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ABSTRACT: The yeast Saccharomyces cerevisiae CCH1 gene encodes a homolog of the pore forming α1 subunit of mammalian voltage-gated calcium channels (VGCCs). Cch1 cooperates with Mid1, a candidate for a putative, functional homologue of the mammalian regulatory subunit α2/δ, and is essential for Ca2+ influx induced by several stimuli. Here, we characterized two mutant alleles of CCH1, CCH1* (or CCH1-star, carrying four point mutations: V49A, N1066D, Y1145H and N1330S) and cch1-2 (formerly designated mid3-2). The product of CCH1* displayed a marked increase in Ca2+ uptake activity in the presence and absence of α-factor, and its increased activity was still dependent on Mid1. Mutations in CCH1* did not affect its susceptibility to regulation by calcineurin. We also showed that not only was the N1066D mutation in the cytoplasmic loop between domains II and III responsible for the increased activity of Cch1*, but also that substitution of another negatively charged amino acid Glu for Asn1066 resulted in a significant increase in the Ca2+ uptake activity of Cch1. This is the first report about a gain-of-function mutation in Cch1. On the other hand, the cch1-2 allele possesses the P1228L mutation located in the extracellular S1-S2 linker of domain III. The Pro1228 residue is highly conserved from fungi to humans, and the P1228L mutation led to a partial loss in Cch1 function, but did not affect the localization and expression of Cch1. Our study extends the understanding of the structure-function relationship and functional regulation of Cch1.
Microbiology 03/2013; · 3.06 Impact Factor
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Ichiro Yasuda,
Naotaka Fujita,
Hiroyuki Maguchi,
Osamu Hasebe,
Yoshinori Igarashi,
Akihiko Murakami,
Hidekazu Mukai,
Tsuneshi Fujii,
Kenji Yamao,
Kensei Maeshiro, Tomoko Tada,
Takeshi Tsujino,
Yutaka Komatsu
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ABSTRACT: Endoscopic sphincterotomy (ES) is a well-established standard method for treating common bile duct stones. However, biliary sphincter function is impaired after the treatment, and this may influence the long-term outcomes. In this study, we aimed to compare the long-term outcomes after ES with those after endoscopic papillary balloon dilation (EPBD) because the latter procedure is expected to preserve biliary sphincter function better than ES.
A prospective follow-up of the original cohort in a previously randomized, controlled trial to compare the early outcomes after ES and EPBD.
Eleven centers, including 6 clinical practices and 5 academic institutions.
A total of 282 patients with common bile duct stones were randomly selected to undergo ES (n = 144) or EPBD (n = 138) in the previous study.
ES or EPBD.
Complications after ES or EPBD occurring during long-term follow-up.
The patients were followed up annually after the treatment. The median duration of the follow-up was 6.7 years. Morbidity was observed in 36 (25.0%) and 14 (10.1%) of the patients who underwent ES and EPBD, respectively (P = .0016). Kaplan-Meier analysis revealed a significantly higher incidence of biliary complications in the ES group than in the EPBD group (P = .0011). Multivariate analysis showed that ES, periampullary diverticulum, and in situ gallbladder stones were independent risk factors for stone recurrence.
During long-term follow-up, patients who underwent ES had significantly more biliary complications than those who underwent EPBD. The biliary sphincter dysfunction after ES results in additional late complications.
Gastrointestinal endoscopy 12/2010; 72(6):1185-91. · 6.71 Impact Factor
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ABSTRACT: Self-expandable metallic stents (SEMSs) are widely used for palliation of malignant gastric outlet obstruction (GOO). A common complication of their use, however, is stent obstruction caused by tumor ingrowth or hyperplasia. The covered SEMS was designed to prevent these problems.
We compared the performance of uncovered and covered SEMSs in patients with GOO.
A retrospective study, single center.
A tertiary-referral center.
Sixty patients with symptomatic malignant GOO.
All patients received an uncovered or covered knitted nitinol stent by using the over-the-wire placement procedure.
Comparison of the clinical outcome, complications, and the reintervention rate between uncovered and covered stents.
Thirty-one patients (mean [+/-SEM] age 72.2 +/- 2.1 years; 16 men) received uncovered SEMSs, and 29 (mean [+/-SEM] age 70.6 +/- 1.7 years; 17 men) received covered SEMSs. The technical success rate was 100% in both groups. No difference in clinical success was seen (90.3% uncovered group vs 86.2% covered group). Regarding early complications (<1 week), one mild case of pancreatitis from the stent covering the papilla occurred in each group. Late complications included reobstruction, migration, bleeding, stent fracture, and perforation. The occurrence of reobstruction did not differ between the 2 groups (3.2% uncovered group vs 10.3% covered group). No difference in migration (0% uncovered group vs 6.9% covered group) was seen. The uncovered group required less frequent reinterventions for stent reobstruction, migration, or stent fracture (3.2% uncovered group vs 20.7% covered group, P = .0490). The uncovered group had 2 major late complications: bleeding and perforation. All 60 patients died, with a median survival time of 51 days and 62 days, respectively.
Small-sized, single-center, retrospective study.
In palliation for malignant GOO, covered stents were associated with a more frequent need for reintervention than uncovered stents, despite similar outcomes and complications. These results require confirmation in a larger randomized comparison.
Gastrointestinal endoscopy 02/2009; 69(4):806-12. · 6.71 Impact Factor
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ABSTRACT: Voltage-gated Ca2+ channels (VGCCs) mediate the influx of Ca2+ that regulates many cellular events, and mutations in VGCC genes cause serious hereditary diseases in mammals. The yeast Saccharomyces cerevisiae has only one gene encoding the putative pore-forming alpha1 subunit of VGCC, CCH1. Here, we identify a cch1 allele producing a completely nonfunctional Cch1 protein with a Gly1265 to Glu substitution present in the domain III S2-S3 cytoplasmic linker. Comparison of amino acid sequences of this linker among 58 VGCC alpha1 subunits from 17 species reveals that a Gly residue whose position corresponds to that of the Cch1 Gly1265 is completely conserved from yeasts to humans. Systematic amino acid substitution analysis using 10 amino acids with different chemical and structural properties indicates that the Gly1265 is essential for Cch1 function because of the smallest residue volume. Replacement of the Gly959 residue of a rat brain Cav1.2 alpha1 subunit (rbCII), positionally corresponding to the yeast Cch1 Gly1265, with Glu, Ser, Lys, or Ala results in the loss of Ba2+ currents, as revealed by the patch clamp method. These results suggest that the Gly residue in the domain III S2-S3 linker is functionally indispensable from yeasts to mammals. Because the Gly residue has never been studied in any VGCC, these findings provide new insights into the structure-function relationships of VGCCs.
Journal of Biological Chemistry 09/2007; 282(35):25659-67. · 4.77 Impact Factor
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ABSTRACT: In patients with gastric outlet obstruction (GOO), palliative enteral stenting is a less invasive procedure compared with gastroenterostomy. Most diseases analyzed in previous studies of such stenting were pancreaticobiliary malignancies.
We reviewed the medical records of patients with GOO secondary to gastric cancer who were admitted to our institution between September 1994 and September 2004. The outcome of stent placement for GOO was compared with the outcome in patients who underwent palliative open gastrojejunostomy during the same period. Enrolled patients from both groups displayed symptomatic GOO. Patients with recurrent gastric cancer were excluded from this study.
Twenty-two patients underwent palliative enteral stenting, and 22 patients were subjected to surgical gastrojejunostomy (bypass). There were no significant differences between the two groups regarding patient baseline characteristics. Technical success and clinical success were obtained in 100% and 77.3%, respectively, of both groups. The operating time was shorter in the stent group (30 vs 118 min; P<0.0001). The time from the procedure to the resumption of food intake was shorter in the stent group than in the bypass group (2 days vs 8 days; P<0.0001). An improvement in performance score after the procedure was observed in both groups (stent group; P=0.0264; bypass group; P=0.0235). No significant differences were observed regarding the possibility of discharge. In patients discharged, the median postoperative hospital stays were 19 days and 28 days (P=0.0558). The median survival periods were 65 days and 90 days. Minor complications were observed in 1 patient in the stent group and in 4 in the bypass group. No mortality or severe complications were observed for either group.
Self-expandable metallic stent placement is a safe and efficacious procedure for palliation, with shorter operating time and more prompt restoration of oral intake, compared to surgical alternatives in patients with GOO caused by gastric cancer.
Journal of Gastroenterology 11/2005; 40(10):932-7. · 4.16 Impact Factor
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ABSTRACT: Self-expandable metallic stents (SEMS) have recently become widely used. A dedicated stent such as the through-the-scope (TTS) stent has enabled easier placement of SEMS by endoscopists for colorectal obstructions. In Japan, however, the TTS stent is not yet available. Therefore, we have to perform non-TTS placement using esophageal, tracheal or vascular stents for colorectal obstructions. We have developed some modifications which aid the placement of esophageal stents, including increasing the length of the delivery system, and the use of a splinting tube or a double splinting tube. These technical modifications allow markedly better placement of a knitted nitinol Ultraflex esophageal stent for a colorectal obstruction. They allowed us to even place SEMS in the proximal colon without difficulty, and in all patients we treated. Therefore, with some technical modifications, it is feasible to use an esophageal stent for proximal colonic obstruction.
Digestive Endoscopy 09/2005; 17(4):334 - 337. · 1.19 Impact Factor
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ABSTRACT: With the conventional pull method of PEG placement, there is a significant risk of wound infection from contamination of the gastrostomy catheter as it passes through the oral cavity. This study compared the occurrence of peristomal wound infection associated with PEG placement with and without use of an overtube.
Consecutive patients with dysphagia were randomized to undergo PEG placement with (Group I) or without (Group II) an overtube. For each patient, the peristomal area was evaluated daily for 1 week after PEG placement. The presence of erythema and of exudate were scored on a scale of 0 to 4; induration was scored on a scale of 0 to 3. Criteria for infection were a maximum combined score of 8 or higher, or the presence of microscopic and microbiologic evidence of suppurating exudate. In each group, cefazolin was administered prophylactically (2 g/d intravenously) for 3 days. For patients who had received an antibiotic(s) before PEG placement, the same antibiotic(s) was used. All procedures in both groups were performed by one of two investigators who used the pull method.
A total of 76 patients were randomized; 3 were excluded from analysis, because death occurred within 1 week after the procedure. Two of 3 deaths were procedure-related (aspiration pneumonia in Group I, peritonitis in Group II). Data for 37 patients in Group I and 36 in Group II were analyzed. There was no significant difference between the groups with respect to baseline characteristics. The occurrence of peristomal infection within 1 week of PEG was significantly lower in Group I compared with Group II (2 vs. 12; p = 0.0029). The mean daily combined scores in Group I also were significantly lower than those in Group II ( p < 0.0001), and the median maximum parameter scores in Group I were significantly lower than those in Group II (erythema, p = 0.0062; induration, p = 0.0390; exudate, p < 0.0001), although the nominal significance for induration was removed by correction for the multiple testing of data. One patient excluded from Group II died from sepsis because of procedure-induced peritonitis. Among the 73 enrolled patients, there was no procedure-related mortality or clinically important wound infections that required surgical intervention in either group.
Use of an overtube during PEG placement reduces the risk of peristomal wound infection.
Gastrointestinal Endoscopy 04/2005; 61(4):522-7. · 4.88 Impact Factor
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ABSTRACT: Saccharomyces cerevisiae has only one gene encoding a putative voltage-gated Ca2+ channel pore-forming subunit, CCH1, which is not possible to be cloned by conventional molecular cloning techniques using Escherichia coli. Here, we report the successful cloning of CCH1 in yeast by in vivo homologous recombination without using E. coli. Overexpression of the cloned CCH1 or MID1 alone, which encodes a putative stretch-activated Ca2+ channel component, does not increase Ca2+ uptake activity, but co-overexpression results in a 2- to 3-fold increase. Overexpression of CCH1 does not substantially complement the lethality and low Ca2+ uptake activity of a mid1 mutant and vice versa. These results indicate that co-overproduction of Cch1 and Mid1 is sufficient to increase Ca2+ uptake activity.
FEBS Letters 11/2004; 576(3):291-6. · 3.54 Impact Factor
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ABSTRACT: The yeast Mid1 protein with an apparent molecular mass of 100 kDa is required for Ca2+ influx stimulated by the mating pheromone and by a capacitative calcium entrylike mechanism acting in response to Ca2+ depletion from the endoplasmic reticulum (ER) and functions as a stretch-activated Ca2+ -permeable channel when expressed in mammalian cells. Our previous work with protease protection experiments has indicated that Mid1 is present in the plasma membrane. In this study, we examined a possible intracellular localization of this protein by indirect fluorescence microscopy and found that Mid1 is present in the ER membrane as well as the plasma membrane. Intracellular fluorescence images for Mid1 were the same as those for the ER marker protein Sec71 but quite different from those of the Golgi protein Ypt1. The results were confirmed by membrane fractionation using Angiografin density gradient analysis. We also investigated the oligomeric structures and protein levels of Mid1 and found that Mid1 forms a 200-kDa oligomer by disulfide bonding. The protein level and modification of Mid1 in the plasma membrane and the ER membrane were unchanged by the mating pheromone. These findings provide new insight into the function of Mid1 in relation to localization, modification, and activation mechanisms.
Experimental Cell Research 03/2004; 293(2):185-95. · 3.58 Impact Factor
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ABSTRACT: The yeast Mid1 protein is an integral membrane protein required for the viability of differentiated cells and Ca2+ influx induced by mating pheromone. Our previous study has identified a loss-of-function mutation, F356S. The F356S mutant is completely unable to maintain viability, but still has Ca2+ accumulation activity near the wild-type level. Here we further examined in detail the F356S mutation to unravel the function of Phe356. After exposure to the pheromone, the F356S mutant was not fully rescued by high extracellular Ca2+, like the mid1 null mutant, suggesting that Phe356 and Mid1 itself are also required for viability maintenance mechanism that does not involve Ca2+ signalling. Substitutions of hydrophilic amino acids for Phe356 caused lethality and low Ca2+ accumulation, but those of hydrophobic amino acids did not. Substitutions of small amino acids for Phe356 caused a significantly reduced viability, but did not affect Ca2+ accumulation. We suggest that the hydrophobicity of the Phe356 residue is important for both viability maintenance and Ca2+ uptake, and that its size for viability maintenance.
Biochemical and Biophysical Research Communications 02/2004; 313(3):752-7. · 2.48 Impact Factor
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ABSTRACT: Stent placement in palliation of unresectable colon cancer is an alternative to surgical treatment. The through-the-scope stent for the exclusive treatment of colorectal cancer is not available in Japan. This report describes the use of an esophageal stent and the technical modifications required for its success in the treatment of colorectal strictures. We describe various technical strategies for colorectal stent placement and report on the outcomes.
Medical records of patients who underwent palliative colonic stenting between June 1997 and March 2003 were reviewed retrospectively, and the clinical outcome was evaluated.
Insertion of a metallic esophageal stent was attempted in 12 patients (mean age, 73.0 years; 5 male, 7 female). Location of the stricture was in the rectum in 4 patients and in the sigmoid, descending, or transverse segments of the colon in 5, 1, and 2 patients, respectively. Two patients had recurrent colon cancer after surgery. The remaining 10 patients did not undergo surgery. Stent placement was technically successful in 11 patients, giving a technical success rate of 92%. Following successful stent placement, all but 1 patient obtained clinical success, generating a clinical success rate of 83%. Late complications occurred in 4 patients and included 2 migrations, 2 bleeds, and 1 obstruction. The complication rate of the procedure was 33.3%. There was no mortality or severe complications. The median survival period was 120 days.
Stent placement can be considered safe and effective palliation for unresectable colorectal cancer. With technical modification of an esophageal stent, this procedure is now feasible. Stent placement in palliation of unresectable colon cancer is an alternative to surgical treatment. The through-the-scope stent for the exclusive treatment of colorectal cancer is not available in Japan. This report describes the use of an esophageal stent and the technical modifications required for its success in the treatment of colorectal strictures. We describe various technical strategies for colorectal stent placement and report on the outcomes.
Journal of Gastroenterology 02/2004; 39(4):334-8. · 4.16 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 12/2003; 48(15 Suppl):2061-7.
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ABSTRACT: PEG by the conventional pull method has the potential drawback of being associated with a higher frequency of wound infection, presumably caused by contamination of the gastrostomy catheter as it passes through the oral cavity. This study investigated the occurrence of peristomal wound infection after PEG placement by using the pull and introducer techniques.
Between September 1999 and May 2002, consecutive patients with dysphagia for whom PEG was recommended were enrolled in the study and randomly assigned to two groups: PEG with the introducer method (Group I) or PEG with the pull method (Group II). The peristomal area of each patient was evaluated on a daily basis for one week after PEG. Erythema and exudate were scored on a scale from 0 to 4 and induration on a scale of 0 to 3. Criteria for infection were a maximum combined score of 8 or higher, or the presence of microscopic and microbiologic evidence of suppurating exudate. In each group, the endoscope was passed once during the procedure, and an antibiotic (piperacillin) was given prophylactically. All procedures were performed by one investigator with the assistance of another physician.
Of the 60 patients enrolled, 30 were assigned to each group. PEG was successful in all patients. One patient was excluded from each group because of death (Group I, stroke; Group II, myocardial infarction) within one week of the procedure. Therefore, 58 patients, 29 in each group, were evaluated. There was no significant difference between the groups in terms of clinical parameters (age, gender, disease, performance score, mode of previous feeding, and recent antibiotic exposure). The occurrence of peristomal infection within one week of PEG was lower in Group I (introducer method) (0 vs. 9; p = 0.00094). The mean daily combined scores in Group I were significantly lower than those in Group II. Median of maximum parameter scores in Group I were significantly lower than those in Group II. There were no procedure-related mortalities or clinically significant wound infections that required surgical intervention.
The risk of peristomal wound infection after PEG is lower with the introducer method compared with the pull method.
Gastrointestinal Endoscopy 07/2003; 57(7):837-41. · 4.88 Impact Factor
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ABSTRACT: Background: Intraductal papillary-mucinous pancreatic tumors (IPMT) are intraductal lesions formed by mucin-producing epithelium, which proliferates in a papillary pattern, and presents a spectrum from hyperplasia to adenocarcinoma. The value of intraductal ultrasonography (IDUS) for excluding malignancy has not been assessed in a case series previously.Methods: Intraductal ultrasonography was performed in 17 patients with IPMT (12 with adenocarcinoma and five with adenoma) between November 1993 and June 2002. Intraductal ultrasonography was used to determine the maximum height and maximum cross-sectional area of protruding lesions. Results were compared after dividing the tumors into three groups: a benign lesion group, a non-invasive cancer group, and an invasive cancer group. The resection line was located over 10 mm from the edge of the protruding lesion visualized by intraductal ultrasonography.Results: All adenocarcinomas had a height ≥ 5 mm and all benign lesions had a height ≤ 3 mm, with this difference being significant (P = 0.0034). The height of non-invasive and invasive cancer was similar. The maximum cross-sectional area of the protrusion was smaller for benign lesions (≤ 15 mm2) than for non-invasive cancer (≥ 34 mm2, P = 0.0034). The cross-sectional area of the protrusion was greater in patients with invasive cancer than in those with non-invasive cancer (P = 0.0367). All surgical margins have remained clear and no patient has suffered from a recurrence during 1 to 8 years of follow-up computed tomography and ultrasonography.Conclusions: Intraductal ultrasonography can distinguish benign from malignant IPMT based on the height and maximum cross-sectional area of the protruding tumor.
Digestive Endoscopy 06/2003; 15(3):196 - 199. · 1.19 Impact Factor
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ABSTRACT: The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca(2+)-permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the alpha1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro site-directed mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca(2+) accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca(2+) accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.
Journal of Biological Chemistry 04/2003; 278(11):9647-54. · 4.77 Impact Factor
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ABSTRACT: gene encodes a novel type of adenylate cyclase. The catalytic domain is located in the carboxyl-terminal half, while the
GAF and PAS domains are conserved in the amino-terminal half. Recombinant CyaB1 and a truncated CyaB1 lacking the amino-terminal
domain (ΔN–CyaB1) were purified and characterized. The purified CyaB1 is activated by divalent cations, such as Mg2+ and Mn2+, like other types of adenylate cyclase. The activity of CyaB1 was slightly elevated by forskolin, but was not affected by
cGMP, irrespective of the presence of the cGMP binding motif in the GAF domain. The specific activity of ΔN–CyaB1 is one-eighteenth
that of CyaB1, whereas the Km values of both proteins are almost the same. The results suggest that the amino-terminal half
has a positive regulatory effect on the catalytic activity.
Journal of Plant Research 11/2001; 114(4):387-394. · 1.75 Impact Factor
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ABSTRACT: Palliative stenting for gastric outlet obstruction (GOO) offers a more rapid resumption of oral intake than surgical gastrojejunostomy. Clinically, delayed gastric emptying is observed less frequently in patients with enteral stenting. The aim of this study was to conduct a functional assessment of gastric emptying after stent placement for GOO using isotope scanning.
Gastric emptying was assessed in 14 patients with GOO (4 female, 10 male; mean age 67.9 years; 8 with gastric cancer; 4 with pancreatic cancer; 1 with biliary cancer; 1 with metastasis) and 10 healthy volunteers (2 female, 8 male; mean age 31.5 years). None of the patients had undergone previous stomach surgery. The patients were studied 1 week after stent placement. Scintigraphy was performed for 2 hours following the ingestion of a labeled liquid meal. Gastric retention was evaluated at 2 hours in both groups.
All patients underwent successful placement of stents and were able to resume an oral diet. All stents were fully deployed and no migration was seen at the time of the investigation. Retained gastric activity at 120 minutes (RGA120) was significantly greater in patients than in controls (65.4% vs. 27.5%, p=0.0128). Median survival time was 179 days in patients with T1/2 of 120 min or less and 75 days in patients with T1/2 of over 120 min.
The results of our study show that although patients with GOO have resumed oral intake 1 week after stent placement, restoration of gastric emptying is often still incomplete.
Hepato-gastroenterology 55(81):298-302. · 0.66 Impact Factor
