Characterization of a processed pseudogene of human psiHSP40 on chromosome 2q32.

A pseudogene for the human Hsp40 gene has been characterized (psiHSP40). The pseudogene sequence shows 90% similarity to the human Hsp40 mRNA at the nucleotide level. No introns were found in the region corresponding to the human Hsp40 cDNA, and two direct repeats flank this same region. Because of these features, the pseudogene can be classified as a processed pseudogene. PsiHSP40 was assigned to chromosome 2q32 by in situ hybridization. This is the first report of a pseudogene for a member of the DnaJ (Hsp40) family protein gene.

Intranuclear arrangement of human chromosome 12 correlates to large-scale replication domains.

The intranuclear arrangement of human chromosome 12 in G0(G1) nuclei from human myeloid leukemia HL60 cells was analyzed by multicolor fluorescence in situ hybridization (FISH) using band-specific cosmid clones as probes. Pairs of differently colored cosmids were detected on paraformaldehyde-fixed HL60 nuclei, and their relative positions, internal or peripheral, in individual nuclei were scored. Our results suggest that the intranuclear arrangement of human chromosome 12 is not random. Some chromosomal domains, including the centromere, were located in the periphery of the nucleus, while other domains, including the telomeres, were positioned in the internal areas of the nucleus in GO(G1) cells. Based on the replication banding patterns of metaphase spreads, human chromosome 12 was divided roughly into five large domains. Interestingly, the clones in late replicating domains were preferentially localized in the nuclear periphery, whereas clones in early replicating domains were arranged in the internal areas of the nuclei. The DNA replication timing of each cosmid determined by FISH-based assay did not reflect the replication bands, but an overall profile of the replication timing was relatively correlated with these domains on chromosome 12. These results suggest that the intranuclear arrangement of a human chromosome is correlated with the large-scale replication domains, even before DNA replication.

Isolation of a novel human canalicular multispecific organic anion transporter, cMOAT2/MRP3, and its expression in cisplatin-resistant cancer cells with decreased ATP-dependent drug transport.

The human multidrug resistance protein (MRP) gene encodes a membrane protein involved in the ATP-dependent transport of hydrophobic compounds. We previously isolated a canalicular multispecific organic anion transporter, cMOAT1/MRP2, that belongs to the ATP binding cassette (ABC) superfamily, which is specifically expressed in liver, and cMOAT1/MRP2 is responsible for the defects in hyperbilirubinemia II/Dubin-Johnson syndrome. In this study, we isolated a new cDNA of the ABC superfamily designated cMOAT2/MRP3 that is homologous to human MRP1 and cMOAT1/MRP2: cMOAT2/MRP3 is 56% identical to MRP1 and 45% identical to cMOAT1/MRP2, respectively. Fluorescence in situ hybridization demonstrated the chromosomal locus of this gene on chromosome 17q22. The human cMOAT2 cDNA hybridized to a 6.5-kb mRNA that was mainly expressed in liver and to a lesser extent in colon, small intestine, and prostate. The cMOAT2/MRP3 gene was not overexpressed in cisplatin-resistant cell lines with increased ATP-dependent transport of cisplatin over their parental counterparts derived from human head and neck cancer and human prostatic cancer cell lines. The human cMOAT2/MRP3, a novel member of the ABC superfamily, may function as a membrane transporter in liver, colon, and prostate.

Mapping of the human genes encoding cyclin H (CCNH) and the CDK-activating kinase (CAK) assembly factor MAT1 (MNAT1) to chromosome bands 5q13.3-q14 and 14q23, respectively.

Cyclin-dependent kinases (CDKs), which play a key role in cell cycle control, are activated by the CDK-activating kinase (CAK), which activates cyclin-bound CDKs by phosphorylation at the specific threonine residue. Mammalian CAK contains three components: CDK7, cyclin H, and an assembly factor called MAT1. The CDK7-cyclin H-MAT1 complex is tightly associated with a multiprotein complex TFIIH, which plays a dual role in transcription and DNA repair. Here, we have determined chromosomal localizations of the human genes encoding cyclin H (CCNH) and MAT1 (HGMW-approved symbol MNAT1) to chromosome bands 5q13.3-q14 and 14q23, respectively, by using fluorescence in situ hybridization, somatic cell hybrid analyses, and mapping to the human YAC contigs.

Molecular cloning of the human methylthioadenosine phosphorylase processed pseudogene and localization to 3q28.

Human methylthioadenosine phosphorylase (MTAP) is a purine and methionine metabolic enzyme present ubiquitously in all normal tissues, but often deleted in many types of cancer. The gene for this enzyme maps to chromosome 9 at band p21 where the cyclin-dependent kinase inhibitor genes for p16 and p15 also reside. During our efforts to clone this gene we also isolated a phage clone containing a processed pseudogene of MTAP. The sequence is 92% homologous to the MTAP cDNA, is flanked at its 3' end by a repetitive element, but does not possess a poly(A) stretch. We localized this processed pseudogene to band 28 on the long arm of chromosome 3 by fluorescence in situ hybridization. All 22 malignant cell lines with deletions at 9p21 screened possessed the pseudogene.

Isolation of human and fission yeast homologues of the budding yeast origin recognition complex subunit ORC5: human homologue (ORC5L) maps to 7q22.

Orc5p is a subunit of the origin recognition complex in the budding yeast Saccharomyces cerevisiae, which has been shown to play a critical role in both chromosomal DNA replication and transcriptional silencing. We have cloned cDNAs from both human and fission yeast Schizosaccharomyces pombe that encode proteins homologous to the budding yeast and Drosophila Orc5p. Human Orc5p showed 35.1, 22.3, and 19.4% identity to the Drosophila, S. pombe, and S. cerevisiae Orc5p, respectively. We have localized the human ORC5 gene (ORC5L) to chromosome 7 using Southern and PCR analysis of DNA isolated from a panel of human/rodent somatic cell hybrids and mapped the gene locus to 7q22 using fluorescence in situ hybridization. We have identified a YAC clone that contains human ORC5L and maps to chromosome band 7q22.1. We have identified the S. pombe ORC5 gene and located it in a cosmid mapped on chromosome II.

Cloning, sequencing, and chromosomal localization of two tandemly arranged human pseudogenes for the proliferating cell nuclear antigen (PCNA).

We have characterized a human genomic clone carrying two pseudogenes for the proliferating cell nuclear antigen (PCNA), which were tandemly arranged on human Chromosome (Chr) 4. One is a processed pseudogene that showed a 73% nucleotide homology to the human PCNA cDNA and possessed none of the introns existing in the functional PCNA gene. This pseudogene presumably arose by reverse transcription of a PCNA mRNA followed by integration of the cDNA into the genome. The other is a 5' and 3' truncated pseudogene that showed a nucleotide homology to a 3' region of the exon 4 and to a 5' region of the exon 5 of the PCNA gene and did not have the intronic sequence between the exons 4 and 5. Both pseudogenes had the same nucleotide deletion as compared with the human functional PCNA gene. A phylogenetic analysis of PCNA gene family, including the functional PCNA gene and another PCNA pseudogene located on a different chromosome, revealed that the truncated pseudogene exhibits the closest evolutionary relationship with the processed pseudogene, suggesting that the truncated pseudogene was generated by duplication of the processed pseudogene after translocation to Chr 4. Furthermore, fluorescence in situ hybridization revealed that these pseudogenes are located on the long arm of Chr 4, 4q24.

A human canalicular multispecific organic anion transporter (cMOAT) gene is overexpressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation.

By targeting the ATP binding conserved domain in three ATP binding cassette superfamily proteins (P-glycoprotein, multidrug resistance protein, and cystic fibrosis transmembrane regulator), we isolated the cDNA of a new ATP binding cassette superfamily that was specifically enhanced in a cisplatin-resistant human head and neck cancer KB cell line. A human clone homologous to rat canalicular multispecific organic anion transporter (cMOAT) was found and designated human cMOAT. Fluorescence in situ hybridization demonstrated the chromosomal locus of the gene on chromosome 10q24. The human cMOAT cDNA hybridized a 6.5-kb mRNA that was expressed 4- to 6-fold higher by three cisplatin-resistant cell lines derived from various human tumors exhibiting decreased drug accumulation. Human cMOAT may function as a cellular cisplatin transporter.

Portsite and intraabdominal metastases of unsuspected gallbladder carcinoma after laparoscopic cholecystectomy: report of a case.

We herein report a rare case of portsite metastasis of gallbladder carcinoma which occurred after laparoscopic cholecystectomy. A 64-year-old man underwent laparoscopic cholecystectomy at another hospital for symptomatic cholecystolithiasis. The histological examination revealed an adenocarcinoma of the gallbladder infiltrating the entire wall. Despite the physician's advice the patient refused any additional treatment. Thirteen months after surgery he visited our hospital because of a palpable mass at the scar of the right trocar incision. The nodule was removed and histological examination confirmed metastasis from the gallbladder carcinoma.

[Wegener's granulomatosis in a woman with asymptomatic primary biliary cirrhosis].

A 60-year-old woman was admitted to our hospital in June 1985, complaining of fever, cough and right lower chest pain, with a five-year history of asymptomatic primary biliary cirrhosis. Chest X-ray on admission showed an infiltrative shadow in the right lower lung field. She was first treated with various antibiotics unsuccessfully. Hemoptysis continued. Dyspnea and anemia appeared. Chest X-ray 17 days after admission showed multiple infiltrative shadows in the both lung fields. She was treated with steroid pulse therapy successfully. During prednisolone treatment decreasing nodular shadows with cavities appeared on chest X-ray. An open lung biopsy was performed in March 1986. The histologic findings showed a necrotizing vasculitis with granuloma and perivascular fibrosis. She was treated with prednisolone and prednisolone-azathioprine therapy unsuccessfully, but successfully with prednisolone-cyclophosphamide therapy. This case was a rare case of Wegener's granulomatosis with transition from fulminant type to granulomatous type. No similar case of Wegener's granulomatosis with asymptomatic primary biliary cirrhosis has been reported in the literature.

Anti-acetylcholine receptor antibody titer with extended thymectomy in myasthenia gravis.

Twenty-four patients with myasthenia gravis of Osserman's generalized type underwent extended thymectomy through a sternal-splitting approach. Their clinical responses to thymectomy and postoperative changes in anti-acetylcholine receptor antibody titers were evaluated. The follow-up time ranged from 1 month to 7 years and 7 months (average, 36 months). Six patients (25%) had remissions and 17 patients (71%) were improved after operation. The preoperative anti-acetylcholine receptor antibody titers dropped significantly after operation (p less than 0.001). The postoperative reduction in these titers correlated with the time course after operation (p less than 0.05). Their postoperative reduction was significantly greater in the six patients having remissions than in the 15 having marked (p less than 0.02) and the six having moderate improvement (p less than 0.005). This study has revealed that anti-acetylcholine receptor antibody titer in plasma declines progressively after thymectomy, and the postoperative reduction of this titer correlates with the clinical effect of thymectomy.

Effects of preoperative duration of symptoms on patients with myasthenia gravis.

In 80 patients with type II nonthymomatous myasthenia gravis who underwent extended thymectomy, we investigated preoperative duration of symptoms, prognosis after thymectomy, immunological findings, and germinal center formation in the thymus. Our findings included the following. First, the palliation rate after thymectomy ranged from 73 to 100% and was independent of the preoperative duration of symptoms. The remission rate was high in patients with a short preoperative duration. Second, the lymphocyte count of peripheral blood decreased as preoperative duration increased. Third, the percentage of positive reactions to purified protein derivative of tuberculin decreased as preoperative duration increased: 100% in the one-year group, 78% in the two-year group, 75% in the three-year group, and 56% in the four-year group. Fourth, the degree of germinal center formation in the thymus was higher in patients with a longer preoperative duration. The correlation between germinal center formation and preoperative duration was significant. Finally, the T-cell population in peripheral blood and immunoglobulin, and antibody to acetylcholine receptor in serum, had no significant relationship with the preoperative duration of myasthenia gravis.

Myasthenia gravis with thymoma: analysis of and postoperative prognosis for 65 patients with thymomatous myasthenia gravis.

Sixty-five patients with thymomatous myasthenia gravis were investigated. Thymomas were present in 44% of the male patients and 19% of the female patients with myasthenia gravis. The incidence of thymomatous disease in male patients was higher than in female patients in all age groups. Eighty percent of men more than 50 years old and women more than 60 years old had myasthenia gravis with thymoma. Germinal center formation in the thymus of patients with thymomatous myasthenia gravis was positive in 91% and was high grade. The prognosis for patients undergoing extended thymectomy of thymomatous myasthenia gravis was significantly better than in those having transsternal simple thymectomy, but it was worse than the prognosis for patients with nonthymomatous myasthenia gravis. No increase in the rate of remission or palliation was seen one year after thymectomy. It is concluded that early thymectomy is effective in control of myasthenia gravis in thymomatous myasthenia gravis.