Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.

Mitral regurgitation after myocardial infarction. Coronary artery bypass grafting and mitral valve replacement with chordae preservation.

A patient with acute ischemic mitral regurgitation after acute myocardial infarction required emergency coronary artery bypass grafting and mitral valve replacement with chordae preservation. For severe mitral regurgitation and heart failure due to myocardial infarction and ischemic papillary muscle dysfunction, mitral valve replacement with chordae preservation was effective. Here, we discuss the etiology of ischemic mitral regurgitation and the operative method for valve repair or replacement.

Coronary artery bypass grafting in dialysis patients.

OBJECTIVE: In dialysis patients, there are two issues to consider, water-electrolyte control and a bypass technique for a calcified aorta. We used continuous hemofiltration for water-electrolyte control and an off-pump bypass with arterial grafting for a calcified aorta. METHODS: We performed coronary artery bypass grafting with extracorporeal circulation in 9 cases and without extracorporeal circulation (off-pump bypass) in 3 cases. In 6 cases, the operation was urgent, and in 6 cases the operation was elective. RESULTS: An average of 3.2 grafts/pt, (the arterial graft: 1.3 grafts/pt) was performed in the pump cases. In the off-pump bypass cases we used arterial grafting only (1.7 grafts/pt). We had 1 early death (sudden death) and 1 hospital death (SLE encephalopathy). One late death due to cerebral bleeding occurred at 2 years later. We used continuous hemofiltration for 2 to 11 days (average 3.9 days) in the pump cases. The off-pump cases could be controlled by conventional hemodialysis. CONCLUSION: Continuous hemofiltration was very easily set up with less interference to the hemodynamics. Using an arterial graft with off-pump bypass, an aortic no-touch technique and water control with conventional hemodialysis were possible.

[Emergency coronary artery bypass grafting in dialysis patients].

Emergency coronary artery bypass surgery was performed in nineteen patients including five chronic hemodialysis patients. In fourteen patients (non-dialysis patients), ten patients required preoperative IABP and four required PCPS for cardiogenic shock. Four patients died (29%); three from LOS and one from sudden death. Most of them (3/4) were preoperative cardiogenic shock. In other five patients (dialysis patients), four patients had conventional CABG, while one received off-pump bypass surgery for calcified aorta and brain complication. All five patients received arterial in situ conduit and continuous hemofiltration in the early postoperative period for controlling water and electrolyte. Two patients died (40%); one from arrhythmia and one from deterioration of original disease (SLE encephalopathy) but no one died from LOS. These methods of surgical modification and perioperative management for chronic hemodialysis patients are considered very useful.

Epidemiological research into nasopharyngeal carcinoma in the Chubu region of Japan.

Although patient death due to nasopharyngeal carcinoma (NPC) is increasing, few epidemiological analyses of NPC in Japan have been conducted since Sawaki's report in 1979. To determine the current incidence of NPC in Japan we examined NPC case in the Chubu area from 1986 to 1995. The leaders and reporting representatives of all otorhinolaryngological groups in the area were asked for their support of this epidemiological research. A total of 607 cases (445 male and 162 female NPC patients) were analyzed epidemiologically, histologically, serologically and clinically in this study. The incidence of NPC gradually increased with age. The mean age of the patients was 54.1 years. The age-standardized annual incidence in the Chubu region was 0.28 per 10(5) persons per year. The incidence in prefectures bordering Japan Sea (0.36) was significantly higher than that of prefectures facing the Pacific Ocean (0.21, P<0.05). On the basis of World Health Organization (WHO) histological criteria, 12%) of the cases were classified as WHO I, 54% as WHO II and 34% as WHO III. As for tumor origin, in 58% of the cases it originated posterosuperiorly, in 32% laterally and in 1% inferiorly. Tumor staging showed 4% to belong to stage I, 9% to stage II, 15% to stage III and 72% to stage IV. The positive rates of serum titers of the antibodies to Epstein Barr virus

Distribution and seasonal variations in levels of three native GnRHs in the brain and pituitary of perciform fish.

Specific and sensitive radioimmunoassays (RIAs) were newly developed for two types of gonadotropin-releasing hormone (GnRH), namely, seabream (sb) GnRH and chicken (c) GnRH-II. We employed these two RIAs together with a previously reported RIA for salmon (s) GnRH to study the presence and regional distribution of these three GnRHs in the brains and pituitaries of four perciform fishes (red seabream, Pagrus major; black seabream, Acanthopagrus schlegeli; striped knifejaw, Oplegnathus fasciatus; and Nile tilapia, Oreochromis niloticus), as well as clarify seasonal changes in levels of these GnRHs in the brain and pituitary of red seabream. All three GnRHs were found in brains of all fishes examined, with regional distributions in the brains of the three GnRHs being rather similar. sbGnRH was abundant in telencephalon and hypothalamus. cGnRH-II was concentrated from the middle to posterior part of the brain and distributed throughout the brain. sGnRH was concentrated in the olfactory bulb and distributed all over the brain, as was cGnRH-II. The dominant form of GnRH in the pituitary was sbGnRH, with levels 500- to 2400-fold higher than those of sGnRH, while cGnRH-II was undetectable in all four species. In the brain and pituitary of female red seabream, levels of both brain and pituitary sbGnRH increased from October (immature phase) and reached a peak in April (spawning phase), reflecting the increase in gonadosomatic index and vitellogenesis. However, levels of sbGnRH remained high only in the pituitary of completely regressed fish in June. Levels of both sGnRH and cGnRH-II in the brain were higher in the regressed phase and remained lower during the spawning phase. From these and previous results, it appears that sbGnRH is physiologically the most important form of GnRH in reproduction in red seabream and, probably, in other perciforms also.

Glucan binding regions of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

We isolated glucan-binding peptides of a dextransucrase from Leuconostoc mesenteroides B-512F. The dextransucrase was bound to DEAE-Sephadex A-50, Sephadex G-100, and mutan from Streptococcus mutans. Mild trypsin digestion dissociated the enzyme and glucan binding. In the presence of ammonium sulfate, several peptides were bound to glucan after trypsin digestion. Four main mutan-binding peptides were obtained by this method, and those amino acid sequences were analyzed. One of them was identical with the dextran-binding peptide that contains lysine, which was previously isolated by differential chemical modification with o-phthalaldehyde. We also found mutan-binding peptides in sucrose- and dextran-binding regions and a lysine-rich region. Also, there was a peptide similar in sequence to glucan-binding A-repeat of streptococcal glucosyltransferases.

Protein Disulfide Isomerase Activity of Some Plant Seeds.

The activity of protein disulfide isomerase, in the extracts of several dormant seeds including soybean, rice, wheat, and maize was assayed. The activity was higher in the extracts of beans than in those of the other seeds. A correlation was significant (R=0.95 and 0.93; p<0.01) between the PDI activity and the concentration of protein soluble in a salt solution.

Flutolanil Resistance as a Genetic Marker of Coprinus cinereus Strains.

All 4 Schizophyllum commune strains among several basidiomycete species were exclusively resistant to flutolanil, which inhibits succinate dehydrogenase complex (SDC) function. Flutolanil resistant strains could be screened from monokaryotic protoplasts of sensitive Coprinus cinereus, by treating them with a cloned genomic SDC iron-sulfur protein (IP) gene from the resistant S. commune. The obtained resistance was stably transferred to the progeny.

Cloning of SEZ-12 encoding seizure-related and membrane-bound adhesion protein.

SEZ-12 is one of the seizure-related cDNAs which was isolated by differential hybridization from primary cultured neurons from the mouse cerebral cortex with or without pentylenetetrazol (PTZ). SEZ-12 expression is transiently down-regulated in the mouse brain by injection of PTZ. To characterize SEZ-12, isolation of full-length cDNA and nucleotide sequence analysis were performed. The deduced amino acid sequence of SEZ-12 revealed that it encodes membrane-bound C-type lectin and has a significant homology to that of human cDNA, DGCR2 and IDD, which were cloned from a balanced translocation breakpoint associated with the DiGeorge syndrome. The isolated cDNA was about 4 kb in length and the message was expressed ubiquitously in various organs with low-abundance. Previously, we also cloned a transmembrane protein which is probably involved in cell-cell interaction by the differential hybridization technique. These findings suggest that transmembrane signaling in neuronal cells may have an important role in PTZ-induced seizure.

Active site peptides with CXXC motif on map-resin can mimic protein disulfide isomerase activity.

Two conserved Trp-Cys-Gly-His-Cys (WCGHC) sequences are assigned to act as catalytic sites for protein disulfide isomerase. Peptides containing the active site sequence, Ala-Pro-Trp-Cys-Gly-His-Cys-Lys(APWCGHCK), were synthesized both in a mono-molecular form and on multiple antigen peptide (MAP) resin or Wang resin by the 9-fluoroenylmethoxycarbonyl (Fmoc)-based solid-phase method. With scrambled RNase as a substrate, the (APWCGHCK)8-MAP was first shown to mimic the PDI activity, which was one thousandth of that of bovine PDI and comparable to that of thioredoxin. APWCGPCK and APWCGHCK, however, did not display a disulfide isomerase activity even at a concentration 8 times higher than that of (APWCGHCK)8-MAP. It was assumed that a sterically proper proximity of at least two active site peptides with CXXC motif was required for the expression of PDI activity.

Cloning and characterization of pentylenetetrazol-related cDNA, PTZ-17.

cDNAs related to pentylenetetrazol-induced bursting activity in neurons were screened by a differential hybridization method using normal and pentylenetetrazol-treated primary cultured neurons from the cerebral cortex of mice. Twenty clones of candidate cDNA with expression increased or decreased by treatment with pentylenetetrazol were obtained. One of them, PTZ-17, was sequenced. Injection of PTZ-17 derived RNA into Xenopus oocytes showed a large calcium inward current with extracellular application of pentylenetetrazol.

Purification and characterization of protein disulfide isomerase from soybean.

Protein disulfide isomerase (PDI), which catalyses the folding of newly synthesized or denatured proteins through correct disulfide formation, was purified from soybean (Glycine max). The enzyme was purified 12,000-fold over crude extracts to apparent homogeneity in six purification steps: 60-70% ammonium sulfate fractionation, and chromatography on DEAE Toyopearl 650M, Q-Sepharose Fast Flow, Hiload Superdex 200 pg, Phenyl Sepharose HP, and TSK G-3000 SW. The native enzyme had a molecular weight of 120 kDa on gel filtration. Subunit molecular weight was estimated as 63 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, thus indicating the enzyme to be comprised of two identical subunits. The enzyme pH optimum was 8.0 with reactivation of scrambled RNase, and the pI 7.65. The N-terminal amino acid sequence of soybean PDI was homologous to that of mature alfalfa as deduced from the cDNA sequence. Two identical active site sequences, APWCGHCK, were obtained from different proteolytic peptide fragments of soybean PDI. Soybean PDI facilitated reactivation not only of scrambled RNase, but denatured and reduced lysozyme and the Bowman Birk soybean trypsin inhibitor as well. This is the first report to appear on the the purification, characterization and amino acid sequence analysis of the active site of a plant PDI.

Identification and characterization of a family of genes for the plasma membrane H(+)-ATPase of Oryza sativa L.

A cDNA clone (cOSA2) encoding a plasma membrane H(+)-ATPase was isolated from rice. Southern blot analysis indicated that the genes that corresponds to cOSA2 was different from that to cOSA1. Northern blots revealed OSA2 mRNA in roots, calli and shoots. OSA1 transcripts were detected only by RT-PCR in these tissues.

Simple and efficient procedure for removing the 34 kDa allergenic soybean protein, Gly m I, from defatted soy milk.

The 34 kDa protein in the salted-out fraction of soy milk is shown to be identical to the allergenic soybean protein, Gly m I, by N-terminal amino acid sequencing and immunoblotting, using the monoclonal antibody against Gly m I. Treatment of soy milk with 1 M Na2SO4, acidifying to pH 4.5 and centrifugation removed more than 90% of the 34 kDa protein with an overall yield of 70% of total soy protein.