopic lithotomy with basket and electrohydraulic lithotripsy is used to remove intrahepatic duct stones, fragments or small stones usually remain in the bile duct that are too small to be captured with a basket. METHODS: An attempt was made to remove stone fragments in 16 patients with intrahepatic duct stones by antegrade balloon dilation of the sphincter of Oddi with a conventional balloon catheter. After balloon dilation, remnant stones and sludge were pushed through the papilla with the choledochoscope. RESULTS: Bile duct stones were completely removed in 12 of 16 patients (75%); stones were removed in 1 session. There was no clinical evidence of procedure-related pancreatitis or fatal complications. CONCLUSIONS: Percutaneous transhepatic papillary balloon dilation of the sphincter of Oddi and clearance of remnant bile duct stones and stone fragments with the tip of choledochoscope is simple and effective in patients undergoing percutaneous transhepatic choledochoscopic lithotomy.

Malignant gastroduodenal obstructions: treatment by means of a covered expandable metallic stent-initial experience.

PURPOSE: To investigate the technical feasibility and clinical effectiveness of a polyurethane-covered expandable nitinol stent in the treatment of malignant gastroduodenal obstructions. MATERIALS AND METHODS: The stent was constructed in-house by weaving a single thread of 0.2-mm nitinol wire in a tubular configuration and was covered with polyurethane solution by means of a dipping method. With fluoroscopic guidance, the stent was placed in 19 consecutive patients with malignant gastric outlet obstruction (n = 15) or duodenal obstruction (n = 4). All patients had severe nausea and recurrent vomiting, and their obstructions were inoperable. RESULTS: Stent placement was technically successful in all but one patient. After stent placement, symptoms improved in all but one patient, who had another stenosis at the proximal jejunum. One patient with stent placement in the second portion of the duodenum became jaundiced. During the mean follow-up of 11 weeks, stent migration occurred in five patients 1-4 days after the procedure. All patients with stent migration were treated by means of placing a second, uncovered nitinol stent. Two of these five patients showed recurrence of stricture because of tumor ingrowth; they underwent coaxial placement of a third, covered nitinol stent with good results. CONCLUSION: Placement of a polyurethane-covered expandable nitinol stent seems to be technically feasible and effective for palliative treatment of inoperable malignant gastroduodenal obstructions. Stent migration, however, is problematic and requires further investigation.

Polyamine synthesis in plants: isolation and characterization of spermidine synthase from soybean (Glycine max) axes.

Spermidine synthase (EC 2.5.1.16) was purified to homogeneity for the cytosol of soybean (Glycine max) axes using ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephacryl S-300, omega-aminooctyl-Sepharose and ATPA-Sepharose. The molecular mass of the enzyme estimated by gel filtration and SDS-PAGE is 74 kDa. Cadaverin and 1,6-diaminohexane could not replace putrescine as the aminopropyl acceptor. Kinetic behaviors of the substrate are consistent with a ping pong mechanism. The kinetic mechanism is further supported by direct evidence confirming the presence of an aminopropylated enzyme and identification of product, 5'-deoxy-5'-methylthioadenosine, prior to adding putrescine. The Km values for decarboxylated S-adenosylmethionine and putrescine are 0.43 microM and 32.45 microM, respectively. Optimum pH and temperature for the enzyme reaction are 8.5 and 37 degrees C, respectively. The enzyme activity is inhibited by N-ethylmaleimide and DTNB, but stimulated by Co2+, Cu2+ and Ca2+ significantly, suggesting that these metal ions could be the cellular regulators in polyamine biosynthesis.

Identification of functionally important residues of Arabidopsis thaliana S-adenosylmethionine decarboxylase.

The Arabidopsis thaliana S-adenosylmethionine decarboxylase (AdoMetDC) cDNA (GenBank(TM) U63633) was cloned, and the AdoMetDC protein was expressed, purified, and characterized. The K(m) value for S-adenosylmethionine (AdoMet) is 23.1 microM and the K(i) value for methylglyoxal bis-(guanylhydrazone) (MGBG) is 0.15 microM. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations at conserved cysteine (Cys(50), Cys(83), and Cys(230)) and lysine(81) residues, chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. The AdoMetDC mutants K81A and C83A retained up to 60 and 10% of wild type activity, respectively, demonstrating that lysyl and sulfhydryl groups are required for full catalytic activity. However, changing Cys(50) and Cys(230) to alanine had minimal effects on the catalytic activity. Changing Lys(81) to alanine produced an altered substrate specificity. When lysine was used as a substrate instead of AdoMet, the substrate specificity for lysine increased 6-fold. The K(m) value for AdoMet is 11-fold higher than that of the wild type, but the V(max) value is more than 60%. Taken together, the results suggest that the lysine(81) residue is critical for substrate binding.