Two-hybrid search for proteins that interact with Sad1 and Kms1, two membrane-bound components of the spindle pole body in fission yeast.

In interphase cells of fission yeast, the spindle pole body (SPB) is thought to be connected with chromosomal centromeres by an as yet unknown mechanism that spans the nuclear membrane. To elucidate this mechanism, we performed two-hybrid screens for proteins that interact with Kms1 and Sad1, which are constitutive membrane-bound components of the SPB that interact with each other. Seven and 26 genes were identified whose products potentially interact with Kms1 and Sad1, respectively. With the exception of Dlc1 (a homolog of the 14-kDa dynein light chain), all of the Kms1 interactors also interacted with Sad1. Among the genes identified were the previously known genes rhp9+ / crb2+, cut6+, ags1+ / mok1+, gst3+, kms2+, and sid4+. The products of kms2+ and sid4+ localize to the SPB. The novel genes were characterized by constructing disruption mutations and by localization of the gene products. Two of them, putative homologues of budding yeast UFE1 (which encodes a t-SNARE) and SFH1 (an essential component of a chromatin-remodeling complex), were essential for viability. Two further genes, which were only conditionally essential, genetically interact with sad1+. One of these was named sif1+ (for Sad1-interacting factor) and is required for proper septum formation at high temperature. Cells in which this gene was overexpressed displayed a wee -like phenotype. The product of the other gene, apm1+, is very similar to the medium chain of an adaptor protein complex in clathrin-coated vesicles. Apm1 appears to be required for SPB separation and spindle formation, and tended to accumulate at the SPB when it was overproduced. It was functionally distinct from its homologues Apm2 and Apm4. Other novel genes identified in this study included one for a nucleoporin and genes encoding novel membrane-bound proteins that were genetically related to Sad1. We found that none of the newly identified genes tested were necessary for centromere/telomere clustering.

Hamster D-amino-acid oxidase cDNA: rodents lack the 27th amino acid residue in D-amino-acid oxidase.

The nucleotide sequence of cDNA that encodes hamster d-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,590 nucleotides and a poly(A) tail. It had an open reading frame for a protein consisting of 346 amino acid residues. The number of the amino acid residues is the same as that of the rat DAO. However, the hamster DAO has one residue more than mouse DAO and one residue less than human, pig, rabbit, and guinea pig DAOs. Amino acid sequence of the hamster DAO was highly similar to those of mouse and rat DAOs: 89% and 88% of the amino acid residues were identical between the hamster and mouse DAOs and between the hamster and rat DAOs, respectively. The homology was slightly less between the hamster DAO and the human (81%), pig (78%), rabbit (78%), or guinea pig DAO (82%). It has been proposed that the mouse and rat DAOs lack an amino acid residue corresponding to the 25th residue of the DAOs of other mammals. However, a detailed comparison of the amino acid sequences as well as the underlying nucleotide sequences by inclusion of the hamster ones revealed that the rodent DAOs does not lack the 25th, but the 27th residue.

Primary intrapelvic seminoma in Klinefelter's syndrome.

Seminoma arising in patients with Klinefelter's syndrome is extremely rare; to our knowledge, only three cases have been reported in the English language literature. We report a case of intrapelvic seminoma in a 39-year-old man with Klinefelter's syndrome. Gross examination revealed that the tumor was a solid and irregular mass measuring 90 mm in diameter. The cut surfaces of this ill-defined tumor were yellow-white with necrotic foci. Histologically, the tumor cells were separated into lobules by branching, fibrous septa containing lymphocytes. In some parts of the tumor, a cord-like arrangement of tumor cells was present. Immunohistochemically, the tumor cells were strongly and diffusely positive for antiplacental alkaline phosphatase antibody along their cytoplasmic membranes, but negative for both chorionic gonadotrophin and alpha-fetoprotein. Based on these findings, we diagnosed this tumor as a seminoma. The testes when examined were found to be atrophic bilaterally, but with no tumor lesions. Chromosomal analysis yielded a 47XXY karyotype, compatible with Klinefelter's syndrome. These findings indicate a case of primary intrapelvic seminoma in Klinefelter's syndrome. The patient underwent intensive radiation therapy postoperatively, and he demonstrated no evidence of recurrence or metastasis during the 13-month period following surgery.

Expression of Bax and apoptosis-related proteins in human esophageal squamous cell carcinoma including dysplasia.

The rate of tumor growth depends on the balance between proliferation and death of tumor cells. It is known that Bax, caspase-3, and p53 proteins are death-promoting factors, whereas Bcl-2 protein is a death antagonist. We immunohistochemically examined the expression of Bax and apoptosis-related proteins such as caspase-3, p53, and Bcl-2 in 76 patients with human esophageal squamous cell carcinoma (SCC) including dysplasia to determine the relationship of expression of each protein to tumor behavior and patients' prognosis. No significant relationships in immunopositivity were found among these proteins in SCCs. Cytoplasmic Bax expression was exhibited in 63 cases of SCCs (82.9%). The apoptotic index of caspase-3-positive lesions was significantly higher than that of caspase-3-negative lesions in both dysplasia and SCC (P =.016, P =.012). On the other hand, the apoptotic index (1.18%) was significantly correlated with Bax overexpression in dysplasia (P =.006), but not in SCC lesions (P =.129). The patients with Bax-positive SCCs were found to have a poor prognosis by the Kaplan-Meier method (P =.043). These findings suggested that Bax expressed in dysplasia may play a role as an apoptotic factor, but that it may be functionally inactive in some cancerous lesions and thus not contribute to suppression of the tumor progression in some cases of human esophageal SCCs.

Complete sequence of the mitochondrial DNA of the primitive opisthobranch gastropod Pupa strigosa: systematic implication of the genome organization.

The complete sequence (14,189 bp) of the mitochondrial DNA of the opisthobranch gastropod Pupa strigosa was determined. The genome contains 13 protein, 2 rRNA, and 22 tRNA genes typical of metazoan mtDNA. The Pupa mitochondrial genome is highly compact and shows the following unusual features, like pulmonate land snails: (1) extremely small genome size, (2) absence of lengthy noncoding regions (with the largest intergenic spacer being only 46 nt), (3) size reduction of encoded genes, and (4) many overlapping genes. Several tRNA genes exhibit bizarre secondary structures with reduced T or D stems, and many tRNA genes have unstable acceptor stems that might be corrected by posttranscriptional RNA editing. The Pupa mitochondrial gene arrangement is almost identical to those of pulmonate land snails but is radically divergent from those of the prosobranch gastropod Littorina saxatilis and other molluscs. Our finding that the unique gene arrangement and highly compact genome organization are shared between opisthobranch and pulmonate gastropods strongly suggests their close phylogenetic affinity.

Association between p53 immunostaining and cigarette smoking in squamous cell carcinoma of the esophagus.

BACKGROUND: It is generally accepted that cigarette smoking is closely associated with esophageal squamous cell carcinoma. This study investigated the molecular targets of cigarette smoke in carcinogenesis of the esophagus. METHODS: Seventy-four patients with esophageal squamous cell carcinoma (SCC) were grouped according to daily cigarette consumption: heavy smoking group (group H) (n = 26), moderate smoking group (group M) (n = 39) and non-smoking group (group N) (n = 9). We compared p53 and retinoblastoma (RB) expression among the three groups by immunohistochemistry. In addition, fresh tumor tissues from 30 smokers with esophageal SCC were tested for p53 mutations in exons 5-8 by direct sequencing. RESULTS: Staining for the p53 product was positive in 65.4% of group H, 38.5% of group M and 44.4% of group N. The frequency of positive staining in the group H was significantly higher than in group M (p = 0.033) and in group M + group N (p = 0.034). The difference with respect to the frequency of overexpression of RB was not significant. The patterns of p53 base-pair mutations in direct sequencing study were of five types, most commonly G:C to T:A transversion (35.3%). CONCLUSIONS: Our study suggests that one of the molecular targets of cigarette smoke is the p53 gene. The pattern of p53 point mutations involved a wide range of base-pair changes.

Guinea pig D-amino-acid oxidase cDNA and phylogenetic position.

The nucleotide sequence of cDNA that encodes guinea pig D-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,399 nucleotides and a poly(A) tail. The cDNA encodes 347 amino acid residues. In contrast to the hamster, rat, and mouse DAOs, guinea pig DAO had the 25th amino acid residue. The homology in amino acid sequences between the guinea pig DAO and the rodent DAOs was not high in comparison to the homology in amino acid sequences between the guinea pig DAO and DAOs of humans, pigs and rabbits. The phylogenetic position of the guinea pig varied depending on the source of sequences (amino acids or nucleotides) and the methods of phylogenetic tree construction. These results suggest that the guinea pig is not a simple rodent.