Dysgerminoma of the Left Ovary in a Patient with Balanced Translocation 46X, t(X:1) (q22;q21): A Case Report.

We report a case of dysgerminoma in a 22-year-old woman diagnosed with chromosomal abnormality, balanced translocation 46X,t(X:1)(q22;q21). She had received hormone replacement therapy for 7 years for primary amenorrhea. She visited a primary care physician because of lower abdominal distension, and a large tumor in the pelvis was discovered. She was admitted to our hospital for further examination of the pelvic tumor. She underwent laparotomy and was diagnosed with stage IIIA1 dysgerminoma (pT3apN0pM0) of the left ovary. Young female patients without the Y chromosome who are treated for primary amenorrhea may also develop malignant germ cell tumors; therefore, gynecologists should provide hormone replacement therapy and periodic pelvic evaluation.

Biological Differences Between Ovarian Cancer-associated Fibroblasts and Contralateral Normal Ovary-derived Mesenchymal Stem Cells.

BACKGROUND/AIM: The aim of this study was to clarify the biological differences between ovarian cancer-associated fibroblasts (OCa-CAFs) and normal ovary-derived mesenchymal stem cells (NO-MSCs). MATERIALS AND METHODS: Surgically resected ovarian cancer and contralateral normal ovarian tissue samples were cut into small pieces for culture as "explants". The number of outgrown cells, their proliferative kinetics, and expression levels of cell surface markers of CAFs, as well as three miRNAs in OCa-CAFs and NO-MSCs were compared directly. Differentially expressed genes between both groups were also investigated. RESULTS: Comparable numbers of outgrown cells were harvested from both groups. Significantly higher expression of α-smooth muscle actin and miR-142 was found in OCa-CAFs, which decreased significantly during ex vivo cell expansion. A total of 21 differentially expressed genes were identified between both groups. CONCLUSION: OCa-CAFs showed different biological properties in direct comparison with NO-MSCs, which might play major roles in the pathogenesis of ovarian cancer.

Malignant transformation from mature cystic teratoma of the ovary.

We present a case of malignant change of an ovarian mature cystic teratoma. Our patient was a 48-year-old woman and she visited a primary care doctor presenting with abdominal pain. At her first visit, her pelvic tumor measured 70 × 50 mm by ultrasonography. She was diagnosed as rupture of the malignant tumor occurred secondary to mature cystic teratoma and she took the surgery (abdominal total hysterectomy, bilateral oophorectomy and partial omentectomy). Pathologic diagnosis was squamous cell carcinoma (SCC) occurred secondary to mature cystic teratoma. Treatment with paclitaxel/carboplatin (TC chemotherapy) and gemcitabine hydrochloride/carboplatin (GC chemotherapy) after operation was not effective, and the refractory ileus resulting from rapid progression of the disease continued. She was died of disease progression 7 months after the diagnosis of ovarian cancer. We discuss about the clinical characteristics of malignant transformation of mature cystic teratoma and considered about the treatment of the ovarian SCC.

Gene disruption of ribosomal protein L5 (RPL5) decreased the sensitivity of CHO-K1 cells to uncoupler carbonylcyanide-3-chlorophenylhydrazone.

Protonophoric uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP) decreases the proton motive force (ΔP) of the mitochondrial inner membrane and results in inhibition of oxidative phosphorylation. In this study, a CCCP-resistant clone was isolated from a random gene trap insertional mutant library of Chinese hamster ovary (CHO)-K1 cells which was constructed by infecting a retrovirus vector, ROSAßgeo. Although we expected the isolation of the mutants defective in nuclear genes responsible for mitochondrial functions, the disrupted gene of the isolated mutant that we named R1 cells was identified as one of the alleles for ribosomal protein 5 of large subunit (RPL5). The R1 cells express as much as 80% RPL5 protein compared with the parental CHO-K1 cells, possibly due to enhanced transcription from a remaining wild-type RPL5 allele in R1 cells. Furthermore, the protein amount is not decreased by CCCP in R1 cells, in contrast to its clear reduction by CCCP in parental cells. Since mutations of RPL5 and other ribosomal proteins are responsible for the ribosomopathies and cancer, the present mutant may be a useful cellular model of such human diseases from a viewpoint of energy metabolism as well as a tool for the study of ribosome biogenesis and extra-ribosomal function of the RPL5 protein.

Molecular dissection of Caenorhabditis elegans ATP-binding cassette transporter protein HAF-4 to investigate its subcellular localization and dimerization.

Caenorhabditis elegans HAF-4 and HAF-9 are half-type ATP-binding cassette (ABC) transporter proteins, which are highly homologous to the human peptide transporter protein, transporter associated with antigen processing-like (TAPL, ABCB9). TAPL forms homodimers and localizes to lysosomes, whereas HAF-4 and HAF-9 form heterodimers and localize to intestine-specific non-acidified organelles. Both TAPL and HAF-4/HAF-9 are predicted to have four amino-terminal transmembrane helices [transmembrane domain 0 (TMD0)] additional to the six transmembrane helices that form the canonical core domain of ABC transporters with a cytosolic ABC region. TAPL requires its amino-terminal domain for localization to lysosomes; however, molecular mechanisms underlying HAF-4 and HAF-9 localization to their target organelles had not been elucidated. Here, we demonstrate that the mechanisms underlying HAF-4 localization differ from those underlying TAPL localization. Using transgenic C. elegans expressing mutant HAF-4 proteins labeled with green fluorescent protein, we reveal that the TMD0 of HAF-4 was not sufficient for proper localization of the protein. The mutant HAF-4, which lacked TMD0, localized to intracellular organelles similarly to the wild-type protein and functioned normally in the biogenesis of its localizing organelles, indicating that the TMD0 of HAF-4 is dispensable for both its localization and function.

Amino-terminal extension of 146 residues of L-type GATA-6 is required for transcriptional activation but not for self-association.

Transcription factor GATA-6 plays essential roles in developmental processes and tissue specific functions through regulation of gene expression. GATA-6 mRNA utilizes two Met-codons in frame as translational initiation codons. Deletion of the nucleotide sequence encoding the PEST sequence (Glu(31)-Cys(46)) between the two initiation codons unusually reduced the protein molecular size on SDS-polyacrylamide gel-electrophoresis, and re-introduction of this sequence reversed this change. The long-type (L-type) GATA-6 containing this PEST sequence self-associated similarly to the short-type (S-type) GATA-6, as determined on co-immunoprecipitation of Myc-tagged GATA-6 with HA-tagged GATA-6. The L-type and S-type GATA-6 also interacted mutually. The L-type GATA-6 without the PEST sequence also self-associated and interacted with the S-type GATA-6. The transcriptional activation potential of L-type GATA-6 is higher than that of S-type GATA-6. When the PEST sequence (Glu(31)-Cys(46)) was inserted into the L-type GATA-6 without Arg(13)-Gly(101), the resultant recombinant protein showed significantly higher transcriptional activity, while the construct with an unrelated sequence exhibited lower activity. These results suggest that the Glu(31)-Cys(46) segment plays an important role in the transcriptional activation, although it does not participate in the self-association.

Involvement of heparan sulfate 3-O-sulfotransferase isoform-1 in the insulin secretion pathway.

UNLABELLED: Aims/Introduction: Heparan sulfate (HS) mediates a variety of molecular recognition events that are essential for differentiation, morphogenesis and homeostasis through various HS forms that result from differential sulfate modification. Recently, we found that HS is localized exclusively around ßß-cells in islets of adult mice and is required for insulin secretion. The aim of this study was to examine the contribution of HS sulfate groups to insulin secretion. MATERIALS AND METHODS: Glucose-induced insulin secretion (GIIS) was examined in mouse pancreatic islets, the mouse pancreatic ß-cell line MIN6 cells and its derivative MIN6T3 cells after removal of sulfate groups by sodium chlorate, a competitive inhibitor of glycosaminoglycan sulfation. Quantitative reverse transcription polymerase chain reaction was used for analyzing messenger ribonucleic acid (mRNA) expression of HS modification enzymes. Expression of HS 3-O-sulfotransferase isoform-1 (Hs3st1) was silenced and GIIS was examined. RESULTS: Impaired insulin secretion by islets, MIN6 cells and MIN6T3 cells was observed after treatment with sodium chlorate. Sodium chlorate-treatment upregulated the mRNA expression of sulfotransferases expressed in MIN6T3 cells. Expression of the Hs3st1 was strongly upregulated by sodium chlorate-treatment, and its silencing by RNA interference reduced GIIS in MIN6T3 cells. CONCLUSIONS: Our data suggest that the 3-O-sulfate group of HS that is modified by Hs3st1 plays a significant role(s) in the insulin secretory pathway, selectively through an interaction with factor(s) upstream of membrane depolarization in ß-cells. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00205.x, 2012).

Conserved GC-boxes, E-box and GATA motif are essential for GATA-4 gene expression in P19CL6 cells.

The promoter of the GATA-4 gene was analyzed in P19CL6 cells. A 124bp segment containing conserved two GC-boxes and E-box was essential for the basal promoter activity, as determined with a transient luciferase reporter gene assay. However, an extended 1312 bp reporter construct but not the 124 bp segment, when ligated to the GFP gene and stably inserted into the chromosome, showed regulated promoter activity since GFP was expressed upon DMSO addition. Mutations of the two GC-boxes and/or E-box significantly impaired the GFP expression. Furthermore, mutation of the distal conserved GATA motif in the 1312 bp sequence decreased the expression of GFP. Chromatin immuno-precipitation assay showed that GATA-6 binds to this conserved GATA motif. These results suggest that the distal GATA motif recognized by GATA-6 together with the GC- and E-boxes may be important for transcriptional activation of the GATA-4 gene in the chromosome.

Normal formation of a subset of intestinal granules in Caenorhabditis elegans requires ATP-binding cassette transporters HAF-4 and HAF-9, which are highly homologous to human lysosomal peptide transporter TAP-like.

TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects.

Functional dissection of transmembrane domains of human TAP-like (ABCB9).

An ABC transporter, TAP-Like (TAPL), was dissected into its amino-terminal transmembrane domain and the following core domain. When these domains were transiently expressed as tagged proteins with a His6- or Myc-epitope tag, the amino-terminal ones (Met(1)-Lys(182)) could not associate with each other, or with the full-length transporter (Met(1)-Ala(766)). However, both the core domain (Arg(141)-Ala(766)) and full-length protein mutually interacted. The amino-terminal domain (Met(1)-Arg(141)) as well as the full-length transporter fused with fluorescent protein GFP was sorted to lysosomal membranes upon their stable expression, as visualized by means of fluorescent microscopy, while the core domain (Arg(141)-Ala(766)) was broadly distributed in the intra-cellular membranes. These results suggest that the sorting signal for lysosomes is present within the amino-terminal transmembrane domain (Met(1)-Arg(141)) of the TAPL molecule.

Cisplatin upregulates Saccharomyces cerevisiae genes involved in iron homeostasis through activation of the iron insufficiency-responsive transcription factor Aft1.

The response of Saccharomyces cerevisiae to cisplatin was investigated by examining variations in gene expression using cDNA microarrays and confirming the results by reverse transcription polymerase chain reaction (RT-PCR). The mRNA levels of 14 proteins involved in iron homeostasis were shown to be increased by cisplatin. Interestingly, the expression of all 14 genes is known to be regulated by Aft1, a transcription factor activated in response to iron insufficiency. The promoter of one of these genes, FET3, has been relatively well studied, so we performed a reporter assay using the FET3 promoter and showed that an Aft1 binding site in the promoter region is indispensable for induction of transcription by cisplatin. The active domain of Aft1 necessary for activation of the FET3 promoter by cisplatin is identical to the one required for activation by bathophenanthroline sulfonate, an inhibitor of cellular iron uptake. Furthermore, we found that cisplatin inhibits the uptake of (55)Fe(II) into yeast cells. These findings suggest that cisplatin activates Aft1 through the inhibition of iron uptake into the cells, after which the expression of Aft1 target genes involved in iron uptake might be induced.

Cisplatin-induced expression of iron-retaining genes FIT2 and FIT3 in Saccharomyces cerevisiae.

cDNA microarray analysis indicated that mRNA levels of Fit2p and Fit3p, proteins involved in iron retention within the yeast cell wall, were markedly increased by treatment of Saccharomyces cerevisiae with cisplatin. Expression of FIT2 and FIT3 is known to be transcriptionally regulated by Aft1p. Northern blotting demonstrated a time- and concentration-dependent increase in the mRNA levels of both proteins following treatment with cisplatin. However, overexpression or disruption of the FIT2 or FIT3 genes had little effect on the susceptibility of yeast to cisplatin. Although Fit2p and Fit3p do not appear to be directly involved in protecting against the toxic effects of cisplatin, the present results suggest the existence of an activation system of gene expression in response to cisplatin within yeast cells.

Examination of drug resistance activity of human TAP-like (ABCB9) expressed in yeast.

A half-type ABC transporter, human TAP-like (hTAPL) tagged with histidine cluster, was expressed in budding yeast protease-deficient strain BJ5457, and the effect of expression for resistance to peptide compounds including antibiotics and proteinase inhibitor was examined. Among these compounds, the yeast expressing hTAPL exhibits high sensitivity to valinomycin, a monovalent cation ionophore. A mutation in Walker A motif, which lost ATP-binding activity of hTAPL, eliminated the enhanced sensitivity to valinomycin. These findings suggest that the transport activity of hTAPL is important for conferring high valinomycin-sensitive phenotype to yeast.

Further extension of mammalian GATA-6.

Mammalian GATA-6, which has conserved tandem zinc fingers (CVNC-X(17)-CNAC)-X(29)-(CXNC-X(17)-CNAC), is essential for the development and specific gene regulation of the heart, gastrointestinal tract and other tissues. GATA-6 recognizes the (A/T/C)GAT(A/T)(A) sequence, and interacts with other transcriptional regulators through its zinc-finger region. The mRNA of GATA-6 uses two Met codons in frame as translational initiation codons, and produces L- and S-type GATA-6 through leaky ribosome scanning. GATA-6 is subjected to cAMP-dependent proteolysis by a proteasome in a heterologous expression system. These protein-based characteristics of GATA-6 will be helpful for the identification of target genes, together with determination of the in vivo binding sites for GATA-6 and understanding of the complex network of gene regulation mediated by GATA-6.

HANABA TARANU is a GATA transcription factor that regulates shoot apical meristem and flower development in Arabidopsis.

We have isolated a new mutant, hanaba taranu (han), which affects both flower and shoot apical meristem (SAM) development in Arabidopsis thaliana. Mutants have fused sepals and reduced organ numbers in all four whorls, especially in the 2nd (petal) and 3rd (stamen) whorls. han meristems can become flatter or smaller than in the wild type. HAN encodes a GATA-3-like transcription factor with a single zinc finger domain. HAN is transcribed at the boundaries between the meristem and its newly initiated organ primordia and at the boundaries between different floral whorls. It is also expressed in vascular tissues, developing ovules and stamens, and in the embryo. han interacts strongly with clavata (clv) mutations (clv1, clv2, and clv3), resulting in highly fasciated SAMs, and we find that WUS expression is altered in han mutants from early embryogenesis. In addition, HAN is ectopically expressed both in clv1 and clv3 mutants. We propose that HAN is normally required for establishing organ boundaries in shoots and flowers and for controlling the number and position of WUS-expressing cells. Ectopic HAN expression causes growth retardation, aberrant cell division patterns, and loss of meristem activity, suggesting that HAN is involved in controlling cell proliferation and differentiation.

Copper(II) protects yeast against the toxicity of cisplatin independently of the induction of metallothionein and the inhibition of platinum uptake.

We have made the unexpected discovery that copper sulfate protects Saccharomyces cerevisiae from the toxic effects of cisplatin. Addition of copper to the culture medium of yeast cells at concentrations above 0.1 microM significantly reduced the toxicity of cisplatin. Since a high-affinity copper transporter, Ctr1, has been reported to play a major role in the uptake of cisplatin, we examined the effects of copper on the cellular uptake of cisplatin. We found that the cellular concentration of platinum was not significantly affected by treatment of cells with 1 microM copper. It is known that mammalian metallothionein is induced by copper and is involved in acquired resistance to cisplatin. Copper significantly increased the level of mRNA for yeast metallothionein at a concentration that has effectively reduced the toxicity of cisplatin. However, the toxicity of cisplatin in cells with a disrupted gene for ACE1, a factor that regulates transcription of the yeast gene for metallothionein, was also significantly reduced by treatment with copper. These results suggest that copper protects yeast cells from cisplatin toxicity independently of induction of the synthesis of metallothionein and of the inhibition of platinum uptake. Since copper is one of the trace elements that are essential for cell function and since a relatively low concentration of copper (0.1 microM) significantly reduced cisplatin toxicity, it is possible that copper might play an important role in the expression of cisplatin toxicity.

Identification and punctate nuclear localization of a novel noncoding RNA, Ks-1, from the honeybee brain.

We identified a novel gene, Ks-1, which is expressed preferentially in the small-type Kenyon cells of the honeybee brain. This gene is also expressed in some of the large soma neurons in the brain and in the suboesophageal ganglion. Reverse transcription-polymerase chain reaction experiments indicated that Ks-1 transcripts are enriched in the honeybee brain. cDNA cloning revealed that the consensus Ks-1 cDNA is over 17 kbp and contains no significant open reading frames. Furthermore, fluorescent in situ hybridization revealed that Ks-1 transcripts are located in the nuclei of the neural cells, accumulating in some scattered spots. These findings demonstrate that Ks-1 encodes a novel class of noncoding nuclear RNA and is possibly involved in the regulation of neural functions.