Differential apoptotic activities of wild-type FOXL2 and the adult-type granulosa cell tumor-associated mutant FOXL2 (C134W).

Some mutations in FOXL2 result in premature ovarian failure accompanied by blepharophimosis, ptosis, epicanthus inversus syndrome type I disease, and FOXL2-null mice exhibit developmental defects in granulosa cells. Recently, FOXL2 c.402C>G, a new somatic mutation that leads to a p.C134W change, was found in the majority of adult-type ovarian granulosa cell tumors (GCTs). In this study, we investigated the possible mechanisms by which the C134W mutation contributes to the development of GCTs. Wild-type (WT) and mutant FOXL2 displayed differential apoptotic activities. Specifically, WT FOXL2 induced significant granulosa cell death, but the mutant exhibited minimal cell death. The FOXL2-induced apoptotic response was greatly dependent on caspase 8, BID and BAK because the depletion of any of these three proteins inhibited FOXL2 from eliciting the full apoptotic response. Activation of caspase 8 and subsequent increased production of truncated BID, and oligomerization of BAK, and release of cytochrome c were all associated with the apoptosis induced by WT FOXL2 expression. In contrast, the mutant FOXL2 was unable to elicit the full array of apoptotic signaling responses. In addition, we found differential TNF-R1 (tumor necrosis factor-receptor 1) and Fas (CD95/APO-1) upregulation between the WT and the mutant, and the silencing of TNF-R1 or Fas and the blockage of the death signaling mediated by TNF-R1 or Fas using TNF-Fc or Fas-Fc, respectively, resulted in significant attenuations of FOXL2-induced apoptosis. Moreover, granulosa cells that expressed either WT FOXL2 or mutant exhibited distinct cell death sensitivities on activation of death receptors and deprivation of serum. Thus, the differential activities of FOXL2 and its mutant may partially account for the pathophysiology of GCT development.

Effect of Lactobacillus gasseri BNR17 on blood glucose levels and body weight in a mouse model of type 2 diabetes.

AIMS: To investigate the effect of Lactobacillus gasseri BNR17 isolated from human breast milk on blood glucose and body weight in type 2 diabetic animals. METHODS AND RESULTS: db/db mice were divided into one control group and five sample groups; the sample groups received BNR17 (10(7), 10(8), 10(9) and 10(10) CFU) or rosiglitazone (8 mg kg(-1)) orally twice a day for 12 weeks. BNR17 groups had a dose-dependent reduction in food, water intake and amount of excrement. Body weight loss was not seen in the BNR17 groups. Fasting and postprandial 2 h blood glucose levels were significantly lower in the BNR17 (10(10) CFU) group compared with the control group. HbA1c decreased in the BNR17 group, although it was not statistically significant. During the oral glucose tolerance test, the BNR17 groups exhibited dose-dependent improvement in glucose sensitivity. CONCLUSIONS: Lactobacillus gasseri BNR17 has a suppressing effect on blood glucose levels and improved diabetic symptoms in db/db mice. SIGNIFICANCE AND IMPACT OF THE STUDY: Blood glucose-lowering lactic acid bacteria are expected to be useful as a therapeutic for treating type 2 diabetes in humans.

Characterization of the TSU-PR1 cell line by chromosome painting and flow cytometry.

TSU-PR1 was originally reported as a prostatic carcinoma cell line derived from a lymph node metastasis. Recently, however, this cell line was reported to be derived from T24 bladder carcinoma cells, and thus further definition of its origin is needed. Conventional cytogenetic study of TSU-PR1 showed aneuploidy, ranging from 65 to 86 chromosome with a modal number of 80, and with 10 marker chromosomes, thus conventional cytogenetics cannot be used to determine which chromosomes or regions of chromosomes are critical in cancer development and progression of this cell line. The present study was conducted to characterize genetic changes of the cell line using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and flow cytometry. CGH results showed that green-to-red fluorescence ratios were within the range of 0.85-1.15, except for a few chromosomes, which reflected near tetraploidy in TSU-PR1. Flow cytometric analysis of TSU-PR1 revealed a DNA index of 3.46n, which is close to the 3.48n calculated from a modal number of 80. The copy numbers of chromosomes 4, 6, 7, 17, and 20 determined by the DNA index and the CGH analyses were 2.85 +/- 0.09, 3.22 +/- 0.77, 3.01 +/- 0.26, 4.05 +/- 0.44, and 4.99 +/- 0.48, respectively. These numbers are also in accordance with the chromosome copy numbers determined with FISH: 2.98 +/- 0.23, 2.91 +/- 0.44, 2.74 +/- 0.44, 3.93 +/- 0.38, and 5.05 +/- 0.78 for chromosomes 4, 6, 7, 17, and 20, respectively (P > 0.05).

A GDP/GTP exchange factor involved in linking a spatial landmark to cell polarity.

In both animal and yeast cells, signaling pathways involving small guanosine triphosphatases (GTPases) regulate polarized organization of the actin cytoskeleton. In the budding yeast Saccharomyces cerevisiae, the Ras-like GTPase Bud1/Rsr1 and its guanosine 5'-diphosphate (GDP)/guanosine 5'-triphosphate (GTP) exchange factor Bud5 are involved in the selection of a specific site for growth, thus determining cell polarity. We found that Bud5 is localized at the cell division site and the presumptive bud site. Its localization is dependent on potential cellular landmarks, such as Bud3 and Axl2/Bud10 in haploid cells and Bud8 and Bud9 in diploid cells. Bud5 also physically interacts with Axl2/Bud10, a transmembrane glycoprotein, suggesting that a receptor-like transmembrane protein recruits a GDP/GTP exchange factor to connect an intrinsic spatial signal to oriented cell growth.

Photochemical and photobiological properties of new bispsoralen derivatives (Bis[PsCn]PIP, n = 4, 6, 8).

Bispsoralen derivatives possessing two psoralens and one piperazine molecule, 1,4-bis[n'-(8-psoralenoxy) alkyl] piperazine (Bis[PsCn]PIP, n = 4, 6, 8), show high water solubility, efficient intercalation into DNA and good photocrosslinking efficiency of DNA. Bis(PsC4)PIP shows high lethality on bacteriophage T7 and can effectively inhibit the amplification of DNA by stopping the polymerase chain reactions in a short period of irradiation time.

Localization of Bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site.

Yeast cells of different cell type exhibit distinct budding patterns that reflect the organization of the actin cytoskeleton. Bud1p (Rsr1p), a Ras-like GTPase, and Bud2p, a GTPase-activating protein for Bud1p, are essential for proper budding pattern. We show that Bud2p is localized at the presumptive bud site in G(1) cells in all cell types and that this localization is independent of Bud1p. Bud2p subsequently localizes to the mother-bud neck after bud emergence; this localization depends on the integrity of the septins. These observations indicate that Bud2p becomes positioned in G(1) cells by recognizing cell type-specific landmarks at the presumptive bud site.

Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity.

Cells of budding yeast organize their cytoskeleton in a highly polarized manner during vegetative growth. Selection of a site for polarization requires a group of proteins including a Ras-like GTPase, Bud1, and its regulators. Another group of proteins, which includes a Rho-like GTPase (Cdc42), its guanine nucleotide exchange factor (Cdc24), and Bem1, is necessary for organization of the actin cytoskeleton and for cell polarization. We have proposed previously that the Bud1 protein, through its GTPase cycle, determines the localization of one or more of the cell polarity proteins to the bud site. Herein we demonstrate that Bud1 directly interacts with Cdc24 and Bem1: Bud1 in its GTP-bound form associates preferentially with Cdc24, whereas the GDP-bound form of Bud1 associates with Bem1. We also present subcellular fractionation data for Bud1 that is consistent with the idea that Bud1 can travel between the site for budding on the plasma membrane and the cytosol. We propose that Bud1 can exist in two active states for association with different partners and that the switch from Bud1-GTP to Bud1-GDP provides a regulatory device for ordered assembly of a macromolecular complex at the bud site.

Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast.

STE20 encodes a protein kinase related to mammalian p65Pak which functions in several signal transduction pathways in yeast, including those involved in pseudohyphal and invasive growth, as well as mating. In addition, Ste20 plays an essential role in cells lacking Cla4, a kinase with significant homology to Ste20. It is not clear how the activity of Ste20 is regulated in response to these different signals in vivo, but it has been demonstrated recently that binding of the small GTP binding protein Cdc42 is able to activate Ste20 in vitro. Here we show that Ste20 functionally interacts with Cdc42 in a GTP-dependent manner in vivo: Ste20 mutants that can no longer bind Cdc42 were unable to restore growth of ste20 cla4 mutant cells. They were also defective for pseudohyphal growth and agar invasion, and displayed reduced mating efficiency when mated with themselves. Surprisingly, however, the kinase activity of such Ste20 mutants was normal when assayed in vitro. Furthermore, these alleles were able to fully activate the MAP kinase pathway triggered by mating pheromones in vivo, suggesting that binding of Cdc42 and Ste20 was not required to activate Ste20. Wild-type Ste20 protein was visualized as a crescent at emerging buds during vegetative growth and at shmoo tips in cells arrested with alpha-factor. In contrast, a Ste20 mutant protein unable to bind Cdc42 was found diffusely throughout the cytoplasm, suggesting that Cdc42 is required to localize Ste20 properly in vivo.

BUD2 encodes a GTPase-activating protein for Bud1/Rsr1 necessary for proper bud-site selection in yeast.

Cells of the budding yeast Saccharomyces cerevisiae bud at either axial or bipolar sites depending on their cell type. Bud-site selection directs both cell polarity and the cytoskeletal orientation during budding and is determined by at least five genes: BUD1/RSR1, BUD2, BUD3, BUD4 and BUD5. Mutants defective in BUD1, BUD2 or BUD5 choose bud sites randomly. Bud1 protein (Bud1p) has sequence similarity to Ras, a small GTP-binding protein, and Bud5p is similar to Cdc25p (refs 4, 5), a GDP-GTP exchange factor. Here we report that Bud2p is a GTPase-activating protein (GAP) for Bud1p with a sequence similar to the catalytic domain of rasGAPs, and that Bud2p purified from yeast stimulates GTP hydrolysis by Bud1p. Chromosomal deletion of BUD2 causes a random budding pattern but no obvious growth defect. Overexpression of BUD2 also causes a random budding pattern by wild-type cells.

Transcriptional regulation of a yeast HSP70 gene by heat shock factor and an upstream repression site-binding factor.

SSA1, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, displays a significant basal level of expression under optimal growth conditions. Although multiple sites related to the heat shock element (HSE) consensus sequences are present in the SSA1 promoter region, one of these, HSE2, plays a key role in basal expression. An upstream repression site (URS) located adjacent to HSE2 causes repression of basal expression but has little effect on heat-inducible expression of SSA1. A series of DNase I footprinting assays suggests that heat shock factor (HSF) and a URS-binding factor (URSF) can bind simultaneously to the adjacent binding sites HSE2 and URS under optimal growth conditions. URSF in extracts from heat-shocked cells does not bind (or binds very poorly) to the URS adjacent to HSE2. However, URSF in these extracts is able to bind the URS if the URS is separated from HSE2 or if the HSE is mutated such that HSF binding is abolished. These in vitro experiments are consistent with in vivo results showing that the URS is able to repress transcription driven by HSE2 both before and after heat shock if it is separated from HSE2. Our results are consistent with a model of repression in which URSF and HSF bind simultaneously to the adjacent binding sites URS and HSE2 prior to heat shock. After heat shock, however, binding of the two proteins to the adjacent sites is exclusive, perhaps due to modification of HSF known to occur upon heat shock. Because HSF binding predominates, repression by URSF is relieved upon heat shock.

Positive and negative regulation of basal expression of a yeast HSP70 gene.

The SSA1 gene, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, also displays a basal level of expression during logarithmic growth. Multiple sites related to the heat shock element (HSE) consensus sequence are present in the SSA1 promoter region (Slater and Craig, Mol. Cell. Biol. 7:1906-1916, 1987). One of the HSEs, HSE2, is important in the basal expression of SSA1 as well as in heat-inducible expression. A promoter containing a mutant HSE2 showed a fivefold-lower level of basal expression and altered kinetics of expression after heat shock. A series of deletion and point mutations led to identification of an upstream repression sequence (URS) which overlapped HSE2. A promoter containing a mutation in the URS showed an increased level of basal expression. A URS-binding activity was detected in yeast whole-cell extracts by a gel electrophoresis DNA-binding assay. The results reported in this paper indicate that basal expression of the SSA1 promoter is determined by both positive and negative elements and imply that the positively acting yeast heat shock factor HSF is responsible, at least in part, for the basal level of expression of SSA1.

Clinical features of Crohn's disease in Korea.

Crohn's disease is a rare disease in Korea, and only 45 cases have been reported during the period of 34 years from 1952 to 1985. The male to female ratio was about 1.3 to 1 with a slight preponderance of males. The age at diagnosis ranged from 8 to 72 (mean 35.5) years, and the peak incidence occurred in the 3rd, 4th and 5th decades and declined thereafter. More than two thirds of the cases had a grossly demonstrable lesion involving the small bowel, including the terminal ileum. The proportion of patients with macroscopic disease continued to the large bowel alone was only 15%. Abdominal pain was common, presenting in 89% of the patients, while such symptoms as fever, hematochezia and diarrhea were not common. Abdominal mass was palpable in more than half the cases, which made it difficult to differentiate Crohn's disease from cancer of the colon, especially in cases with a predominant infiltration of the bowel wall and a secondary ulcer formation. That is one of the reasons why most cases in Korea have been reported by surgeons. A wide variety of complications were present, of which small bowel obstruction was the most common. Other complications were free perforation, malnutrition, fistula formation, hemorrhage and abscess formation, in decreasing order. The incidence of symptomatic perianal disease was only 11%, and this might be due to the small proportion of the disease confined to large bowel. Extraintestinal manifestations were also rare, and only three patients presented symptoms of arthritis. Other systemic features such as liver disease, skin lesion, eye complications were absent.