Feasibility study of axillary reverse mapping for patients with clinically node-negative breast cancer.

BACKGROUND: The axillary reverse mapping (ARM) procedure was developed to identify and preserve arm lymphatic drainage during axillary lymph node dissection (ALND), thereby theoretically reducing the incidence of arm lymphedema. However, the oncological safety of this procedure has not yet been determined. METHODS: Two hundred ninety-two patients with clinically negative nodes (cN0) underwent both sentinel lymph node (SLN) biopsy and ARM. SLN was identified by dye and gamma probe methods, and ARM nodes were identified using a fluorescence imaging system. If SLN was histologically positive, ALND was performed with removal of ARM nodes. Otherwise, identified ARM nodes were preserved unless they coincided with SLN. Postoperatively, SLN as well as ARM nodes were histologically examined with H&E staining. RESULTS: SLN was identified in 286 of 292 patients, and ARM nodes were identified in 90 patients. In 54 patients with positive SLN, SLN was the same as the ARM node in 19 patients (the concordance type), whereas it was not an ARM node in the remaining 35 patients (the separate type). Non-SLN and ARM node was not involved in 51 of 54 patients with positive SLN, while it was involved in 3 patients of the concordance type. CONCLUSIONS: When ARM nodes were involved in patients with cN0, these were most often the SLN-ARM nodes. Therefore, it may be concluded that ARM nodes that do not coincide with SLNs might be preserved during ALND in SLN-positive patients.

New methods for species and sex determination in three sympatric Mustelids, Mustela itatsi, Mustela sibirica and Martes melampus.

We developed novel species and sex determination methods for three Japanese mustelid species. We used DDX3Y to determine sex and generated a primer set to amplify both DDX3X and DDX3Y DNA in Mustela itatsi, M. sibirica and Martes melampus. To determine species and sex simultaneously, we generated fluorescence-labelled primers that give different fragment lengths at D-loop, DDX3X and DDX3Y of these three species using a DNA sequencer.

Proposal for a new clinical entity, IgG4-positive multiorgan lymphoproliferative syndrome: analysis of 64 cases of IgG4-related disorders.

BACKGROUND: Mikulicz's disease (MD) has been considered as one manifestation of Sjögren's syndrome (SS). Recently, it has also been considered as an IgG(4)-related disorder. OBJECTIVE: To determine the differences between IgG(4)-related disorders including MD and SS. METHODS: A study was undertaken to investigate patients with MD and IgG(4)-related disorders registered in Japan and to set up provisional criteria for the new clinical entity IgG(4)-positive multiorgan lymphoproliferative syndrome (IgG(4)+MOLPS). The preliminary diagnostic criteria include raised serum levels of IgG(4) (>135 mg/dl) and infiltration of IgG(4)(+) plasma cells in the tissue (IgG(4)+/IgG+ plasma cells >50%) with fibrosis or sclerosis. The clinical features, laboratory data and pathologies of 64 patients with IgG(4)+MOLPS and 31 patients with typical SS were compared. RESULTS: The incidence of xerostomia, xerophthalmia and arthralgia, rheumatoid factor and antinuclear, antiSS-A/Ro and antiSS-B/La antibodies was significantly lower in patients with IgG(4)+MOLPS than in those with typical SS. Allergic rhinitis and autoimmune pancreatitis were significantly more frequent and total IgG, IgG(2), IgG(4) and IgE levels were significantly increased in IgG(4)+MOLPS. Histological specimens from patients with IgG(4)+MOLPS revealed marked IgG(4)+ plasma cell infiltration. Many patients with IgG(4)+MOLPS had lymphocytic follicle formation, but lymphoepithelial lesions were rare. Few IgG(4)+ cells were seen in the tissue of patients with typical SS. Thirty-eight patients with IgG(4)+MOLPS treated with glucocorticoids showed marked clinical improvement. CONCLUSION: Despite similarities in the involved organs, there are considerable clinical and pathological differences between IgG(4)+MOLPS and SS. Based on the clinical features and good response to glucocorticoids, we propose a new clinical entity: IgG(4)+MOLPS.

Fecal DNA analysis for identifying species and sex of sympatric carnivores: a noninvasive method for conservation on the Tsushima Islands, Japan.

Fecal analysis is a useful tool for the investigation of food habits and species identity in mammals. However, it is generally difficult to identify the species based on the morphological features and contents of feces deposited by mammals of similar body size. Therefore we developed noninvasive DNA analysis methods using fecal samples for identification of the species and sex of four small sympatric carnivores living on the Tsushima Islands of Japan: the leopard cat (Felis bengalensis), Japanese marten (Martes melampus), Siberian weasel (Mustela sibirica), and feral cat (Felis catus). Based on DNA sequence data from previous phylogenetic studies, we designed species-specific primers for polymerase chain reaction (PCR) amplification of the partial mitochondrial cytochrome b gene (112-347 bp) to identify the species and primers for the partial SRY gene (135 bp) to determine the sex. Due to the adjustment of PCR conditions, those specific DNA fragments were successfully amplified and then applied for species and sex identification. Nucleotide sequences obtained from the PCR products corresponded with cytochrome b sequences of the carnivore species expected. The protocol developed could be a valuable tool in the management and conservation of the four carnivore species occurring on the Tsushima Islands.

Multicentric occurrence of esophageal cancer after gastrectomy: a preliminary report.

The effect of gastrectomy on the subsequent development of esophageal cancer was investigated, focusing on its multicentric occurrence. We retrospectively evaluated 28 patients who underwent subtotal esophagectomy for intrathoracic esophageal cancer between 1985 and 1999. They were divided into two groups according to whether or not they had previously undergone a gastrectomy: group 1, comprising 7 patients who had undergone gastrectomy and group 2, comprising 21 patients who had not. Clinical profiles of the patients were obtained from the medical records and the whole resected esophagus was histopathologically examined. The interval between gastrectomy and esophagectomy in group 1 was significantly shorter in the patients who had undergone gastrectomy for gastric cancer than in those who had undergone gastrectomy for a peptic ulcer, and also in the patients for whom anastomosis had been performed by Billroth I compared with Billroth II. The patients in group 1 were significantly younger than those in group 2. The multiple occurrence of esophageal cancer was found in 4 of 5 patients (80%) in group 1, and in 2 of 18 patients (11%) in group 2, with significantly higher frequency being seen in group 1. More than two coexisting cancer lesions apart from the primary tumor were detected in all four patients. Histological examination of all the coexisting cancer lesions showed well-differentiated squamous cell carcinoma confined within the superficial mucosal layer. No significant differences were noted in the location of the coexisting lesions between the oral and anal side of the primary tumors. Squamous dysplasia was randomly observed, especially around the cancer lesions. These findings suggest that gastrectomy precipitated subsequent chronic gastroesophageal reflux which in turn induced the development of squamous dysplasia and carcinoma at multiple locations in the esophagus.

A spontaneously ruptured gastric stromal tumor presenting as generalized peritonitis: report of a case.

Among the diverse clinical presentations of gastrointestinal stromal tumor (GIST), spontaneous rupture with peritonitis is extremely rare. We report herein the unusual case of a 75-year-old man found to have a spontaneously ruptured gastric stromal tumor after presenting with generalized peritonitis. The patient was brought to the emergency department of our hospital by ambulance, with generalized severe abdominal pain. On examination, his abdomen was extensively distended with generalized severe rebound tenderness. Abdominal computed tomography scan showed a giant mass arising from the anterior gastric wall with an irregular internal low-density area and a small amount of ascites. An emergency laparotomy revealed a ruptured gastric tumor with dissemination of its necrotic tissue throughout the peritoneal cavity. The tumor was excised together with normal gastric tissue around its base. The tumor, which was 15 x 11 x 4.4cm in size, had a coarse laceration over its well-capsulated smooth serosal surface with massive necrosis and clotted blood inside. Immunohistochemical examination revealed positive reactivity to C-kit protein, which was consistent with the newly introduced diagnostic criteria of GIST. The patient had an uneventful postoperative course and remains well.

Isolation of high-malate-producing sake yeasts from low-maltose-assimilating mutants.

High-malate-producing sake yeasts were isolated from low-maltose-assimilating mutants. A sake yeast Kyokai no. 701 (K-701) was mutagenized with ethyl methanesulfonate. After nystatin treatment, low-maltose-assimilating mutants were selected using a replica plating method. The forty-two mutants obtained were fermented in koji extract medium and almost all of them produced more malate than the parental strain. In a small-scale sake brewing test, several of these mutants produced 2.3 to 6.7 times higher concentrations of malate and 1.5 to 2.1 times higher total acidity than the parental strain. However, these mutants exhibited neither resistance to cycloheximide nor sensitivity to dimethyl succinate, which are used as the phenotypes for isolation of high-malate-producing sake yeasts. The expression levels of the MDH and FUM1 genes in one mutant (M20), which produced the highest amount of malate among the mutants obtained, were analyzed by Northern hybridization. The transcriptional level of the FUM1 gene in strain M20 during sake brewing had a similar profile to that in strain K-701. However, the transcriptional level of the MDH2 gene in strain M20 for days 4 and 8 of malate formation during sake brewing was higher than that in strain K-701.

Frequency of a 30-base pair deletion in the latent membrane protein-1 gene in Epstein-Barr virus-associated lymphomas in Japan.

The association of Epstein-Barr virus (EBV) with various lymphoid malignancies has been reported. The precise pathogenesis of EBV in malignancies has not yet been elucidated. Latent membrane protein-1 (LMP-1) and Epstein-Barr nuclear antigen-2 (EBNA-2) genes are suspected to be tumorigenic genes. Previous studies suggest that a deletion within the LMP-1 gene may increase the oncogenic potential of EBV. In this study, we analyzed the sequence within the carboxy terminal end of the LMP-1 gene in paraffin-embedded specimens from T-cell lymphomas, Hodgkin's disease (HD), and the buffy coat of peripheral blood from healthy individuals in Japan. Polymerase chain reaction (PCR) was performed using primers spanning the carboxy terminal region of the LMP-1 gene, and sequence analysis was performed to show the exact location of the deletion. The PCR product of the Raji cell line was 161 base pairs (bp), and the LMP-1 gene with deletion was 30 bp shorter in a direct sequence of PCR products. The 30-bp deletion was located in position 168285-168256 of the Raji cell. A deletion within the LMP-1 gene was found in 4 of 25 cases (16%) of EBV-positive T-cell lymphomas, 4 of 10 cases (40%) of EBV-positive HD cases, and 2 of 13 specimens (15%) with amplified PCR products from 49 healthy individuals. The incidence of the 30-bp deletion within the LMP-1 gene in HD was comparable to that of subjects in the United States and Brazil, but the deletion was not found in a high proportion of EBV-positive T-cell lymphoid malignancies. No statistical significance was found regarding the clinical outcome between patients with a deletion within the LMP-1 gene and patients with wild-type LMP. This deletion cannot be considered as simply causing the pathogenesis of EBV-associated lymphoid malignancies in Japan.

Effect of the FAA1 gene disruption of sake yeast on the accumulation of ethyl caproate in sake mash.

Fatty acid activation gene (FAA1) in sake yeast Kyokai no. 701 (K701) was disrupted to investigate the accumulation of ethyl caproate in sake mash. Ethyl caproate, recognized as an important apple-like flavor in sake, is generated by fatty acid synthesis in yeast cells. The disruptant for the FAA1 gene (K701deltafaa1) exhibited a reduced growth rate in a medium containing cerulenin and myristic acid or oleic acid compared with that of the parental strain (K701). In a sake brewing test in which the rice used was polished to 60% of its original size, the fermentation ability of K701deltafaa1 was inferior to that of K701 but the production of ethyl caproate by K701deltafaa1 was 1.6-fold higher than that by K701. These results suggest that the FAA1 gene in sake yeast plays an important role in sake brewing and the accumulation of ethyl caproate.

Intrageneric diversity of the cytochrome B gene and phylogeny of eurasian species of the genus mustela (mustelidae, carnivora).

To illuminate molecular phylogenetic relationships among Eurasian species of the genus Mustela (Mustelidae, Carnivora), we determined nucleotide sequences of the complete mitochondrial cytochrome b gene region (1,140 base pairs). Molecular phylogenetic trees, constructed using the neighbor-joining and the maximum likelihood methods, showed the common topology of species relationships to each other. The American mink M. vison first branched off and was positioned very remotely from the other species of Mustela. Excluding M. vison, the ermine M. erminea first split from the rest of the species. Two small body-sized weasels, the least weasel M. nivalis and the mountain weasel M. altaica, comprised one cluster (named "the small weasel group"). The other species formed another cluster, where the remarkably close relationships among the domestic ferret M. furo, the European polecat M. putorius, and the steppe polecat M. eversmanni were noticed with 87-94% bootstrap values (named "the ferret group"), supporting the history that the ferret was domesticated from M. putorius and/or M. eversmanni. The European mink M. lutreola was the closest to the ferret group. The genetic distance between the Siberian weasel M. sibirica and the Japanese weasel M. itatsi corresponded to differences of interspecific level, while the two species were relatively close to M. lutreola and the ferret group. These results provide invaluable insight for understanding the evolution of Mustela as well as for investigating the hybridization status between native and introduced species for conservation.

Improvement of isoamyl acetate productivity in sake yeast by isolating mutants resistant to econazole.

Sake yeast strains were improved so as to produce larger amounts of isoamyl acetate than the parental strain by isolating econazole-resistant mutants. Econazole, an imidazole antimycotic, directly interacts with unsaturated fatty acids in the yeast cell membrane, where it also inhibits the synthesis of ergosterol and decreases the ratio of unsaturated to saturated fatty acids. In contrast, alcohol acetyltransferase (AATase), which catalyzes the synthesis of isoamyl acetate, is inhibited by unsaturated fatty acids. Fifty econazole-resistant mutants were isolated from a sake yeast, Kyokai no. 701, several of which produced approximately 1.4 to 2.4 times more isoamyl acetate and an almost equal amount of isoamyl alcohol compared with the parental strain. The AATase activities of the mutants in koji extract were 1.2 to 1.4 times higher, and the unsaturated to saturated fatty acid ratios were lower, than in the parental strain.

Effect of NAD+-dependent isocitrate dehydrogenase gene (IDH1, IDH2) disruption of sake yeast on organic acid composition in sake mash.

The ratio of organic acids in sake mash is a very important factor affecting the taste of alcoholic beverages. To alter the organic acid composition in sake and investigate the mechanism of producing organic acids in sake mash, we examined the effect of NAD+-dependent isocitrate dehydrogenase (IDH) activity deficiency in sake yeast by disrupting the IDH1 or IDH2 gene. Two haploid strains (MATa or MATa genotype) isolated from sake yeast Kyokai no. 701 (K701) were disrupted using the aureobasidin A resistant gene (AUR1-C) as a selection marker. These disruptants were defective in the activity of IDH and failed to grow on medium containing glycerol as a sole carbon source. Sake meter, alcohol concentration, and glucose consumption in sake brewed with the disruptants were reduced in comparison with those of the parental strains. The production of citrate (including isocitrate), malate, and acetate by the disruptants was increased, but succinate production was reduced to approximately half in comparison with the parental strains. These results indicate that approximately half the amount of succinate in sake mash is produced via the oxidative pathway of the TCA cycle in sake yeast. While the diploid strain constructed by mating haploid disruptants for the IDH gene exhibited stronger fermentation ability than the haploid disruptants, almost similar profiles of components in sake were obtained for both strains.

Lambert-Eaton myasthenic syndrome associated with Sjögren's syndrome and discoid lupus erythematosus.

A 65-year-old woman with facial erythema and hypergammaglobulinemia developed excessive fatigability. A diagnosis of Lambert-Eaton myasthenic syndrome (LEMS) was made from electrophysiological studies. She had symptoms and laboratory data compatible with probable Sjögren's syndrome. Skin biopsy revealed the histological findings of discoid lupus erythematosus. Treatment with 3,4-diaminopyridine resulted in the improvement of fatigability. LEMS should be recognized as a treatable complication of systemic autoimmune diseases.

Systemic elastolytic granulomatosis with cutaneous, ocular, lymph nodal, and intestinal involvement. Spectrum of annular elastolytic giant cell granuloma and sarcoidosis.

A 15-year-old Japanese girl had widespread annular serpiginous erythematous plaques, bilateral granulomatous uveitis, bloody diarrhea, and seronegative arthralgia. She also had anemia and leukopenia. The histopathologic findings were compatible with those of annular elastolytic giant cell granuloma. Elastolytic granulomas were also found in the cervical lymph nodes, terminal ileum, parietal peritoneum, and mesentery. Bilateral hilar lymphadenopathy, hypercalcemia, and an increased level of angiotensin converting enzyme were not observed throughout the clinical course. To the best of our knowledge, systemic elastolytic granulomatosis has not been previously described in annular elastolytic giant cell granuloma or sarcoidosis. This case may represent a type of granulomatosis in the broad spectrum of annular elastolytic giant cell granuloma and sarcoidosis.

Nucleotide sequence of the gene responsible for D-xylose uptake in Escherichia coli.

The nucleotide sequence of the cloned DNA, 363 bp in length, has been determined. It can complement the mutation of Escherichia coli having a decreased activity of D-xylose uptake at low temperature. Nucleotide sequence analysis found one possible reading frame coding for a polypeptide consisting of 61 amino acids. Several signal sequences conserved in the promoter regions of E. coli were found in the upstream regions of the open frame. This included the Shine-Dalgarno sequence, the Pribnow box, and the sequence conserved in the "-35 region" with a preferable spacing from each other for an efficient transcription. Downstream from the termination codon, the inverted repeat sequence was present, followed by 3 successive T's.