Msps protein is localized to acentrosomal poles to ensure bipolarity of Drosophila meiotic spindles.

The female meiotic spindle is commonly formed in a centrosome-independent manner. Here we report the identification of proteins at acentrosomal poles in the female meiotic spindle of Drosophila. The acentrosomal poles contain at least two proteins, Mini-spindles (Msps) and D-TACC, which are also associated with mitotic centrosomes. These proteins interact with one another and are both required for maintaining the bipolarity of acentrosomal spindles. The polar localization of Msps is dependent on D-TACC and Ncd, a kinesin-like microtubule motor. We propose that the polar localization of Msps mediated by D-TACC and Ncd may be crucial for the stabilization of meiotic spindle bipolarity.

mini spindles: A gene encoding a conserved microtubule-associated protein required for the integrity of the mitotic spindle in Drosophila.

We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.

Metaphase arrest with centromere separation in polo mutants of Drosophila.

The Drosophila gene polo encodes a conserved protein kinase known to be required to organize spindle poles and for cytokinesis. Here we report two strongly hypomorphic mutations of polo that arrest cells of the larval brain at a point in metaphase when the majority of sister kinetochores have separated by between 20-50% of the total spindle length in intact cells. In contrast, analysis of sister chromatid separation in squashed preparations of cells indicates that some 83% of sisters remain attached. This suggests the separation seen in intact cells requires the tension produced by a functional spindle. The point of arrest corresponds to the spindle integrity checkpoint; Bub1 protein and the 3F3/2 epitope are present on the separated kinetochores and the arrest is suppressed by a bub1 mutation. The mutant mitotic spindles are anastral and have assembled upon centrosomes that are associated with Centrosomin and the abnormal spindle protein (Asp), but neither with gamma-tubulin nor CP190. We discuss roles for Polo kinase in recruiting centrosomal proteins and in regulating progression through the metaphase-anaphase checkpoint.

Isolation and characterization of the fission yeast protein phosphatase gene ppe1+ involved in cell shape control and mitosis.

We isolated a fission yeast putative protein serine/threonine phosphatase gene designated ppe1+ by hybridization. The predicted amino acid sequence is similar to those of the fission yeast ppa2 (53% identity) and dis2 (39%) phosphatases, and highly similar to those of the budding yeast SIT4 (72%), Drosophila PPV (68%) and rabbit PPX (61%) phosphatases. Antibodies against ppe1 protein identified a 37-kd polypeptide in fission yeast. A gene disruption (designated delta ppe1) caused cold-sensitive lethality and short, pear-shaped cells. These phenotypes were fully suppressed by a plasmid carrying ppe1+. Three classes of multicopy suppressor genes for delta ppe1 were identified as follows: 1) ppa1+ and ppa2+ encoding type 2A-like phosphatases, 2) mitotically essential dis3+ similar to the budding yeast SSD1/SRK1, a suppressor for sit4, and 3) pck1+ coding for a protein kinase C-like kinase. Consistently, the budding yeast SIT4 gene was also a multicopy suppressor for delta ppe1. Phosphatase ppe1 may play a role in cell morphogenesis and mitosis by either regulating or being regulated by these multicopy suppressor gene products. Consistent with this hypothesis, double mutants ppe1-ppa2 and ppe1-pck1 are lethal at the permissive temperature.

Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe.

Polo kinases execute multiple roles during cell division. The fission yeast polo related kinase Plo1 is required to assemble the mitotic spindle, the prophase actin ring that predicts the site for cytokinesis and for septation after the completion of mitosis (Ohkura et al., 1995; Bahler et al., 1998). We show that Plo1 associates with the mitotic but not interphase spindle pole body (SPB). SPB association of Plo1 is the earliest fission yeast mitotic event recorded to date. SPB association is strong from mitotic commitment to early anaphase B, after which the Plo1 signal becomes very weak and finally disappears upon spindle breakdown. SPB association of Plo1 requires mitosis-promoting factor (MPF) activity, whereas its disassociation requires the activity of the anaphase-promoting complex. The stf1.1 mutation bypasses the usual requirement for the MPF activator Cdc25 (Hudson et al., 1990). Significantly, Plo1 associates inappropriately with the interphase SPB of stf1.1 cells. These data are consistent with the emerging theme from many systems that polo kinases participate in the regulation of MPF to determine the timing of commitment to mitosis and may indicate that pole association is a key aspect of Plo1 function. Plo1 does not associate with the SPB when septation is inappropriately driven by deregulation of the Spg1 pathway and remains SPB associated if septation occurs in the presence of a spindle. Thus, neither Plo1 recruitment to nor its departure from the SPB are required for septation; however, overexpression of plo1+ activates the Spg1 pathway and causes transient Cdc7 recruitment to the SPB and multiple rounds of septation.

Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner.

Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium.

A human cholangiocarcinoma cell line, designated as HChol-Y1, was established in a protein-free, chemically defined medium after a very short period of primary culture in 0.1% fetal bovine serum (FBS)-containing medium. The cell line has been propagated in this medium for 2 years. The cells grew as a monolayer and the doubling time was about 52 hours. Addition of FBS did not stimulate cell growth (population-doubling time = 50 hr) or increase saturation density. The cells grown in a protein-free medium secreted small amounts of carcinoembryonic antigen (CEA) and large amounts of carbohydrate antigen (CA) 19/9 (CEA: 12.5 +/- 2.1 ng/10(6) cells/48 hr; CA 19/9: 760 +/- 52 IU/10(6) cells/48 hr); these tumor markers were immunohistochemically demonstrated in HChol-Y1 cells. Addition of FBS slightly stimulated the production of CEA and CA 19/9. The HChol-Y1 cell line was xenotransplantable in athymic nude mice and increased the serum CEA and CA 19/9 levels in the tumor-bearing nude mice. For determination as to whether a human carcinoma cell line can proliferate and secrete CEA and CA 19/9 in synthetic medium without any protein supplements, the cells were cultivated long term (2 yr) in a protein-free, chemically defined medium. When this method of cultivation is used, it is easy to purify these substances from spent medium, because contaminating antigens such as FBS or other substances usually added to cultures are absent.

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast.

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.

A rapid anticancer drug screening assay by [14C] thymidine uptake in cultured human cancer cells.

A rapid screening test by suppression of DNA synthesis in cancer cells was developed with [methyl-14C]-thymidine (14C-TdR), a microculture filtration plate and a radiochromatoscanner. Mitomycin, tamoxifen and 5-fluorouracil (5-FU) were tested against four human gastric cancer cell lines and HeLa cells. The tetrazolium-based colorimetric (MTT) assay underestimated cell inactivation by mitomycin in three cell lines compared with the cell count and the 14C-TdR assays. Inactivation by 5-FU in one cell line by 14C-TdR uptake was considerably lower than that by other methods. Thus neither the radio-labelled DNA precursor uptake nor the MTT assay is suitable for every anticancer drug but they are complementary.

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin from a patient with extragonadal germ cell tumour.

Human chorionic gonadotropin (hCG) purified from the urine of a male patient with extragonadal germ cell tumour contained four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively released from the polypeptide moiety by hydrazinolysis and recovered as oligosaccharides after N-acetylation. The oligosaccharide mixture was separated into a neutral (N) and three acidic (A1, A2 and A3) fractions by anion-exchange column chromatography. By sequential exoglycosidase digestion, methylation analysis and lectin column chromatography, the structures of these oligosaccharides were found to be the same as those of female gestational choriocarcinoma hCGs. Both contain eight kinds of sugar chains: triantennary, abnormal and normal biantennary, and monoantennary complex-type sugar chain with or without a fucosylated core portion.

Identification of mycobacteria by nonradioisotopic single-strand conformation polymorphism analysis.

Clinical isolates of mycobacteria were identified to species levels using nonradioisotopic single-strand conformation polymorphism (non-RI SSCP) analysis of 16S rRNA gene fragments amplified by polymerase chain reaction with primers common to all of mycobacterial species. The method is based on a hypervariable region within the 16S rRNA in mycobacteria, which is characterized by species-specific nucleotide sequences. A total of 92 mycobacterial strains (Mycobacterium tuberculosis, M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. nonchromogenicum, M. xenopi, and unidentified strain) were studied. They were classified into nine types of pattern showing single-strand DNA bands having different mobilities. Each strain was shown in the species-specific mobility by non-RI SSCP analysis. The results of non-RI SSCP analysis were identical to those of standard biochemical methods and 16S rRNA sequencing.

DNA gyrase gyr A gene mutations in ofloxacin-resistant Staphylococcus aureus.

A total of 106 clinical isolates of Staphylococcus aureus were classified into nine pattern types correlated with gyrA mutations. In 62 strains, mutations were found at a single codon (84, 85, 86 and 88), while 22 strains showed double mutations either at codons 84 and 85 or at codons 84 and 88. The double mutations led to the highest levels of ofloxacin resistance (MIC, > or = 128 microg/ml). All isolates with a single Ser-84--> Leu change had an ofloxacin MIC of 8-128 microg/ml, whereas others showed an MIC range of 8-16 microg/ml. Twenty-two wild type strains and one strain with a single mutation at codon 86 (silent mutation) were ofloxacin-susceptible. Thus, gyrA mutations seem to play a definite role in the high-level of resistance to ofloxacin.

Serum placental alkaline phosphatase (PLAP) in gynecologic malignancies--with special reference to the combination of PLAP and CA54/61 assay.

Serum placental alkaline phosphatase (PLAP) levels in patients with gynecological tumors were measured by two kinds of enzyme-antigen immunoassay kits provided by Innogenetics (PLAP-I) and Sangtec Medical (PLAP-S), and the combination assays for PLAP with other tumor markers were studied. None of the healthy women studied were PLAP positive, and the positive rate in patients with benign ovarian tumors was less than 6%. The positive rate in patients with ovarian cancers was about 35%, which was higher than the rates for other cancers. It was significantly higher in patients with ovarian serous cystadenocarcinoma (60%). Remarkably high PLAP-I values were observed in patients with dysgerminoma. By the combination assay for PLAP with CA54/61 antigen in ovarian cancers, the diagnostic efficiency increased compared with that for PLAP and CA125. We conclude that PLAP is useful in the diagnosis of ovarian serous cystadenocarcinoma and dysgerminoma and that CA54/61 is the better partner for the combination assay.

Semiautomated 14C-thymidine incorporation assay for in vitro screening of anticancer drug.

We developed a rapid screening test for anticancer drugs by using 14C-thymidine (14C-TdR), a microculture filtration plate and a radiochromatoscanner. Mitomycin C (MMC), tamoxifen and 5-fluorouracil (5FU) were used as the anticancer drugs against four human gastric cancer cell lines. The rates of cell inactivation (14C-TdR uptake) determined with the radiochromatoscanner were almost the same as those determined with the liquid scintillation counter. Comparing the rate of cell inactivation obtained by our method with that obtained by proliferative activity of cells derived from the cell count, both assays showed the approximately same results by tamoxifen and MMC but the rates of cell inactivation by 5FU in two cell lines obtained by the 14C-TdR uptake assay was considerably lower than those obtained by the cell count. These results show that the radio-labeled DNA precursor uptake assay is not suitable for metabolic inhibitors of DNA synthesis. Ninety six samples on a plate, however, were assayed semiautomatically and rapidly just as well as in the tetrazolium-based calorimetric (MTT) assay. Therefore, our 14C-TdR uptake assay system is useful for the cancer chemotherapeutic agents except the metabolic inhibitors of DNA precursors.

Immunohistochemical detection of c-erbB-2 oncoprotein in patients with breast cancer.

Using a polyclonal antibody against the c-erbB-2 gene product, an immunohistochemical study on the expression of c-erbB-2 oncoprotein was performed in routine formalin-fixed, paraffin-embedded tissue sections from 119 patients with primary breast cancer. Overexpression of c-erbB-2 protein was detected in 35 cases (29%). There was a strongly negative association between c-erbB-2 protein overexpression and estrogen receptor status (p < 0.001). Expression of c-erbB-2 protein was found to be related to clinical stage and tumor size (p < 0.05) but not to the number of involved nodes or age of patients at diagnosis. In addition, this study demonstrated that the c-erbB-2 protein over-expression was an effective independent prognostic indicator in patients with breast cancer.

[Evaluation of time-resolved fluorometric immunoassay of CA 50].

Serum CA 50 was determined by a time resolved fluorometric immunoassay (TR-FIA) with CANAG CA-50 DELFIA kit. Evaluation of the assay system gave satisfactory results in its sensitivity, accuracy, reproducibility, dynamic range and easy handling. No prozone phenomenon was observed up to 347,000 U/ml. From a histogram of 134 normal sera, the cut off point was determined at 34 U/ml. CA 50 in 202 patients' sera was determined with this assay. Nineteen of 20 patients pancreatic cancer, 6 of 21 gastric cancer, 14 of 25 hepatoma gave positive values. In comparison with CA 19-9, higher values and higher rates of positive CA 50 were observed in benign and malignant liver diseases, suggesting its non-cancerous origin in the liver. A high correlation was observed between the level of CA 50 and CA 19-9 of 157 patients' sera. Serum CA 50 was completely correlated with CA 19-9 in the clinical course of patients with pancreatic cancer, but not in patients with hepatoma. Thus we conclude that the CANAG CA-50 DELFIA System is useful for the diagnosis and monitoring cancer patients but must be used with care because of its elevation in benign liver diseases.

[Development of sandwich radioimmunometric assay for serum c-erbB-2 oncogene product and its significance in diagnosing breast carcinoma].

A sandwich radioimmunometric assay using monoclonal antibodies 6G10 and SV2-61 gamma directed to the extracellular domain of the c-erbB-2 oncogene product was developed and the antigen levels in sera from normal donors and patients with breast carcinoma were determined. The antigen levels in normal donors were uniformly low (9.52 +/- 0.91 ng/ml) and 3.0% (2/66 cases) slightly exceeded the cutoff value (11.4 ng/ml). In patients with breast carcinoma, serum c-erbB-2 protein levels increased and the positivity was as high as 45.7% (16/35 cases) in recurrent cases. Determination of c-erbB-2 protein may be a useful marker for serological diagnosis of breast carcinoma.

[Sensitive detection of K-ras oncogene codon 12 mutations by nested PCR using mismatched primers and selective digestion of non-mutated PCR fragments with restriction enzyme].

Sensitive detection of K-ras oncogene mutation at codon 12 using nested polymerase chain reactions (PCR) with mismatched primers combined with selective digestion of nonmutated DNA fragments after the first PCR amplification was reported by Levi S, et al. (Cancer Res 51:3497-3502, 1991). We examined the usefulness of this novel technique in cell lines carrying various K-ras codon 12 mutations and clinical samples such as colon carcinoma tissue, corresponding normal colonic mucosa and pancreatic juice obtained from the patients with pancreatic carcinoma undergoing endoscopic retrograde pancreatography (ERP). In analysis of tumor cell lines carrying mutated K-ras at codon 12, the fragment length of the amplified DNA was 135 bp after digestion using restriction enzyme BstN1, whereas those of nonmutated cell lines were 106 bp. This method was highly sensitive to detect mutant DNA diluted at a ratio of 2048 fold with normal DNA samples obtained from peripheral blood lymphocytes. In analysis of clinical samples, 4 out of 10 colorectal carcinoma tissues were positive for K-ras gene mutation at codon 12, however, no mutations were detected in corresponding normal colonic mucosa. In analysis of pancreatic juice taken from the patients with pancreatic carcinoma, 1 out of 3 samples were positive for K-ras mutation at codon 12. Thus, this novel approach was thought to be useful for detection of minimum number of cancer cells from compound samples containing larger amounts of normal cells.

[Analysis of genomic instability by fluorescence DNA sequencer].

Within the recent four years, there have been substantial advances in understanding the molecular pathogenesis of HNPCC. As one of the result of investigation, microsatellite instability has been observed in hereditary non-polyposis colorectal cancer (HNPCC) and other sporadic cancers. Moreover, there is strong supporting evidence that mismatch repair genes play a role in HNPCC. Here, we present our investigational results and discuss possible molecular mechanisms governing DNA mutation and genomic instability, leading to the development of neoplasm. We investigated replication error (RER) of 4 microsatellite markers (dinucleotide repeats) in 131 patients with colorectal cancer (10 met the criteria of HNPCC group B), using fluorescence-based DNA sequencer. We detected RER positivity (at more than two loci) in 12 of 131 patients (9.2%). PCR-SSCP and direct sequencing of the mismatch repair genes, hMSH2 and hMLH1, revealed that two patients in HNPCC group B had germline mutations of hMSH2. The fluorescence-based techniques, such as the present RER analysis do not require radioactive materials and specialized rooms, and can easily be performed in a clinical laboratory.