Alu-Alu fusion sequences identified at junction sites of copy number amplified regions in cancer cell lines.

Alu elements are short, ∼300-bp stretches of DNA and are the most abundant repetitive elements in the human genome. A large number of chromosomal rearrangements mediated by Alu-Alu recombination have been reported in germline cells, but only a few in somatic cells. Cancer development is frequently accompanied by various chromosomal rearrangements including gene amplification. To explore an involvement of Alu-Alu fusion in gene amplification events, we determined 20 junction site sequences of 5 highly amplified regions in 4 cancer cell lines. The amplified regions exhibited a common copy number profile: a stair-like increase with multiple segments, which is implicated in the breakage-fusion-bridge (BFB) cycle-mediated amplification. All of the sequences determined were characterized as head-to-head or tail-to-tail fusion of sequences separated by 1-5 kb in the genome sequence. Of these, 4 junction site sequences were identified as Alu-Alu fusions between inverted, paired Alu elements with relatively long overlapping sequences of 17, 21, 22, and 24 bp. Together with genome mapping data of Alu elements, these findings suggest that when breakages occur at or near inverted, paired Alu elements in the process of BFB cycle-mediated amplification, sequence homology of Alu elements is frequently used to repair the broken ends.

Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-kappa b-inducing kinase.

The alymphoplasia (aly) mutation of mouse is autosomal recessive and characterized by the systemic absence of lymph nodes (LN) and Peyer's patches (PP) and disorganized splenic and thymic structures with immunodeficiency. Although recent reports have shown that the interaction between lymphotoxin (LT) and the LT beta-receptor (Ltbeta r, encoded by Ltbr) provides a critical signal for LN genesis in mice, the aly locus on chromosome 11 is distinct from those for LT and its receptor. We found that the aly allele carries a point mutation causing an amino acid substitution in the carboxy-terminal interaction domain of Nf-kappa b-inducing kinase (Nik, encoded by the gene Nik). Transgenic complementation with wild-type Nik restored the normal structures of LN, PP, spleen and thymus, and the normal immune response in aly/aly mice. In addition, the aly mutation in a kinase domain-truncated Nik abolished its dominant-negative effect on Nf-kappa b activation induced by an excess of Ltbeta r. Our observations agree with previous reports that Ltbeta r-deficient mice showed defects in LN genesis and that Nik is a common mediator of Nf-kappa b activation by the tumour necrosis factor (TNF) receptor family. Nik is able to interact with members of the TRAF family (Traf1, 2, 3, 5 and 6), suggesting it acts downstream of TRAF-associating receptor signalling pathways, including Tnfr, Cd40, Cd30 and Ltbeta r. The phenotypes of aly/aly mice are more severe than those of Ltbr-/- mice, however, indicating involvement of Nik in signal transduction mediated by other receptors.

Coamplification of multiple regions of chromosome 2, including MYCN, in a single patchwork amplicon in cancer cell lines.

Coamplification of multiple segments of chromosome 2, including an MYCN-bearing segment, was examined in 2 cancer cell lines, NCI-H69 (lung cancer) and IMR-32 (neuroblastoma). High-resolution array-CGH analysis revealed 13 and 6 highly amplified segments located at different sites in chromosome 2 in NCI-H69 and IMR-32, respectively. FISH analysis demonstrated that these segments were co-localized in double minutes in NCI-H69 and in homogeneously staining regions in IMR-32. Connectivity of the segments was determined by a PCR assay using designed primer sets. It was found that all the segments were connected to each other irrespective of their order and orientation against the genome sequence, and a single chain-like cluster was configured in both cell lines. Such patchwork structures of the amplicons suggest the possibility that massive genomic rearrangements, explained by the single catastrophic event model, are involved in the formation of the amplicons, enabling the coamplification of different chromosomal regions including the MYCN locus. The model comprises massive fragmentation of chromosomes and random rejoining of the fragments.

Chemotherapy-related hypersensitivity reaction in Japanese patients with gynecologic malignancy.

PURPOSE OF INVESTIGATION: Chemotherapy-related hypersensitivity reaction seems to be problematic in the safe management of chemotherapy. In this study we investigated chemotherapy-related hypersensitivity reaction in patients with gynecologic malignancy. METHODS: Between January 2009 and December 2010, we examined hypersensitivity reaction (> or = grade2) using the Common Terminology Criteria for Adverse Events (CTCAE) v.4.0. We analyzed the incidence, clinical features, management, and outcome. RESULTS: We administered over 1,057 infusions (24 regimens) to 205 patients. We found a total of four hypersensitivity reactions (> or = grade 2) cases (carboplatin: 2; nedaplatin: 1; docetaxel: 1). Signs and symptoms were varied. In two cases, the same regimen was rechallenged by using anti-allergic drugs. The docetaxel case was successful. The carboplatin case was not successful. CONCLUSION: Chemotherapy-related hypersensitivity reaction (> or = grade2) does not occur frequently. In the case of platinum, especially, carboplatin, re-administering after hypersensitivity reaction should be done carefully though platinum is a key drug in patients with gynecologic malignancies.

Response training shortens visuo-motor related time in athletes.

In the present study, we aimed to determine whether response training shortens visuo-motor related time in athletes performing a simple reaction task. 14 healthy male athletes were included in the study. Subjects were randomly divided into 2 groups: a training group, which underwent response training consisting of a mastication task in response to a visual signal, and a non-training (control) group, which did not undergo response training. Pre-motor time and transcranial magnetic stimulation over the primary motor cortex for recording motor evoked potentials were measured in the control group, and before and after the response training session in the training group. Both pre-motor time and visuo-motor related time, but not motor evoked potential latency, were significantly reduced after response training in the training group. Subjects who had a longer visuo-motor related time before training showed a greater reduction in visuo-motor related time after training. These results suggest that visuo-motor related time before training could be useful as a predictor of the reduction in reaction time following response training.

Increase in detectable opportunistic bacteria in the oral cavity of orthodontic patients.

OBJECTIVES: This study was performed to detect the opportunistic bacteria and fungi from the oral cavities of orthodontic patients and examine the ability of the organisms to adhere to saliva-coated metallic brackets. METHODS: Opportunistic bacteria and fungi were isolated from 58 patients (orthodontic group: 42; non-orthodontic group: 16) using culture methods and were identified based on their biochemical and enzymatic profiles. Seven opportunistic and four streptococcal strains were tested for their ability to adhere to saliva-coated metallic brackets. RESULTS: More opportunistic bacteria and fungi were detected in the orthodontic group than in the non-orthodontic group (P < 0.05). Opportunistic bacteria adhered to saliva-coated metallic brackets to the same degree as oral streptococci. CONCLUSIONS: The isolation frequencies of opportunistic bacteria and fungi increase during orthodontic treatment, suggesting the importance of paying special attention to oral hygiene in orthodontic patients to prevent periodontal disease and the aggravation of systemic disease in immunocompromised conditions.

Enhancing oral moisture using an extract of Capparis masaikai Levl.

The purpose of this study was to examine the effect of a Capparis masaikai Levl. extract on enhancing oral moisture. Solutions of Capparis masaikai extract, citric acid, sodium chloride, and sucrose were dropped on the tongue dorsum of 20 healthy subjects aged 23-34 years. After swallowing each solution, the oral moisture was measured for 60 min using a saliva wetness tester and a moisture checker. The subjects recorded the degree of taste using a visual analog scale to examine the stimulating effect of each solution on salivation. The Capparis masaikai extract had a long-lasting moistening effect on both the tongue dorsum and buccal mucosa for up to 60 min. The weakly bittersweet taste of the extract was perceived stronger than the other taste elements. The results suggest that the Capparis masaikai extract is useful for enhancing oral moisture.

A non-MHC locus essential for autoimmune type I diabetes in the Komeda Diabetes-Prone rat.

The Long-Evans Tokushima Lean (LETL) rat, characterized by rapid onset of insulin-dependent (type I) diabetes mellitus (IDDM), no sex difference in the incidence of IDDM, autoimmune destruction of pancreatic beta cells, and no significant T cell lymphopenia, is a desirable animal model for human IDDM. We have established a diabetes-prone substrain of the LETL rat, named Komeda Diabetes-Prone (KDP) rat, showing a 100% development of moderate to severe insulitis within 220 d of age. The cumulative frequency of IDDM was 70% at 120 d of age, and reached 82% within 220 d of age. Here, we performed the first genome-wide scan for non-MHC IDDM susceptibility genes in this strain. The analysis of three crosses has led to the revelation of a major IDDM susceptibility gene, termed Iddm/kdp1, on rat chromosome (Chr) 11. Homozygosity for the KDP allele at this locus is shown to be essential for the development of moderate to severe insulitis and the onset of IDDM. Comparative mapping suggests that the homologues of Iddm/ kdp1 are located on human Chr 3 and mouse Chr 16 and would therefore be different from previously reported IDDM susceptibility genes.

Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.

A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5.

We have isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying mutations of DBF4, a gene that is required for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae and that interacts with the CDC7 protein kinase. Nucleotide sequence analysis of the suppressor gene, provisionally named MSD2, revealed an open reading frame encoding a protein with a calculated M(r) of 81,024, with amino acid sequence similarity to the catalytic domains of protein kinases. Both genetic linkage and complementation analyses indicated that MSD2 is identical to the cell division cycle gene CDC5. An activity that phosphorylated exogenously added casein was immunoprecipitated by antiserum against a TrpE-Cdc5 fusion protein from lysates of wild-type cells containing CDC5 on a multicopy plasmid but not of cells bearing a small deletion in the predicted protein kinase domain of CDC5 on the plasmid. Deletion of CDC5 was lethal and resulted in a dumbbell-shaped terminal morphology, with the nuclei almost divided but still connected. Consistent with the function at the G2/M boundary, the CDC5 transcript accumulated periodically during the cell cycle, peaking at the G2/M boundary. CDC5 on a multicopy plasmid also suppresses temperature-sensitive cdc15, cdc20, and dbf2 mutations which affect mitosis during the cell cycle.

[Malignant solitary fibrous tumor of the pleura; report of a case].

A 68-year-old female was admitted to our hospital for further examination of abnormal shadow on chest X-ray. Needle biopsy could not establish pathological diagnosis. Three years later, chest computed tomography (CT) revealed the tumor was enlarged. We suspected it was a malignant tumor, and resected by video-assisted thoracoscopy. The tumor occurred from the right middle lobe, and intraoperative diagnosis was malignant tumor. We added middle lobectomy. Histological examination revealed that tumor was malignant solitary fibrous tumor.

A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3.

We have investigated how point mutations in the two ATP-binding motifs (G(463)PNGCGK(469)ST and G(701)PNGAGK(707)ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine(463 and 701) and lysine(469 and 707) were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine(463 and 701) was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine(469 and 707) and glycine(463) was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.

Genetic Control of Meiosis in Rice, ORYZA SATIVA L. III. Effect of DS Genes on Genetic Recombination.

The recombination frequency as influenced by five independent recessive ds genes was measured on three segments of different chromosomes of rice, Oryza sativa L. Each ds gene in the homozygous condition resulted in an almost equally reduced recombination frequency in the three segments. When the mean reduction in recombination frequency was related to the reduction of chiasma frequency, the five ds genes were divided into two types: in one type the reduction of chiasma frequency almost corresponded to the mean reduction of recombination frequency, and in the other the chiasma frequency was greatly reduced in comparison with the mean reduction of recombination frequency. Three of the five ds genes were found to belong to the former group. In both types, normal synaptonemal complexes were observed in pachytene cells homozygous for ds genes. This finding suggests that ds genes do not affect the formation of synaptonemal complexes which are regarded as the prerequisite structure for crossing over.

Temperature-sensitive cdc7 mutations of Saccharomyces cerevisiae are suppressed by the DBF4 gene, which is required for the G1/S cell cycle transition.

When present on a multicopy plasmid, a gene from a Saccharomyces cerevisiae genomic library suppresses the temperature-sensitive cdc7-1 mutation. The gene was identified as DBF4, which was previously isolated by complementation in dbf4-1 mutant cells and is required for the G1----S phase progression of the cell cycle. DBF4 has an open reading frame encoding 695 amino acid residues and the predicted molecular mass of the gene product is 80 kD. The suppression is allele-specific because a CDC7 deletion is not suppressed by DBF4. Suppression is mitosis-specific and the sporulation defect of cdc7 mutations is not suppressed by DBF4. Conversely, CDC7 on a multicopy plasmid suppresses the dbf4-1, -2, -3 and -4 mutations but not dbf4-5 and DBF4 deletion mutations. Furthermore, cdc7 mutations are incompatible with the temperature-sensitive dbf4 mutations. These results suggest that the CDC7 and DBF4 polypeptides interact directly or indirectly to permit initiation of yeast chromosome replication.

Molecular and phenotypic analysis of Attractin mutant mice.

Mutations of the mouse Attractin (Atrn; formerly mahogany) gene were originally recognized because they suppress Agouti pigment type switching. More recently, effects independent of Agouti have been recognized: mice homozygous for the Atrn(mg-3J) allele are resistant to diet-induced obesity and also develop abnormal myelination and vacuolation in the central nervous system. To better understand the pathophysiology and relationship of these pleiotropic effects, we further characterized the molecular abnormalities responsible for two additional Atrn alleles, Atrn(mg) and Atrn(mg-L), and examined in parallel the phenotypes of homozygous and compound heterozygous animals. We find that the three alleles have similar effects on pigmentation and neurodegeneration, with a relative severity of Atrn(mg-3J) > Atrn(mg) > Atrn(mg-L), which also corresponds to the effects of the three alleles on levels of normal Atrn mRNA. Animals homozygous for Atrn(mg-3J) or Atrn(mg), but not Atrn(mg-L), show reduced body weight, reduced adiposity, and increased locomotor activity, all in the presence of normal food intake. These results confirm that the mechanism responsible for the neuropathological alteration is a loss--rather than gain--of function, indicate that abnormal body weight in Atrn mutant mice is caused by a central process leading to increased energy expenditure, and demonstrate that pigmentation is more sensitive to levels of Atrn mRNA than are nonpigmentary phenotypes.

Famotidine vs. omeprazole: a prospective randomized multicentre trial to determine efficacy in non-erosive gastro-oesophageal reflux disease.

BACKGROUND: Several studies in Western countries showed that proton-pump inhibitors are superior to histamine2-receptor antagonists or placebo in the treatment of non-erosive gastro-oesophageal reflux disease. The efficacy of acid-suppressive drugs for non-erosive gastro-oesophageal reflux disease in Japan, in which the prevalence of Helicobacter pylori infection is higher compared with Western countries, is unknown. AIM: To compare the efficacy of famotidine and omeprazole in Japanese patients with non-erosive gastro-oesophageal reflux disease by a prospective randomized multicentre trial. METHODS: A total of 98 patients received either famotidine 20 mg b.d. (n = 48) or omeprazole once daily (n = 50). Frequency of gastro-oesophageal reflux disease symptoms and health-related quality of life were evaluated at baseline and after 4 weeks of treatment. Complete relief was defined as no gastro-oesophageal reflux disease symptoms during the 7-day interval in week 4. RESULTS: Complete relief was achieved in 23 (48%) of patients receiving famotidine and 28 (56%) of patients treated with omeprazole. In the famotidine group, complete relief rate in H. pylori-negative patients was significantly lower than H. pylori-positive patients (35% vs. 64%). Both famotidine and omeprazole improved most scales of health-related quality of life. Omeprazole significantly improved reflux score irrespective of H. pylori infection while famotidine significantly improved reflux score in H. pylori-positive patients but not in H. pylori-negative patients. CONCLUSIONS: Omeprazole is more effective than famotidine for the control of gastro-oesophageal reflux disease symptoms in H. pylori-negative patients, while similar efficacy is observed in H. pylori-positive patients with non-erosive gastro-oesophageal reflux disease.

Effects of ranitidine on quality of gastric ulcer healing compared with famotidine: a randomized, controlled, multicenter trial.

Ranitidine has been found to have anti-inflammatory action as well as antisecretory action in experimental models. However, there are no reports in human gastric ulcer. The aim of this study was to investigate the effects of ranitidine compared with those of famotidine on the quality of gastric ulcer healing. We randomly assigned 69 consecutive patients with gastric ulcers to ranitidine (n = 34) or famotidine (n = 35) for 12 weeks, with endoscopic assessment of the quality of gastric ulcer healing and histological assessment of gastric mucosa 12 weeks after treatment started. Ulcer healing rates of over 95% were very similar in the two groups. The rates of ulcer scars with a flat pattern (good-quality healing) were significantly higher in the ranitidine group than in the famotidine group (per protocol, 63.0% and 34.5%, p = 0.033). The neutrophil infiltration score in the body mucosa treated with famotidine, but not ranitidine, significantly increased after treatment. In contrast, the mononuclear cell infiltration score in the antral mucosa treated with ranitidine, but not in that treated with famotidine, had significantly decreased. In conclusion, initial therapy with ranitidine significantly improved the quality of gastric ulcer healing and the histological scores of gastric mucosa compared with famotidine.

Multi-gene family of major surface glycoproteins of Pneumocystis carinii: full-size cDNA cloning and expression.

The major surface glycoprotein (MSG) of Pneumocystis carinii plays a crucial role in the fatal pneumonia caused by this organism in AIDS patients. A cDNA encoding a full-length MSG polypeptide was isolated from a lambda library of rat-derived P. carinii cDNAs. The deduced MSG, referred to as the MSG5 subtype, is a 120,765-Da protein composed of 1,076 amino acids and contains an anchoring hydrophobic sequence at the C-terminus of the protein. Sequence analyses of cloned MSG-cDNAs revealed an MSG-gene family with approximately 70% protein sequence identity between subtypes. P. carinii karyotype hybridization analyses indicated that the MSG gene family members are scattered throughout most of the P. carinii chromosomes. These recombinant MSG proteins reacted with the antiserum from P. carinii-infected rats, as expected, and antiserum generated against P. carinii-infected mice, indicating the existence of common determinants in MSG polypeptides. The family of MSG proteins is rich in cysteine residues and these cysteines are highly conserved in all MSG subtypes regardless of species specificity, suggesting the structural and/or functional importance of these cysteines. The pathobiological significance of the MSG gene family and its sequence diversity in P. carinii is discussed.

Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation.

The Candida glabrata (Cg) TRP1 and HIS3 genes have been isolated by complementation of the Saccharomyces cerevisiae (Sc) trp1 and his3 mutants, respectively. Cg TRP1 encodes a polypeptide of 217 amino acids (aa), whose aa sequence is 58% identical to that of Sc TRP1. Cg HIS3 encodes a polypeptide of 210 aa, whose aa sequence is 73% identical to that of the Sc HIS3. Both Cg TRP1 and HIS3 were disrupted by sequential integrative transformation where the Sc URA3 was used as a selection marker for transformation. The resulting auxotrophic strain of his3- and trp1- was used to examine the ability of the Sc genes to complement the Cg mutations; Sc HIS3 and TRP1 complemented the Cg his3- and trp1- mutations, respectively.