Increased expression of glial cell line-derived neurotrophic factor and neurturin in a case of colon adenocarcinoma associated with diffuse ganglioneuromatosis.

Intestinal ganglioneuromatosis (GN) is an uncommon disease of the enteric nervous system (ENS) and its pathogenesis remains unclear. Here we describe a unique case of diffuse GN of the intestinal wall associated with colon adenocarcinoma occurring in a 38-year-old female. Because it is well-known that glial cell line-derived neurotrophic factor (GDNF) and its receptor components, GDNF family receptor-alpha 1 (GFR-alpha 1) and RET receptor tyrosine kinase, play a crucial role in the development of ENS, their expression was analyzed by immunohistochemistry. Interestingly, GDNF as well as a related neurotrophic factor, neurturin (NTN), were expressed at high levels in adenocarcinoma cells whereas expression of GFR alpha 1 and RET was undetectable in them. In contrast, GFR-alpha 1 showed positive staining in both proliferating ganglion cells and glial cells, and RET immunoreactivity was found mainly in ganglion cell bodies. These findings suggested that GDNF and NTN expression in adenocarcinoma cells may play an important role in the pathogenesis of GN.

Characterization of virus-induced gene silencing in tobacco plants infected with apple latent spherical virus.

Apple latent spherical virus (ALSV) expressing green fluorescent protein (GFP-ALSV) was used for analysis of virus-induced gene silencing (VIGS) in tobacco plants expressing GFP (GFP-tobacco). In GFP-tobacco inoculated with GFP-ALSV, small dark spots appeared on inoculated leaves at 5 days post-inoculation (dpi), then expanded, and finally covered the whole area of the leaves after 12 dpi. Most of the fluorescence of upper leaves above the 12th true leaf disappeared at 21 dpi. Thus, GFP-ALSV infection efficiently triggered VIGS of a transgene (GFP gene) in tobacco plants. Analysis of GFP-silenced leaves showed that viral RNAs and proteins accumulated in all leaves where most GFP mRNA had been degraded. The siRNAs derived from ALSV-RNAs were not detected in samples from which siRNA of GFP mRNA could be easily detected. Direct tissue blot analysis showed that the spread of GFP-ALSV always preceded the induction of VIGS in infected leaves of GFP-tobacco. GFP leaf patch tests using Nicotiana benthamiana line 16c showed that Vp20, one of the three capsid proteins, is a silencing suppressor which interferes with systemic silencing.

Interference of Long-Distance Movement of Grapevine berry inner necrosis virus in Transgenic Plants Expressing a Defective Movement Protein of Apple chlorotic leaf spot virus.

ABSTRACT Transgenic Nicotiana occidentalis plants expressing a movement protein (P50) and partially functional deletion mutants (DeltaA and DeltaC) of the Apple chlorotic leaf spot virus (ACLSV) showed resistance to Grapevine berry inner necrosis virus (GINV). The resistance is highly effective and GINV was below the level of detection in both inoculated and uninoculated upper leaves. In contrast, GINV accumulated in inoculated and uninoculated leaves of nontransgenic (NT) plants and transgenic plants expressing a dysfunctional mutant (DeltaG). On the other hand, in some plants of a transgenic plant line expressing a deletion mutant (DeltaA', deletion of the C-terminal 42 amino acids), GINV could spread in inoculated leaves, but not move into uninoculated leaves. In a tissue blot hybridization analysis of DeltaA'-plants inoculated with GINV, virus could be detected in leaf blade, midribs, and petiole of inoculated leaves, but neither in stems immediately above inoculated leaves nor in any tissues of uninoculated leaves. Immunohistochemical analysis of GINV-inoculated leaves of DeltaA'-plants showed that GINV could invade into phloem parenchyma cells through bundle sheath of minor veins, suggesting that the long-distance transport of GINV might be inhibited between the phloem cells and sieve element (and/or within sieve element) rather than bundle sheath-phloem interfaces. Immunogold electron microscopy using an anti-P50 antiserum showed that P50 accumulated on the parietal layer of sieve elements and on sieve plates. The results suggested that resistance in P50-transgenic plants to GINV is due to the interference of both long-distance and cell-to-cell movement of the virus.

A movement protein and three capsid proteins are all necessary for the cell-to-cell movement of apple latent spherical cheravirus.

Immunoblot analysis of apple latent spherical cheravirus (ALSV)-infected leaves using a polyclonal antibody against the 21 C-terminal amino acids of a 53 K/42 K movement protein (MP) showed that a protein with an Mr of 42 kDa (42KP) is the dominant form found in vivo, which could indicate that the second AUG is used as an initiation codon of a ORF in RNA2. Co-expression of GFP with 42KP in tobacco epidermal cells showed that 42KP is able to facilitate cell-to-cell trafficking of GFP that is expressed in the same cells. The analysis of deletion mutants on each of MP, Vp24, Vp20, or Vp25 using an ALSV vector that stably expresses GFP indicated that an MP and three capsid proteins are all indispensable for the cell-to-cell movement of the virus. In ultrathin sections of infected leaves, a file of virus-like particles passing through the plasmodesmata connecting neighboring cells and tubular structures containing virus-like particles extending into the cytoplasm were observed. These results show that ALSV moves from cell to cell as virus particles.

3C-like protease encoded by Rice tungro spherical virus is autocatalytically processed.

The 3C-protease of Rice tungro spherical virus (RTSV) was previously identified as a cis- and trans-acting protease. In vitro translation of the protease resulted in several protein products, demonstrating that the protease is cleaved by itself. The protease was then produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Two forms of the protease were purified after MBP affinity chromatography in the column buffer. After analyses of the purified proteins, we speculated that a major internal cleavage site was in the C-terminal half. A point mutation was introduced at a potential major self-cleavage site (C(2763)). The mutation abolished the catalytic activity, suggesting that the mutation site is important for the recognition of the protease.

Stable expression of foreign proteins in herbaceous and apple plants using Apple latent spherical virus RNA2 vectors.

Infectious cDNA clones of Apple latent spherical virus (ALSV)-RNA1 (pEALSR1) and -RNA2 (pEALSR2) were constructed using an enhanced 35S promoter. A viral vector was constructed from pEALSR2 by creating artificial protease processing sites by duplicating the Q/G protease cleavage site between 42KP and Vp25. Eight RNA2-derived vectors expressing GFP with varied sizes of duplications around the 42KP/Vp25 junction were constructed and tested for infectivity in Chenopodium quinoa. The results indicated that greater than five aa from the C-terminus of 42KP and N-terminus of Vp25 in duplication are necessary for systemic infection. In infected C. quinoa plants, GFP fluorescence was observed in both inoculated and upper leaves. Serial passages of the viruses derived from the above vectors in C. quinoa showed that the size of duplications affected the stability of the GFP gene. The version of the RNA2-vector (pER2L5R5GFP) with the shortest duplications and its silent mutant version could stably express GFP in leaves even after at least nine serial passages. ALSV-RNA2 vector has a capacity to maintain a DNA insert as long as 1300 bp because Apple chlorotic leaf spot virus movement protein (50KP) gene could be expressed in C. quinoa. Inoculation of a virus derived from pER2L5R5GFP to apple seedlings resulted in the expression of GFP fluorescence in uninoculated upper leaves, indicating that the vector is available for the expression of foreign genes in apple trees.

The 50-kDa protein of Apple chlorotic leaf spot virus interferes with intracellular and intercellular targeting and tubule-inducing activity of the 39-kDa protein of Grapevine berry inner necrosis virus.

To understand why transgenic Nicotiana occidentalis plants expressing a functional movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) show specific resistance to Grapevine berry inner necrosis virus (GINV), the MPs of ACLSV (50KP) and GINV (39KP) were fused to green, yellow, or cyan fluorescent proteins (GFP, YFP, or CFP). These fusion proteins were transiently expressed in leaf cells of both transgenic (50KP) and nontransgenic (NT) plants, and the intracellular and intercellular trafficking and tubule-inducing activity of these proteins were compared. The results indicate that in epidermal cells and protoplasts from 50KP plant leaves, the trafficking and tubule-inducing activities of GINV-39KP were specifically blocked while those of ACLSV-50KP and Apple stem grooving virus MP (36KP) were not affected. Additionally, when 39KP-YFP and 50KP-CFP were coexpressed in the leaf epidermis of NT plants, the fluorescence of both proteins was confined to single cells, indicating that 50KP-CFP interferes with the cell-to-cell trafficking of 39KP-YFP and vice versa. Mutational analyses of 50KP showed that the deletion mutants that retained the activities described above still blocked cell-to-cell trafficking of 39KP, but the dysfunctional 50KP mutants could no longer impede cell-to-cell movement of 39KP. Transgenic plants expressing the functional 50KP deletion mutants showed specific resistance against GINV. In contrast, transgenic plants expressing the dysfunctional 50KP mutants did not show any resistance to the virus. From these results, we conclude that the specific resistance of 50KP plants to GINV is due to the ability of the 50KP to block intracellular and intercellular trafficking of GINV 39KP.

Supravesical hernia: CT diagnosis.

We report two cases of surgically proven supravesical hernia, one an internal supravesical hernia and the other an external supravesical hernia. Abdominal computed tomography showed the relation of the incarcerated intestine anterior to and compressing the urinary bladder. Although neither case was diagnosable preoperatively, we believe that the preoperative diagnosis of supravesical hernia by abdominal computed tomography is possible.

[A case of ruptured distal aortic arch aneurysm with bronchiolitis obliterans organizing pneumonia].

We report a case of histologically proved bronchiolitis obliterans organizing pneumonia (BOOP) associated with ruptured distal aortic arch aneurysm (DAAA) into the lung. A 63-years-old male with preoperative episode of hemosputum and hemoptysis was diagnosed DAAA. Preoperative computed tomographic scanning demonstrated that the aneurysm was surrounded with the structure of 2 layers of the enhanced high density external layer and the not enhanced low density internal layer. Combined resection of the left upper lobe and the aneurysm was performed safely because of marked adhesion between the lung and the aneurysm. Postoperative histological examination revealed that the perianeurysmal structure was due to BOOP.

Hepatectomy for cholangiocarcinoma complicated with right umbilical portion: anomalous configuration of the intrahepatic biliary tree.

The right umbilical portion (right-sided round ligament) has been discussed as an intrahepatic portal venous anomaly associated with "left-sided gallbladder" in several reports. We treated two patients with right umbilical portion (RUP) associated with cholangiocarcinoma. Left hepatectomies were performed, preserving the residual hepatic blood flow and biliary continuity. From our experience in these patients we propose the presence of anomalous configuration of the intrahepatic biliary tree in RUP, because both patients showed medial segmental bile ducts ramified from the right and left hepatic ducts. In general, although the medial segmental bile duct ramified from the left, we surmised that this abnormal bilateral drainage pattern may not be a rare phenomenon in RUP. Special attention may be required to focus on the anatomy of the portal tributaries and biliary ramifications in RUP.

Complete nucleotide sequence of the rice tungro spherical virus genome of the highly virulent strain Vt6.

The complete nucleotide sequence of rice tungro spherical virus (RTSV) strain Vt6, originally from Mindanao, the Philippines, with higher virulence to resistant rice cultivars, was determined and compared with the published sequence for the Philippine-type strain A (RTSV-A-Shen). It was reported that RTSV-A was not able to infect a rice resistant cultivar TKM 6 (10). RTSV-Vt6 and RTSV-A-Shen share 90% and 95% homology at nucleotide and amino-acid levels, respectively. The N-terminal leader sequence of RTSV-Vt6 contained a 39-amino acids-region (positions 65 to 103) which was totally different from that of RTSV-A-Shen; the difference resulted from frame shifting by nucleotide insertions and deletions. To confirm the amino-acid sequence differences of the leader polypeptide, the same region was cloned and sequenced using a newly obtained variant of RTSV-type 6, which had been collected in the field of IRRI, and seven field isolates from Mindanao, the Philippines. Since all the sequences of the target region are identical to that of the Vt6 leader polypeptide, the sequence difference in the leader region seems not to correlate with the virulence of Vt6.

Hepatic vein reconstruction by external iliac vein graft using vascular clips.

The utility of hepatic vein reconstruction following resection of segments VII and VIII plus the right hepatic vein (RHV) is still controversial. The purpose of this study was to investigate the surgical benefits of hepatic vein reconstruction using stapled vascular clips and the draining area of hepatic vein using angiographic computed tomography (CT) to determine strict indications for hepatic vein reconstruction. Five patients underwent RHV reconstruction by external iliac vein graft using stapled vascular clips (VCS clips) following resection of segments VII and VIII, regardless of whether an inferior right hepatic vein (IRHV) was present. In eight other patients CT during arterial portography (CTAP) under temporary RHV occlusion using a balloon catheter was performed to determine the drainage area of the RHV. Operating times were 240 to 400 minutes (mean 336 +/- 59 minutes), and the mean hepatic vein reconstruction time was 26 +/- 5 minutes. There were no complications related to the surgery. Follow-up examinations showed patency of the graft in all cases; three patients are still alive with long-term graft patency of 10 to 24 months. CTAP under RHV occlusion demonstrated that segment VI and part of segment V were almost hypoattenuated in cases of absent or small IRHV, although those segments were hyperattenuated in thick IRHV and RHV-IRHV communicating patients. In conclusion, this anastomotic technique using vascular clips resulted in sound patency of the graft, which was accomplished by a simple technique. Preoperative CT AP with the RHV occlusion method can be useful for determining whether hepatic vein reconstruction is necessary.

Successful transcatheter embolization of a pancreaticoduodenal artery aneurysm in association with celiac axis occlusion: a case report.

We report a case of a pancreaticoduodenal artery (PDA) aneurysm in association with celiac axis occlusion. A 54 year-old female complaining of abrupt onset of abdominal pain was admitted to our hospital. On admission, abdominal CT examination revealed a hematoma in the retroperitoneal space. Selective superior mesenteric artery (SMA) angiography disclosed an aneurysm in the anterior inferior pancreaticoduodenal artery (AIPDA). The celiac axis was occluded and blood was flowing to the liver and spleen via the enlarged pancreaticoduodenal arcade from the SMA. Transcatheter embolization of the aneurysm was performed successfully. Up to 1996, there have been 37 reported cases of PDA aneurysm in association with celiac axis stenosis or occlusion, including this one. Transcatheter embolization was performed successfully in only 5 of these cases. The formation of this type of PDA aneurysm is thought to be a result of the increased blood flow in the pancreaticoduodenal arcade due to celiac axis stenosis or occlusion. The transcatheter embolization performed in our report produced a far greater blood flow, which may lead to further aneurysmal formation. Careful follow-up is therefore necessary.

[Effects of propiverine hydrochloride (propiverine) on the muscarinic receptor binding affinity in guinea pig tissues and on salivation in conscious dogs].

Propiverine is a drug for the treatment of incontinence and pollakiuria. Such micturitional disorders are principally caused by a hyperactive bladder. The effects of propiverine, its active metabolite, 1-methyl-4-piperidyl benzilate N-oxide (DPr-P-4 (N-->O)), oxybutynin and terodiline on muscarinic receptors in guinea pig urinary bladder, salivary glands, cerebral cortex, ileal longitudinal muscle and heart were compared. Both propiverine and DPr-P-4 (N-->O) competitively inhibited specific binding of 3H-quinuclidinyl benzilate (3H-QNB) to membrane fractions of these tissues. Oxybutynin, terodiline, pirenzepine and atropine also competitively inhibited the binding of 3H-QNB. The order of these drugs in terms of their affinity for muscarinic receptors was as follows: atropine > oxybutynin > pirenzepine, DPr-P-4 (N-->O), terodiline > propiverine. Propiverine and DPr-P-4 (N-->O) had no selectivity for muscarinic receptors in these tissues, the same as atropine. In contrast, pirenzepine, a M1-selective drug, had 10.1 times greater affinity for muscarinic receptors in the cerebral cortex than in urinary bladder, and the affinity of oxybutynin for muscarinic receptors in salivary glands and in cerebral cortex was 10.9 times and 13.9 times higher, respectively, than in urinary bladder. The affinity of terodiline for muscarinic receptors in the cerebral cortex was 4.4 times higher than in urinary bladder. In this study, the effect of propiverine and oxybutynin on pilocarpine (1 mg/kg, s.c.)-induced salivation in conscious dogs was also compared. Propiverine (5 mg/kg, i.v.) had no effect on pilocarpine-induced salivation, whereas oxybutynin (0.1 mg-0.5 mg/kg, i.v.) inhibited it significantly and dose-dependently. The ID50 values (95% confidence limits) for propiverine and oxybutynin during the 20 min after intravenous administration were 6.88 mg/kg (4.71-15.67) and 0.154 mg/kg (0.115-0.205), respectively. These findings suggest that although propiverine, its active metabolite DPr-P-4 (N-->O), oxybutynin and terodiline competitively inhibit the binding of 3H-QNB to muscarinic receptors, the affinity of these drugs for the muscarinic receptors of these tissues is very different and that propiverine has less effect on salivation than oxybutynin.

Thirty-six cases of obturator hernia: does computed tomography contribute to postoperative outcome?

Obturator hernia is relatively rare and occurs mostly in elderly, thin, multiparous women. Recent reports have highlighted the importance of pelvic computed tomography (CT) for the preoperative diagnosis. Thirty-six patients with an obturator hernia operated in our hospital were divided retrospectively into two groups (group A: 18 operations from 1973 to 1986, before we used CT; group B: 18 CT cases from 1987 to 1995). Preoperative diagnoses, operative procedures, and postoperative course were reviewed. No statistically significant differences were found between groups A and B in terms of patient characteristics. Rates of accurate preoperative diagnoses were significantly higher in group B: 39% (7/18) in group A and 78% (14/18) in group B (p = 0.018). The intraoperative findings, occurrence of postoperative complications, and overall mortality rates were similar between the two groups. There were four postoperative deaths (mortality rate 11%). Three of four patients who died had panperitonitis because of small bowel perforation. The correct preoperative diagnosis of obturator hernia was facilitated by CT of the pelvis, but it has no impact on patient outcome. Early diagnosis and surgical intervention are essential for this rare entity.

Potentiation by neurotensin of carbachol-induced tension development in beta-escin-skinned smooth muscle of guinea-pig ileum.

Effect of neurotensin (NT) on carbachol(CCh)-induced tension development due to Ca2+ release from intracellular stores was investigated in beta-escin-skinned smooth muscle of guinea-pig ileum. NT (10 nM) increased the tension development in response to CCh. NT also increased the tension response to caffeine, another store-Ca2+ releaser. NT did not exert such an effect in pertusis toxin (PTX)-treated preparations. Treatment with isoprenaline to elevate endogenous cyclic AMP levels or with dibutyryl cyclic AMP did not affect the effect of NT. A nonpeptide NT antagonist, SR 48692, failed to block the effect of NT. NT shifted the pCa-tension relationship in the lower direction of Ca2+ concentration. NT was incapable of releasing Ca2+ from intracellular stores. The results suggest that NT may cause an increase in Ca2+ sensitivity of contractile elements to potentiate the CCh-induced tension development due to release of stored Ca2+ and that the effect is mediated by SR 48692-insensitive NT receptors linked to a PTX-sensitive G protein which works with no relation to a change in cytosolic cyclic AMP levels.

A case of obstructive colitis caused by possible colostomy dysfunction.

A case of obstructive colitis caused by possible stricture of colostomy is herein reported. A 58 year old female with an obstructive sigmoid colon cancer underwent an emergency descending decompression colostomy. At laparotomy, the colon proximal to the carcinoma was markedly distended and the bowel wall was thin, but the serosa appeared normal. Postoperatively, however, abdominal pain and distension persisted and low grade fever developed. Diarrhea through the colostomy continued. Nine days after the initial surgery, she underwent a left hemicolectomy. An abnormally thickened segment was identified in the resected specimen; normal mucosa was lost and several pseudopolyps were scattered. Histopathological findings of the abnormal segment were consistent with obstructive colitis. A preserved segment of normal mucosa intervened between the site of colostomy and the abnormal segment of obstructive colitis. A possible stenosis of the colostomy was considered to have caused colostomy dysfunction and subsequent obstructive colitis. She was complicated with anastomotic leakage due to the diseased colon being used for anastomosis. Obstructive colitis should be kept in mind in patients with obstructive colonic carcinomas who complain of persistent abdominal pain, distension and diarrhea in the early postoperative period after colostomy.

Intraportal stent placement combined with right portal vein embolization against advanced gallbladder carcinoma.

We describe herein the first successful implementation of intraportal stent placement combined with right portal vein embolization as preoperative management against far advanced gallbladder carcinoma. The patient was a 66-year-old woman with obstructive jaundice, in whom computed tomography confirmed that gallbladder carcinoma had invaded the liver and that massive lymph node metastases involved the hepatoduodenal ligament. Portography also revealed severe stenosis of the main portal trunk to less than 2 mm in diameter. To prevent the contribution of intraportal thrombosis and ensure postoperative liver functional reserve, an intraportal metallic stent implantation was conducted simultaneously with right portal vein embolization via a single route using the percutaneous transhepatic approach. There were no complications following this technique, and the patient subsequently underwent hepato-ligament-pancreatoduodenectomy. The resected specimen disclosed a well-expanded stent containing no thrombus. This method could therefore be an amenable strategy for the preoperative treatment of far advanced biliary malignancies in selected patients.

The protective effect of Clostridium novyi type B alpha-toxoid against challenge with spores in guinea pigs.

Clostridium novyi (C. novyi) Type B alpha-toxin was purified from culture supernatant by column chromatography, and was inactivated by formalin. A purified alpha-toxoid vaccine was prepared by mixing it with an aluminum phosphate gel adjuvant. Guinea pigs immunized twice with 4 micrograms or more of alpha-toxin survived against challenge with C. novyi Type B spores. Anti-alpha-toxin (antitoxin) titer was measured by toxin neutralization test using Vero cells. All of the guinea pigs having antitoxin titers of 10 units (U) or more at challenge were survived. In another experiment, guinea pigs were immunized with crude alpha-toxoid vaccines prepared by inactivated culture supernatant or by adding broken bacterial cells to the former. In this experiment, 10 U of antitoxin titer was the border of survival or death after challenge. Guinea pigs with antitoxin titers of less than 5 U, 5 U and 10 U died at 2, 3 to 4 and 4 days, respectively, after challenge. These results suggest that C. novyi alpha-toxin was the main protective antigen against challenge exposure to spores in guinea pigs.