Characterization of a fission yeast mutant which displays defects in cell wall integrity and cytokinesis.

The fission yeast cps6-153 mutant was originally isolated based on its hypersensitivity to the spindle poison isopropyl N-3-chlorophenyl carbamate (CIPC). The mutant also shows defects in both cell wall integrity and cytokinesis, resulting in the accumulation of unseparated cells with weakened cell walls. The arrested cells display a disoriented alignment of cytoplasmic microtubules. When the mutant cells are cultivated at high temperature (35 degrees C), both cell walls and septa become very thick. Electron microscopy revealed the disorganized structure of the thickened cell walls and septa, in which fibrillar components were not completely masked with an amorphous matrix. rad25+ was cloned from a genomic library by complementation of the mutant phenotypes, suggesting the involvement of Rad25p, one of two 14-3-3 proteins in S. pombe, in the pathway of cell wall integrity and cytokinesis.

Abnormal formation of the glucan network from regenerating protoplasts in Schizosaccharomyces pombe cps8 actin point mutant.

To study the close relationship between the actin cytoskeleton and cell wall formation, the process of cell wall formation in reverting protoplasts of the fission yeast, Schizosaccharomyces pombe, cps8 actin point mutant was investigated by ultra-high-resolution low-voltage scanning electron microscopy (UHR-LVSEM) and transmission electron microscopy (TEM). The protoplast of the cps8 mutant began to form a glucan network in a unipolar manner and to secrete alpha-galactomannan. The site of cell wall formation grew in a cylindrical shape in the wild-type protoplast. The alpha-galactomannan did not fill in the intrafibrillar spaces completely, however, and the fibrils were exposed on the cell surface. UHR-LVSEM images indicated that the glucan fibrils were thin and rope-shaped, forming a looser network than the wild-type. TEM images indicated the finest fibrils were approximately 1.5 nm in diameter, the same diameter as the wild-type. These results suggest that the cps8 mutant was insufficient in developing cross-linkage with the glucan fibrils up to the wide ribbon shape as found in the wild-type [Osumi M et al. (1989) J. Electron Microsc. 38: 457-468; Osumi M (1998) Micron 29: 207-233]. These findings appear to indicate that the actin cytoskeleton controls formation of the glucan network and secretion of beta-1,6-glucan, and confirm the close relationship of the actin cytoskeleton and glucan formation.

Ultrastructure of cell wall of the cps8 actin mutant cell in Schizosaccharomyces pombe.

A Schizosaccharomyces pombe cps8 mutant, of which the gene encodes a mutant actin with an amino acid substitution of Asp for Gly(273) [J. Ishiguro and W. Kobayashi (1996) FEBS Lett. 392, 237-241], was used to determine the role of the actin cytoskeleton in cell wall formation. In the cps8 mutant cells, atomic force microscopic and scanning electron microscopic images showed abnormal depolarized and branched morphology. Fibrous material covered a part of the surface of growing cps8 cells. Transmission electron microscopic images showed variable thickness of the cell wall due to multilayering of cell wall materials, and aberrant multisepta due to diagonal growth of the primary septum, whereas the normal primary septum grows at a right angle from the cortex. This abnormal septum formation may induce abnormality of the cell with multinuclei and/or multisepta, caused by non-separation of daughter cells. These results indicate that actin plays an important role in cell wall and septum formation.

Percutaneous choledochoscopic treatment of intrahepatic stones, including management of associated biliary stenoses.

BACKGROUND AND STUDY AIMS: Choledochoscopic lithotomy is a useful non-surgical treatment for intrahepatic stones. In patients with stenoses, the procedure often fails, and recurrence rates are high. PATIENTS AND METHODS: The efficacy and risks of choledochoscopic lithotomy using our procedure were investigated in 15 patients with intrahepatic stones, with and without strictures. Long-term follow-up results in patients after successful clearance were also reviewed. The follow-up period ranged from one month to 127 months (mean 75 months). RESULTS: Complete removal of stones was achieved in a mean of 2.0 sessions in all cases. The relationship between the number of sessions and the presence of the stenosis was not significant. One patient (6.7%) who had recurrent stones after complete clearance was successfully treated by repeat choledochoscopy. With regard to the rate of recurrence, there was no difference between patients with stenosis and those without. There was no procedure-related mortality. The rate of procedure-related complications was 6.7%. CONCLUSIONS: Choledochoscopic lithotomy for hepatolithiasis is an effective and safe procedure in most patients, even those with severe biliary stenosis. The choice of the appropriate route for lithotomy and appropriate management of stenoses offers a higher success rate and a lower rate of recurrence.

Clinical evaluation of the peroral cholangioscopy using a new videoscope.

Peroral cholangioscopy (PCS) has been performed in 22 cases using XCHF-B200 (Olympus Optical Co.) since June 1995 and in 77 cases using CHF-B20 (Olympus Optical Co.) after EST from Jan. 1989. XCHF-B200 has a longer rigid portion of distal end and a smaller channel diameter than CHF-B20. The successful rate of PCS using XCHF-B200 (82%) was lower than that of CHF-B20 (89%). The vascular pattern and fine vertical groove of the bile duct mucosa were shown more clearly on the photographs obtained with XCHF-B200 than those obtained with CHF-B20. However, not enough biopsy specimens could be obtained because the channel diameter of XCHF-B200 was rather small.If the length of rigid portion and biopsy channel of XCHF-B200 are improved, PCS using XCHF-B200 will be more useful for the diagnosis of bile duct disorders.

Successful endoscopic resection of an early carcinoma of the duodenum.

We describe a patient in whom an early carcinoma of the duodenum was able to be resected endoscopically. A 77-year-old man presented with epigastric pain. Upper gastrointestinal endoscopy revealed a mass in the duodenum, and the patient was admitted. A whitish nodular aggregated lesion, measuring 20 mm in diameter, was found in the second portion of the duodenum. Examination of a biopsy specimen showed a Group III tubular adenoma. Endoscopic ultrasonography showed that the lesion was confined to the mucosa. The large size of the lesion suggested the possibility of malignancy. Endoscopic mucosal resection was therefore performed. Histopathologically, the diagnosis was carcinoma in adenoma. The depth of invasion was mucosal. We conclude that endoscopic muosal resection can be used to treat mucosal lesions arising in the duodenum.

Effects of photoperiod and temperature on seasonal morph development and diapause egg oviposition in a bivoltine race (Daizo) of the silkmoth, Bombyx mori L.

A bivoltine race (Daizo) of the silkmoth, Bombyx mori, has two types of adults which are classified on the basis of the number of dark-brown and brown scales distributed along the outer lines of the anterior wings. Development of these two types of adults is determined by photoperiod and temperature experienced during the embryonic and larval stages. The two types of adults are thought to be seasonal (summer and autumn) morphs. All adults developed under short days at low temperature (15 degrees C and 23 degrees ) were classified as summer morphs and oviposited only nondiapause eggs (ND-eggs), and those that developed under long days at high temperature (28 degrees C and 25 degrees C) were autumn morphs ovipositing only diapause eggs (D-eggs), only ND-eggs or a mixture of D- and ND-eggs. Larval exposure to long days at 25 degrees C shifted the wing pattern towards the autumn morphs and decreased the incidence of female adults ovipositing only D-eggs. Larval exposure to long days elicits an opposite effect on D-egg oviposition to that observed when long days are received only during the embryonic stage.

Genetic control of fission yeast cell wall synthesis: the genes involved in wall biogenesis and their interactions in Schizosaccharomyces pombe.

The fungal cell wall is an essential structure which protects cells from various environmental stresses such as hyper- or hypo-osmosis, and endows them with specific morphology in response to their life or cell division cycle. In addition, the cell wall has a variety of enzymatic activities per se, which are required for nutritional uptake, secretion, and cell adhesion including mating processes. In addition to these cytological interests, clinical demands to clarify the regulatory mechanisms of cell wall synthesis have been increasing, since the cell wall is a unique and effective target of antifungal agents. However, the molecular mechanisms are poorly understood at present, although the role of several signal transduction pathways have recently been implicated in regulation. In this review, the author focuses on genes and their interactions which are involved in fission yeast cell wall biogenesis.

Biliary bacteria as an indicator of the risk of recurrence of choledocholithiasis after endoscopic sphincterotomy.

Bacteria have been implicated in recurrent choledocholithiasis associated with endoscopic sphincterotomy (EST). This study was designed to clarify whether bacterial examination of bile provides information useful in predicting the risk of recurrence of choledocholithiasis in patients undergoing EST. Bacteria in bile collected via a duodenoscope before cholangiography were cultured. We compared bacterial isolates and quantity among 41 patients with choledocholithiasis (7 with and 34 without a history of recurrent choledocholithiasis) who had undergone EST more than 3 months previously and 13 control patients with no evidence of pancreatobiliary disease. The bile samples were cultured under aerobic and anaerobic conditions. The bacterial quantity was expressed as the mean logarithm of the number of colony forming units (CFU)/ml. Furthermore, cholescintigraphic studies of bile flow were performed with the use of (99 m)TC-HIDA to study the clinical implication of these variables. No bacteria were detected in 10 of the 13 patients in the control group. In the other three control patients the bacterial count was 2.2 log CFU/ml or less. The mean bacterial count was significantly higher in patients with recurrence than in those without recurrence. Cholescintigraphy revealed a trend toward a higher number of isolates and a higher bacterial count in bile in patients with delayed bile passage than in those with good passage. The results suggest that an increased number of biliary isolates and an increased bacterial count indicate decreased bile flow in patients with choledocholithiasis who are being followed up after EST. These variables may potentially serve as indicators of the risk of stone recurrence. Especially when the bacterial count is higher than 7.0 log CFU/ml, the risk of a decrease in bile flow and an increased stone recurrence would be possibly found.

cps1+, a Schizosaccharomyces pombe gene homolog of Saccharomyces cerevisiae FKS genes whose mutation confers hypersensitivity to cyclosporin A and papulacandin B.

The Schizosaccharomyces pombe cps1-12 (for chlorpropham supersensitive) mutant strain was originally isolated as hypersensitive to the spindle poison isopropyl N-3-chlorophenyl carbamate (chlorpropham) (J. Ishiguro and Y. Uhara, Jpn. J. Genet. 67:97-109, 1992). We have found that the cps1-12 mutation also confers (i) hypersensitivity to the immunosuppressant cyclosporin A (CsA), (ii) hypersensitivity to the drug papulacandin B, which specifically inhibits 1,3-beta-D-glucan synthesis both in vivo and in vitro, and (iii) thermosensitive growth at 37 degrees C. Under any of these restrictive treatments, cells swell up and finally lyse. With an osmotic stabilizer, cells do not lyse, but at 37 degrees C they become multiseptated and multibranched. The cps1-12 mutant, grown at a restrictive temperature, showed an increase in sensitivity to lysis by enzymatic cell wall degradation, in in vitro 1,3-beta-D-glucan synthase activity (173% in the absence of GTP in the reaction), and in cell wall biosynthesis (130% of the wild-type amount). Addition of Ca2+ suppresses hypersensitivity to papulacandin B and septation and branching phenotypes. All of these data suggest a relationship between the cps1+ gene and cell wall synthesis. A DNA fragment containing the cps1+ gene was cloned, and sequence analysis indicated that it encodes a predicted membrane protein of 1,729 amino acids with 15 to 16 transmembrane domains. S. pombe cps1p has overall 55% sequence identity with Fks1p or Fks2p, proposed to be catalytic or associated subunits of Saccharomyces cerevisiae 1,3-beta-D-glucan synthase. Thus, the cps1+ product might be a catalytic or an associated copurifying subunit of the fission yeast 1,3-beta-D-glucan synthase that plays an essential role in cell wall synthesis.

Metabolic fate of ulinastatin (2); Pharmacokinetics in rabbits following intra-articular administration.

The absorption and distribution of ulinastatin following intra-articular administration of [125I]ulinastatin to rabbits were determined and kinetic analysis using a multi-compartment model was performed. At 4 h after administration, the content of radioactivity in the synovial fluid, which was comparable to that of the immunoreactive ulinastatin, was 14.51% of the dose and decreased in a biphasic manner. The highest level of radioactivity was observed in the synovial membrane, followed by the meniscus, ligament, cartilage and patella, the radioactivities of which also declined biphasically. After intra-articular administration, the plasma concentration of total radioactivity increased slowly and reached maximum at 4.3 h, and then declined slowly in a monophasic manner with a half-life of 10.8 h. The radioactivity of the high molecular weight fraction in plasma, which reached maximum at 1.7 h after administration and then declined with a half-life of 11.8 h, was consistent with the time curve for immunoreactive ulinastatin in the plasma through 24 h after the administration. Within 8 and 24 h after administration, respectively, 1.48 and 4.66% of the administered radioactivity were transferred to the lymphatic fluid. The pharmacokinetics of [125I]ulinastatin after an intra-articular administration could be explained using a multi-compartment model in which a portion of the administered ulinastatin was absorbed via the lymphatic system. This finding suggested that ulinastatin was rapidly distributed and retained for a long period of time in the joint tissues. In addition, the pharmacokinetics of ulinastatin following intra-articular administration indicated typical flip-flop kinetics.

An actin point-mutation neighboring the 'hydrophobic plug' causes defects in the maintenance of cell polarity and septum organization in the fission yeast Schizosaccharomyces pombe.

The fission yeast cps8 mutation gives rise to abnormally enlarged and dispolarized cells, each of which contains several nuclei with aberrant multisepta. Molecular cloning and sequence analysis of the cps8 gene indicated that it encodes an actin with an amino acid substitution of aspartic acid for glycine at residue 273 in the hydrophobic loop that is located between actin subdomains 3 and 4. Fluorescence microscopy using phalloidin and anti-actin antibody revealed changes in the F-actin structure and distribution in the mutant cells. These results indicate that the hydrophobic loop plays an essential role for creating normal F-actin structure, only by which cell polarity and the late mitotic events can be maintained properly.

Is diabetic heart muscle disease related to cardiac ischemia or autonomic neuropathy?

Whether diabetic heart muscle disease is related to cardiac ischemia or autonomic neuropathy was investigated employing thallium-201 and [123I]meta-iodobenzylguanidine (MIBG) myocardial scintigraphy. Among ischemic hearts, displayed by TI201 scintigraphy, a distinctly dilated and hypokinetic left ventricle could not be detected. In autonomically denervated hearts, however, well demonstrated by MIBG imaging, distinct dysfunction was found. But, even in these hearts, interacting effects of some concomitant complications could not be excluded.

A practical method for fission yeast transformation by electroporation.

High-voltage shock within a very short duration under the proper conditions causes cells to incorporate exogenous macromolecules. This technique, electroporation, has been widely used in recent years to transform many organisms. We determined optimum conditions for fission yeast transformation using this method. Of nineteen combinations of electric field strength and pulse time examined, 1.75 kV/0.2 cm, 4 msec pulse was found to provide approximately 4.0 x 10(5) transformants per micrograms of DNA. Other factors responsible for the transformation efficiency in fission yeast are also discussed.

Molecular cloning and characterization of a fission yeast gene responsible for supersensitivity to the spindle poison, isopropyl N-3-chlorophenyl carbamate.

The cps3 gene of the fission yeast, Schizosaccharomyces pombe, was previously identified as a mutation conferring supersensitivity to the spindle poison, isopropyl N-3-chlorophenyl carbamate (CIPC). A 3.2 kb DNA fragment that complements the mutant phenotype was cloned from a S. pombe genomic library. The base sequence analysis showed that the fragment contains a maximum 1086 nucleotide open reading frame and that the putative product consists of 362 amino acids, having a molecular weight of 39.3 KDa. No significant homology of the potential product with known proteins could be found by database searches. A disruptant of the gene, produced by insertion of a ura4+ fragment was able to germinate, but not to undergo cell division, suggesting that the gene to be essential for the cell cycle progression. The disruption experiment suggests that the gene is an extragenic suppressor of cps3 mutation.

[Clinical evaluation of patients with right ventricular infarction detected by dual SPECT imaging of thallium-201 and technetium-99m pyrophosphate].

We evaluated clinical significance of dual-nuclide SPECT imaging (D-SPECT) of thallium-201 and technetium-99m pyrophosphate (PYP) in patients of acute inferior left ventricular infarction with PYP uptake in the right ventricle (PYP (+) group) in comparison with those without PYP uptake (PYP (-) group). There was no difference in coronary risk factors, history of angina, blood pressure, heart rate, and hemodynamics on admission between PYP (+) group and PYP (-) group. The duration from onset to admission was longer in PYP (+) group and coronary reperfusion therapies were carried out in few cases. In 7 of 8 PYP-positive patients, the diagnosis of right ventricular infarction was made only by D-SPECT. Four of 8 were complicated with shock within three days, and the duration of hospitalization was longer. Coronary angiography demonstrated many proximal lesions (50%) in PYP (+) group but few ones (18%) in PYP (-) group. D-SPECT was very useful for diagnosing acute right ventricular infarction, and it might contribute to the prevention of shock if performed within a few days.

Combination therapy of transcatheter chemoembolization and percutaneous ethanol injection therapy for unresectable hepatocellular carcinoma.

The therapeutic effectiveness of a combination therapy--pretreatment with transcatheter arterial chemoembolization (TACE) followed by percutaneous ethanol injection (PEI) therapy--for large (> 3 cm in diameter) unresectable hepatocellular carcinoma (HCC) was compared with that of TACE alone. PEI therapy was performed in 24 cases of unresectable HCC that had previously been treated with TACE using doxorubicin 30-60 mg or epirubicin 50-90 mg. In all, 2-10 ml of 90% ethanol mixed with carbocaine was repeatedly injected through a 21-gauge, closed-end needle (PEIT needle) for a median of 3.6 injections and 31.1 ml of ethanol. As adverse effects, transient localized pain and a burning sensation were observed in 75.0% of the cases; fever, in 66.7%; and transient hypotension, in two cases. A small unresectable tumor is a good indication for PEI therapy. In cases with a larger tumor, i.e., measuring more than 3 cm in diameter, or multiple tumors, the 1-year survival rate obtained with this combination therapy, i.e., TACE and PEI, was 87.0%, and the 2-year survival rate was 65.2%. These rates were greater than those obtained with TACE alone. Accordingly, additional PEI therapy was effective for larger tumors and multiple tumors previously treated with TACE.

Orthodromic capture of the atrial electrogram during transient entrainment of atrioventricular nodal reentrant tachycardia.

BACKGROUND: The reentry circuit of atrioventricular nodal reentrant tachycardia (AVNRT) has not been fully demonstrated. We hypothesized that if an upper common pathway was present, the atrial electrogram could not be captured orthodromically during transient entrainment of AVNRT by rapid atrial pacing. Based on this hypothesis, the presence of an upper common pathway was investigated. METHODS AND RESULTS: The atrial electrogram at the recording site of the His bundle potential was identified during induced AVNRT in 9 patients. To entrain AVNRT transiently, rapid pacing from the high right atrium and coronary sinus was applied at a cycle length 10 milliseconds shorter than that of AVNRT and repeated after a decrement of the paced cycle length in steps of 5 milliseconds until AVNRT was interrupted. In 5 of 7 patients, orthodromic capture of the atrial electrogram at the recording site of the His bundle potential was observed during transient entrainment of AVNRT by coronary sinus pacing, ie, the first postpacing interval of the atrial electrogram at the recording site of the His bundle potential was the same as the paced cycle length. In these 5 patients, the mean minimum paced cycle length capable of orthodromic atrial capture was 349 milliseconds, and the mean difference from the cycle length of AVNRT was only 16 milliseconds. During transient entrainment of AVNRT by high right atrial pacing, the atrial electrogram could not be captured orthodromically. CONCLUSIONS: Observation of orthodromic capture of the atrial electrogram at the recording site of the His bundle potential by coronary sinus pacing ruled out the presence of an upper common pathway in AVNRT, and the concept that perinodal atrial tissue is involved in the reentry circuit of AVNRT was supported.

A novel mutation involved in the mitotic checkpoint in the fission yeast Schizosaccharomyces pombe.

The cps8 mutation which confers supersensitivity to a spindle poison, Isopropyl N-3-chlorophenyl carbamate (CIPC), in the fission yeast Schizosaccharomyces pombe was investigated. The cps8 mutant accumulated enlarged multinucleate cells in the stationary phase under normal growth conditions. The mutant was highly lethal at 36.5 degrees C in a fresh growth medium but not in a saline solution where the cell cycle ceases quickly. Lethality at high temperature was significantly suppressed by cdc1 or nda2 mutation which blocks nuclear division, but not by hydroxyurea treatment or cdc22 mutation which blocks DNA synthesis. A cdc10 cps8 double mutant remained lethal to high temperature, suggesting this double mutant to bypass the requirement for cdc10+ indispensable for the cell cycle start in a wild-type cell. After being transferred to a fresh medium at 36.5 degrees C, the multinucleate cells rapidly divided with aberrant nuclear segregation. Thus, cps8 mutation allows cells to undergo mitosis without DNA replication at the restrictive temperature. The cps8 gene was mapped on the left arm of chromosome II closely linked to but distinct from cdc2 locus.