Observation of three-dimensional internal structure of steel materials by means of serial sectioning with ultrasonic elliptical vibration cutting.

A three-dimensional (3D) internal structure observation system based on serial sectioning was developed from an ultrasonic elliptical vibration cutting device and an optical microscope combined with a high-precision positioning device. For bearing steel samples, the cutting device created mirrored surfaces suitable for optical metallography, even for long-cutting distances during serial sectioning of these ferrous materials. Serial sectioning progressed automatically by means of numerical control. The system was used to observe inclusions in steel materials on a scale of several tens of micrometers. Three specimens containing inclusions were prepared from bearing steels. These inclusions could be detected as two-dimensional (2D) sectional images with resolution better than 1 mum. A three-dimensional (3D) model of each inclusion was reconstructed from the 2D serial images. The microscopic 3D models had sharp edges and complicated surfaces.

Extensive bleeding during surgical treatment for gingival overgrowth in a patient on haemodialysis--a case report and review of the literature.

Before performing renal transplantation, a most important concern is to control any infection, including oral infections before transplantation. The bleeding diathesis of patients with uraemia is a significant clinical concern, especially when surgery is required. A 44-year-old female patient on haemodialysis was referred for evaluation of gingival overgrowth. The patient was planning a renal transplantation two months later. As the lesions were not considered successfully treatable before transplantation, a gingivectomy and teeth extraction was performed. In pre-operative examinations, an abnormal bleeding time was not detected and other coagulation tests were normal. Under general anaesthesia, 19 teeth were extracted and overgrown gingiva was removed. During the operation, extensive blood loss of 1650ml occurred and four units of concentrated red blood cells were transfused. This study suggests that patients with renal failure undergoing dental surgery require careful pre-surgical evaluation including assessment of their coagulation ability.

Antisense Bcl-x oligonucleotide induces apoptosis and prevents arterial neointimal formation in murine cardiac allografts.

OBJECTIVE: Cardiac allograft arteriosclerosis, which limits long-term survival of recipients, cannot be prevented by conservative therapies. The arteriopathy is characterized by diffuse intimal thickening comprised of proliferative smooth muscle cells (SMCs). Cell death is a prominent feature of atherosclerosis; Bcl-x is one of the anti-apoptotic mediators. METHODS: To test the hypothesis that antisense bcl-x oligodeoxynucleotide (ODN) is effective in preventing intimal hyperplasia through enhancing apoptosis after cardiac transplantation, we performed single intraluminal delivery of antisense bcl-x ODN into murine cardiac allografts (n = 9). DBA/2 (H-2d) hearts were transplanted into B10.D2 (H-2d) mice. Sense bcl-x ODN (n = 8) and no treatment (n = 8) studies were also performed. RESULTS: Allografts were harvested at 4 weeks after transplantation; all allografts kept beating throughout the period. Coronary intimal thickening had developed in nontreated and sense ODN transfected allografts at 4 weeks after transplantation with enhanced expression of Bcl-x and cell adhesion molecules, and suppressed apoptosis. However, antisense bcl-x ODN prevented neointimal formation through enhanced apoptosis. CONCLUSION: These results indicate that apoptosis of vascular SMCs induced by Bcl-x is associated with initial hyperplasia after heart transplantation. Antisense bcl-x ODN inhibits SMC proliferation by inducing apoptosis in graft coronary arteries.

Compound heterozygosity for a point mutation and a deletion located at splice acceptor sites in the LAMB3 gene leads to generalized atrophic benign epidermolysis bullosa.

An autosomal recessive disorder, generalized atrophic benign epidermolysis bullosa, is a rare form of nonlethal type junctional epidermolysis bullosa. It is associated not only with skin fragility but also with other unique clinical features including widespread atrophic skin changes, alopecia, reduced axillary and pubic hair, dysplastic teeth, and dystrophic nails. The majority of generalized atrophic benign epidermolysis bullosa cases are caused by mutations in the COL17A1 gene coding for type XVII collagen (or the 180 kDa bullous pemphigoid antigen). Another candidate gene for mutations in some forms of generalized atrophic benign epidermolysis bullosa is LAMB3 encoding the beta3 chain of laminin 5. This report documents compound heterozygosity for novel mutations in LAMB3 of a Japanese patient showing typical clinical features of generalized atrophic benign epidermolysis bullosa. One is an A-to-G transversion at the splice acceptor site of intron 14, which is designated as a 1977-2A-->G mutation; the other is a deletion of 94 bp located at the junction of intron 18 and exon 19, which is a 2702-29del94 mutation. Reverse transcriptase polymerase chain reaction analysis suggested skipping of exon 19 in LAMB3 mRNA produced from the allele with 2702-29del94 and impaired stability of the aberrant mRNA transcribed from the second allele with the 1977-2A-->G mutation.

Experimental study on the role of osteoclasts and free radicals in the mandibular invasion of VX2 carcinoma in Japanese white rabbits.

Oxygen-derived free radicals are stimulators of bone resorption in vitro and in vivo. We hypothesized that oxygen-derived free radicals or reactive oxygen species (ROS) generated around bone matrix invaded by cancer cells might be associated with the activation or formation of osteoclasts in bone metastasis, and thus the administration of an ROS scavenger or a free-radical scavenger might control osteoclastic bone destruction in tumor metastasis. We examined the ability of VX2 carcinoma cells to generate superoxide anion (O2-) as well as the effects of the O2- scavenger superoxide dismutase (SOD) and the hydrogen peroxide (H2O2) scavenger catalase (CAT) on the activation and formation of osteoclasts in VX2 carcinoma tissue of rabbits. VX2 carcinoma cells generated more O2- than tartrate-resistant acid phosphatase-positive (TRAP+) cells. The TRAP activity from TRAP+ cells in the presence of VX2 carcinoma cells was higher than that from TRAP+ cells in the absence of the carcinoma cells. The addition of SOD but not of CAT into the incubation medium inhibited the TRAP activity from TRAP+ cells in the presence or absence of VX2 carcinoma cells. Similar inhibitory effects were also observed with SOD plus CAT on TRAP+ cells in the presence or absence of carcinoma cells. Intravenous administration of SOD and SOD plus CAT, but not of CAT, caused a decrease in the number of osteoclasts in mandibles implanted with VX2 carcinoma cells, and showed a predominant occupation by osteoclasts with poorly developed ruffled borders in lesions of bone resorption. In contrast, the administration of CAT, but not of SOD and SOD plus CAT, decreased the more mature forms of osteoclasts in implanted mandibles. From the results obtained, it is suggested that O2- may stimulate bone resorption by increasing the activity and number of osteoclasts, and H2O2 may stimulate resorption by enhancing the formation of mature osteoclasts in tumor metastasis. The administration of some ROS or free-radical scavengers to patients with cancer may provide a defense against bone destruction in bone metastatic lesions.

Dentinal response against carious invasion: localization of antibodies in odontoblastic body and process.

The pulpal origin of dentinal immunoglobulins was demonstrated by means of immunohistological methods. Immunoglobulins were located both in the cytoplasm of odontoblasts in pulp and at odontoblastic processes in dentin. Positive reactivity of the immunoglobulins to antigens was confirmed using peroxidase-immunized rabbits. IgG, IgA, IgM, C3, and C4 were observed on some invasive bacteria in human carious dentin.

Role of the autonomic nervous system in regulating salivary mucin secretion by the canine submandibular gland in vivo.

The secretion of mucin was assessed by measuring changes in protein and sialic acid concentrations in saliva. Electrical stimulation of the sympathetic nerve increased protein and sialic acid concentrations greatly but stimulation of the parasympathetic nerve by bethanechol (5-20 micrograms/kg, intravenously) caused a slight increase. Sympathomimetic drugs (1-10 micrograms/kg, intravenously) also increased protein and sialic acid concentrations (isoproterenol greater than adrenaline greater than noradrenaline). Mucin secretion was increased by H133/22, a beta 1 adrenoceptor agonist, or terbutaline, which is roughly equipotent. Adrenaline-induced mucin secretion was inhibited by propranolol, but not by tolazoline. These data suggest that mucin secretion is predominantly regulated by the sympathetic nervous system via beta adrenoceptors, both beta 1 and beta 2.

Involvement of prostaglandin E-like material in the purgative action of rhein anthrone, the intraluminal active metabolite of sennosides A and B in mice.

Intracaecal administration of rhein anthrone, the intraluminally active metabolite of sennosides A and B, to mice quickly induced severe diarrhoea. Pretreatment with the prostaglandin (PG) biosynthesis inhibitor, indomethacin, and PGE2 antagonist, SC-19220, prevented the onset of diarrhoea induced by rhein anthrone, but the PGE2 antagonist polyphoretin phosphate (PPP) showed only a weak inhibitory effect. Rhein anthrone stimulated the production of PGE-like material only in the colon and its large intestinal propulsive activity was depressed by indomethacin and SC-19220, but not by PPP which suggests that the release of PGE-like material has some role in its purgative action.

A novel modification of a bone repositioning device and a new technique for reestablishing facial contours after mandibular resection surgery.

A novel modification of a bone repositioning device previously published by the same authors is introduced. A flexible tube to define the intersegmental bony relationship is filled with light-cured resin. It solidifies following exposure to strong visible light for about 1 min. This technique can be used for bone positioning after mandibular resection surgery and during positioning of the proximal segment after sagittal split ramus osteotomy. The authors also propose a simple method for determining the contour of the reconstructed mandible to regain the original shape and form. The advantage of this technique is its simplicity and flexibility compared with other methods of bone positioning during mandibular segmental surgery.

[Re-examination of the masticatory function of the first permanent molar].

The biting force of the first permanent molar, from children in the first grade of elementary school to adults in their seventies, was measured in 1959 and in 1987. Comparison between these two sets of data showed no significant difference in each age. The biting force of the second milk molar of kindergarteners aged from 4 to 5 years old, showed a decrease of nearly 5 kilograms on the average and in particular. The number of the children whose biting force was less than 15 kilograms increase. There was a negative correlation between the depth of the periodontal pocket of every kind of tooth and the biting force, and the average of the correlation coefficient was about minus 0.3. Especially, there was negative correlation between the depth of the periodontal pocket of the first permanent molar of patients in their forties and their biting force.

Characterization of 5,7-dichlorokynurenate-insensitive D-[3H]serine binding to synaptosomal fraction isolated from rat brain tissues.

To explore target sites for endogenous D-serine that are different from the glycine site of the N-methyl-D-aspartate (NMDA) type glutamate receptor, we have studied the binding of D-[3H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 microM) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 mM unlabeled D-serine. Association, dissociation, and saturation experiments indicated that D-[3H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K(D) of 614 nM and a Bmax of 2.07 pmol/mg of protein. D-Serine, L-serine, and glycine produced a total inhibition of the specific DCK-insensitive D-[3H]serine binding to the cerebellum with similar Ki values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 microM. The profiles of displacement of the DCK-insensitive D-[3H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive D-[3H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive D-[3H]serine binding site could be a novel candidate for a target for endogenous D-serine in mammalian brains.

Adenylate cyclase activity and cyclic AMP production in the outer and inner zones of the adrenal cortex.

It has been reported that cells isolated from the inner zone of the guinea pig adrenal cortex fail to have a steroidogenic response to ACTH. To further explore this, adenylate cyclase activity of membrane particles and cAMP production by cells prepared from the inner and outer adrenocortical zones were determined. The cAMP response to ACTH and forskolin was similar for cells from both zones. Basal adenylate cyclase activity was significantly higher in the inner zone; and while absolute responses to ACTH, GppNHp, GTP, NaF, and forskolin were greater for the inner zone, relative responses were similar for the two zones. These observations suggest that the inner zone of the guinea pig adrenal cortex may have a defect in ACTH action at a step(s) beyond cAMP formation.

Regulation of translocation of the desmoyokin/AHNAK protein to the plasma membrane in keratinocytes by protein kinase C.

Desmoyokin was identified as a desmosomal plaque protein. We previously demonstrated that desmoyokin is identical to a protein encoded by a human gene, AHNAK, whose expression is suppressed in neuroblastoma cells. Although this protein is distributed in the cytoplasm and the nucleus in various cells, it is associated closely with the plasma membrane in keratinocytes. In keratinocytes, desmoplakin translocates from the cytoplasm to the plasma membrane following both high calcium switch and protein kinase C (PKC) activation by 12-O-tetradecanoylphorbol-13-acetate (TPA). In the low calcium medium, the desmoyokin/AHNAK protein resides diffusely in the cytoplasm and the nucleus. However, 2 h after shift to the high calcium medium, the desmoyokin/AHNAK protein localized to the cell boundary in all cells in a pattern similar to that of desmoplakin. Selective PKC inhibitors completely inhibited the calcium-induced translocation of the desmoyokin/AHNAK protein, but the inhibition of desmoplakin translocation by these inhibitors was only partial. TPA also induced translocation of both the desmoyokin/AHNAK protein and desmoplakin, which was completely inhibited by PKC inhibitors. The calcium-induced phosphorylation of the desmoyokin/AHNAK protein was confirmed by immunoprecipitation using [32P]orthophosphate-labeled keratinocytes. Furthermore, the study of extractability with non-ionic detergent indicated that desmoplakin, but not the desmoyokin/AHNAK protein, is associated with the cytoskeleton. These results suggested an involvement of PKC in the translocation of the desmoyokin/AHNAK protein in keratinocytes. It was, however, also suggested that different mechanisms are likely involved in the translocation of the desmoyokin/AHNAK protein and desmoplakin.

Coexistent urticaria pigmentosa, acromegaly and acanthosis nigricans.

We report a case of urticaria pigmentosa, acromegaly and acanthosis nigricans in a 25-year-old male. The patient exhibited multiple pigmented papules on the trunk and the extremities. Histological examinations of the papules revealed infiltrates of mast cells in the upper dermis. Ultrastructurally, the mast cells were fully matured and exhibited no atypical features. A typical appearance of acromegaly, frontal bossing, prominence of the jaw and bony overgrowth and cutaneous changes of acanthosis nigricans on the neck, the axillae and the groins were observed. Growth hormone hypersecretion and insulin resistance were detected in the patient. A pituitary tumor was found and resected surgically. After the operation, endocrinological abnormalities and cutaneous manifestations of acanthosis nigricans improved markedly. As far as we know, this is the first report of the coexistence of urticaria pigmentosa, acromegaly and acanthosis nigricans.

Enantioselective determination of bromoisovalerylurea by liquid chromatography on chiral stationary phase in reversed- or normal-phase partition mode.

Bromoisovalerylurea (bromvalerylurea) is a sedative-hypnotic given orally as a racemate. Enantiomers of this drug could be separated by high-performance liquid chromatography on the three chiral stationary phases (a vancomycin-bonded, beta-cyclodextrin derivative-bonded, or urea derivative-bonded phase). Biological fluids of human subjects who had ingested toxic or therapeutic doses of the racemate were chromatographed after liquid-liquid extraction. The (+)-enantiomer concentration was almost equal to the (-)-enantiomer concentration in the serum of one overdosed patient. In all the other subjects, the (+)-enantiomer was less than the (-)-enantiomer in their sera and saliva. The data suggest that the drug is absorbed non-stereoselectively from the gastrointestinal tract and eliminated from the blood stereoselectively.

A Case of pemphigus foliaceus which occurred after five years of remission from pemphigus vulgaris.

A 77-year-old Japanese female developed pemphigus foliaceus (PF) after 5 years of remission from pemphigus vulgaris (PV). The patient had painful erosions in her mouth and flaccid blisters of the skin and was diagnosed as having PV, which responded well to corticosteroid treatment. She was then free from any lesion of PV for 5 years with a low dose of corticosteroid. Then she developed scaly erythematous lesions on the skin and was diagnosed as suffering from PF. Enzyme-linked immunosorbent assay (ELISA) using recombinant desmoglein 1 (Dsg-1) and Dsg-3 revealed that she had anti-Dsg-3 IgG in the PV stage, no antibodies during remission and anti-Dsg-1 IgG in the PF stage. These findings indicate that the target antigen was shifted from Dsg-3 to Dsg-1 along with the phenotype after a 5-year interval in this patient.

Epidermolysis bullosa simplex associated with muscular dystrophy: phenotype-genotype correlations and review of the literature.

BACKGROUND: Epidermolysis bullosa simplex associated with muscular dystrophy (EBS-MD; OMIM# 226670) is an autosomal recessive disorder caused by genetic defects in the plectin gene. Because EBS-MD is relatively rare, and gene defects have been elucidated only in a limited number of patients, the precise phenotype-genotype correlations have not yet been fully elucidated. OBJECTIVE: The purpose of this study was to define clinical features of EBS-MD and to clarify its phenotype-genotype correlations. METHODS: Clinical, ultrastructural, immunohistochemical, and molecular features of 4 unrelated Japanese patients with EBS-MD were recorded. In addition, 6 cases with defined plectin gene mutations reported in the literature were reviewed. RESULTS: In skin of the EBS-MD patients, the blister formation always occurs just above the hemidesmosomes, and expression of plectin is absent or markedly reduced in all cases examined. All 10 patients, including 6 cases in the literature, showed generalized blistering at birth or soon thereafter, and experienced nail deformities. In addition, decayed teeth (5 cases), urethral strictures (3), mild palmoplantar hyperkeratosis (2), infantile respiratory complications (2), alopecia (1), and laryngeal webs (1) were present. All 8 patients who were older than 9 years demonstrated considerable muscle weakness, and the majority of them ended up being wheelchair bound. Among the 10 patients, 7 were products of consanguineous marriage, 9 have premature termination codon (PTC) mutations in both alleles of the plectin gene, and 7 cases were homozygous for the mutation. One patient who is homozygous for a 2719del9 in-frame deletion mutation that resulted in elimination of 3 amino acids, QEA, could still walk at the age of 46 and showed milder clinical severity. CONCLUSION: EBS-MD reveals clinical features not only characteristic of EBS and MD, but also other manifestations including urethral, dental, and respiratory complications. The majority of patients are products of consanguineous marriage and have homozygous plectin gene mutations. Whereas patients with PTC mutations in both alleles typically showed severe clinical features of EBS-MD and ended up being wheelchair bound, a homozygous patient for an in-frame deletion mutation showed positive, yet attenuated, plectin expression and milder clinical phenotype. Thus plectin immunofluorescence, combined with identification of the underlying plectin mutations, is of value in predicting the severity of the muscle involvement that occurs later in life of patients with EBS-MD.