Free glycans derived from O-mannosylated glycoproteins suggest the presence of an O-glycoprotein degradation pathway in yeast.

In eukaryotic cells, unconjugated oligosaccharides that are structurally related to N-glycans (i.e. free N-glycans) are generated either from misfolded N-glycoproteins destined for the endoplasmic reticulum-associated degradation or from lipid-linked oligosaccharides, donor substrates for N-glycosylation of proteins. The mechanism responsible for the generation of free N-glycans is now well-understood, but the issue of whether other types of free glycans are present remains unclear. Here, we report on the accumulation of free, O-mannosylated glycans in budding yeast that were cultured in medium containing mannose as the carbon source. A structural analysis of these glycans revealed that their structures are identical to those of O-mannosyl glycans that are attached to glycoproteins. Deletion of the cyc8 gene, which encodes for a general transcription repressor, resulted in the accumulation of excessive amounts of free O-glycans, concomitant with a severe growth defect, a reduction in the level of an O-mannosylated protein, and compromised cell wall integrity. Our findings provide evidence in support of a regulated pathway for the degradation of O-glycoproteins in yeast and offer critical insights into the catabolic mechanisms that control the fate of O-glycosylated proteins.

Yeast Rim11 kinase responds to glutathione-induced stress by regulating the transcription of phospholipid biosynthetic genes.

Glutathione (GSH), a tripeptide composed of glycine, cysteine, and glutamic acid, is an abundant thiol found in a wide variety of cells, ranging from bacterial to mammalian cells. Adequate levels of GSH are essential for maintaining iron homeostasis. The ratio of oxidized/reduced GSH is strictly regulated in each organelle to maintain the cellular redox potential. Cellular redox imbalances cause defects in physiological activities, which can lead to various diseases. Although there are many reports regarding the cellular response to GSH depletion, studies on stress response to high levels of GSH are limited. Here, we performed genome-scale screening in the yeast Saccharomyces cerevisiae and identified RIM11, BMH1, and WHI2 as multicopy suppressors of the growth defect caused by GSH stress. The deletion strains of each gene were sensitive to GSH. We found that Rim11, a kinase important in the regulation of meiosis, was activated via autophosphorylation upon GSH stress in a glucose-rich medium. Furthermore, RNA-seq revealed that transcription of phospholipid biosynthetic genes was downregulated under GSH stress, and introduction of multiple copies of RIM11 counteracted this effect. These results demonstrate that S. cerevisiae copes with GSH stress via multiple stress-responsive pathways, including a part of the adaptive pathway to glucose limitation.

Mtc6/Ehg2 is a novel endoplasmic reticulum-resident glycoprotein essential for high-pressure tolerance.

Hydrostatic pressure is a common mechanical stressor that modulates metabolism and reduces cell viability. Eukaryotic cells have genetic programs to cope with hydrostatic pressure stress and maintain intracellular homeostasis. However, the mechanism underlying hydrostatic pressure tolerance remains largely unknown. We have recently demonstrated that Maintenance of telomere capping protein 6 (Mtc6) plays a protective role in the survival of the budding yeast Saccharomyces cerevisiae under hydrostatic pressure stress by supporting the integrity of nutrient permeases. The current study demonstrate that Mtc6 acts as an endoplasmic reticulum (ER) membrane protein. Mtc6 comprises two transmembrane domains, a C-terminal cytoplasmic domain, and a luminal region with 12 Asn (N)-linked glycans attached to it. Serial mutational analyses showed that the cytoplasmic C-terminal amino acid residues GVPS are essential for Mtc6 activity. Multiple N-linked glycans in the luminal region are involved in the structural conformation of Mtc6. Moreover, deletion of MTC6 led to increased degradation of the leucine permease Bap2 under hydrostatic pressure, suggesting that Mtc6 facilitates proper folding of nutrient permeases in the ER under the stress condition. We propose a novel model of molecular function in which the glycosylated luminal domain and cytoplasmic GVPS sequences of Mtc6 cooperatively support the nutrient permease activity.

Action of multiple endoplasmic reticulum chaperon-like proteins is required for proper folding and polarized localization of Kre6 protein essential in yeast cell wall ß-1,6-glucan synthesis.

Saccharomyces cerevisiae Kre6 is a type II membrane protein essential for cell wall ß-1,6-glucan synthesis. Recently we reported that the majority of Kre6 is in the endoplasmic reticulum (ER), but a significant portion of Kre6 is found in the plasma membrane of buds, and this polarized appearance of Kre6 is required for ß-1,6-glucan synthesis. An essential membrane protein, Keg1, and ER chaperon Rot1 bind to Kre6. In this study we found that in mutant keg1-1 cells, accumulation of Kre6 at the buds is diminished, binding of Kre6 to Keg1 is decreased, and Kre6 becomes susceptible to ER-associated degradation (ERAD), which suggests Keg1 participates in folding and transport of Kre6. All mutants of the calnexin cycle member homologues (cwh41, rot2, kre5, and cne1) showed defects in ß-1,6-glucan synthesis, although the calnexin chaperon system is considered not functional in yeast. We found synthetic defects between them and keg1-1, and Cne1 co-immunoprecipitated with Keg1 and Kre6. A stronger binding of Cne1 to Kre6 was detected when two glucosidases (Cwh41 and Rot2) that remove glucose on N-glycan were functional. Skn1, a Kre6 homologue, was not detected by immunofluorescence in the wild type yeast, but in kre6Δ cells it became detectable and behaved like Kre6. In conclusion, the action of multiple ER chaperon-like proteins is required for proper folding and localization of Kre6 and probably Skn1 to function in ß-1,6-glucan synthesis.

Molecular mechanisms of the localization of membrane proteins in the yeast Golgi compartments.

The Golgi apparatus of the eukaryotic cell is an essential organelle at the center of the network of vesicular transport delivering proteins and lipids to the correct locations in the cell. There are several Golgi compartments that have distinct resident proteins and functions, but the mechanism creating and maintaining the differences has long been an unsolved mystery in cell biology. After the discovery and molecular characterization of the transport vesicles and their coat proteins, we realized that the Golgi is an extremely dynamic organelle existing as repeating cycles of appearance, maturation, and disappearance. In this review, we describe essential findings as to the Golgi apparatus uncovered by work on an excellent model microorganism, the yeast Saccharomyces cerevisiae, with special reference to the results of our studies.

Kre6 protein essential for yeast cell wall beta-1,6-glucan synthesis accumulates at sites of polarized growth.

Saccharomyces cerevisiae Kre6 is a type II membrane protein with amino acid sequence homology with glycoside hydrolase and is essential for ß-1,6-glucan synthesis as revealed by the mutant phenotype, but its biochemical function is still unknown. The localization of Kre6, determined by epitope tagging, is a matter of debate. We raised anti-Kre6 rabbit antiserum and examined the localization of Kre6 and its tagged protein by immunofluorescence microscopy, subcellular fractionation in sucrose density gradients, and immunoelectron microscopy. Integration of the results indicates that the majority of Kre6 is in the endoplasmic reticulum; however, a small but significant portion is also present in the secretory vesicle-like compartments and plasma membrane. Kre6 in the latter compartments is observed as strong signals that accumulate at the sites of polarized growth by immunofluorescence. The truncated Kre6 without the N-terminal 230-amino acid cytoplasmic region did not show this polarized accumulation and had a severe defect in ß-1,6-glucan synthesis. This is the first evidence of a ß-1,6-glucan-related protein showing the polarized membrane localization that correlates with its biological function.

Jerveratrum-Type Steroidal Alkaloids Inhibit ß-1,6-Glucan Biosynthesis in Fungal Cell Walls.

The limited number of available effective agents necessitates the development of new antifungals. We report that jervine, a jerveratrum-type steroidal alkaloid isolated from Veratrum californicum, has antifungal activity. Phenotypic comparisons of cell wall mutants, K1 killer toxin susceptibility testing, and quantification of cell wall components revealed that ß-1,6-glucan biosynthesis was significantly inhibited by jervine. Temperature-sensitive mutants defective in essential genes involved in ß-1,6-glucan biosynthesis, including BIG1, KEG1, KRE5, KRE9, and ROT1, were hypersensitive to jervine. In contrast, point mutations in KRE6 or its paralog SKN1 produced jervine resistance, suggesting that jervine targets Kre6 and Skn1. Jervine exhibited broad-spectrum antifungal activity and was effective against human-pathogenic fungi, including Candida parapsilosis and Candida krusei. It was also effective against phytopathogenic fungi, including Botrytis cinerea and Puccinia recondita. Jervine exerted a synergistic effect with fluconazole. Therefore, jervine, a jerveratrum-type steroidal alkaloid used in pharmaceutical products, represents a new class of antifungals active against mycoses and plant-pathogenic fungi. IMPORTANCE Non-Candida albicans Candida species (NCAC) are on the rise as a cause of mycosis. Many antifungal drugs are less effective against NCAC, limiting the available therapeutic agents. Here, we report that jervine, a jerveratrum-type steroidal alkaloid, is effective against NCAC and phytopathogenic fungi. Jervine acts on Kre6 and Skn1, which are involved in ß-1,6-glucan biosynthesis. The skeleton of jerveratrum-type steroidal alkaloids has been well studied, and more recently, their anticancer properties have been investigated. Therefore, jerveratrum-type alkaloids could potentially be applied as treatments for fungal infections and cancer.

Svp26 facilitates endoplasmic reticulum to golgi transport of a set of mannosyltransferases in Saccharomyces cerevisiae.

Svp26 is a polytopic integral membrane protein found in the ER and early Golgi compartment. In the Deltasvp26 cell, the Golgi mannosyltransferase Ktr3 remains in the ER. Here, we report that two other Golgi mannosyltransferases, Mnn2 and Mnn5 are also mislocalized and found in the ER in the absence of Svp26 and that localization of other mannosyltransferases including Mnn1 are not affected. Mnn2 and Mnn5 bind to Svp26 in vivo as Ktr3 does. Using an in vitro budding assay, the incorporation of Ktr3 and Mnn2 in the COPII vesicles is greatly stimulated by the presence of Svp26. As Svp26 itself is an efficient cargo, Svp26 is likely to support selective incorporation of a set of mannosyltransferases into COPII vesicles by working as their adaptor protein. The domain switching between Svp26-dependent Mnn2 or Ktr3 and Svp26-independent Mnn1 suggests that the lumenal domain of mannosyltransferases, but not the cytoplasmic or transmembrane domain, is responsible for recognition by Svp26.

Ypt11 functions in bud-directed transport of the Golgi by linking Myo2 to the coatomer subunit Ret2.

A yeast class V myosin Myo2 transports the Golgi into the bud during its inheritance. However, the mechanism that links the Golgi to Myo2 is unknown. Here, we report that Ypt11, a Rab GTPase that reportedly interacts with Myo2, binds to Ret2, a subunit of the coatomer complex. When Ypt11 is overproduced, Ret2 and the Golgi markers, Och1 and Sft2, are accumulated in the growing bud and are lost in the mother cell. In a ret2 mutant that produces the Ret2 protein with reduced affinity to Ypt11, no such accumulation is observed upon overproduction of Ypt11. At a certain stage of budding, it is known that the late Golgi cisternae labeled with Sec7-GFP show polarized distribution in the bud. We find that this polarization of late Golgi cisternae is not observed in the ypt11Delta mutant. Indeed, analyses of Sec7-GFP dynamics with spatio-temporal image correlation spectroscopy (STICS) and fluorescence loss in photobleaching (FLIP) reveals that Ypt11 is required for the vectorial actin-dependent movement of the late Golgi from the mother cell toward the emerging bud. These results indicate that the Ypt11 and Ret2 are components of a Myo2 receptor complex that functions during the Golgi inheritance into the growing bud.

ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles.

Rho-associated kinase (ROCK) I mediates signaling from Rho to the actin cytoskeleton. To investigate the in vivo functions of ROCK-I, we generated ROCK-I-deficient mice. Loss of ROCK-I resulted in failure of eyelid closure and closure of the ventral body wall, which gave rise to the eyes open at birth and omphalocele phenotypes in neonates. Most ROCK-I(-/-) mice died soon after birth as a result of cannibalization of the omphalocele by the mother. Actin cables that encircle the eye in the epithelial cells of the eyelid were disorganized and accumulation of filamentous actin at the umbilical ring was impaired, with loss of phosphorylation of the myosin regulatory light chain (MLC) at both sites, in ROCK-I(-/-) embryos. Stress fiber formation and MLC phosphorylation induced by EGF were also attenuated in primary keratinocytes from ROCK-I(-/-) mice. These results suggest that ROCK-I regulates closure of the eyelids and ventral body wall through organization of actomyosin bundles.

Pga1 is an essential component of Glycosylphosphatidylinositol-mannosyltransferase II of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae essential gene YNL158w/PGA1 encodes an endoplasmic reticulum (ER)-localized membrane protein. We constructed temperature-sensitive alleles of PGA1 by error-prone polymerase chain reaction mutagenesis to explore its biological role. Pulse-chase experiments revealed that the pga1(ts) mutants accumulated the ER-form precursor of Gas1 protein at the restrictive temperature. Transport of invertase and carboxypeptidase Y were not affected. Triton X-114 phase separation and [(3)H]inositol labeling indicated that the glycosylphosphatidylinositol (GPI)-anchoring was defective in the pga1(ts) mutants, suggesting that Pga1 is involved in GPI synthesis or its transfer to target proteins. We found GPI18, which was recently reported to encode GPI-mannosyltransferase II (GPI-MT II), as a high-copy suppressor of the temperature sensitivity of pga1(ts). Both Gpi18 and Pga1 were detected in the ER by immunofluorescence, and they were coprecipitated from the Triton X-100-solubilized membrane. The gpi18(ts) and pga1(ts) mutants accumulated the same GPI synthetic intermediate at the restrictive temperature. From these results, we concluded that Pga1 is an additional essential component of the yeast GPI-MT II.

Both Svp26 and Mnn6 are required for the efficient ER exit of Mnn4 in Saccharomyces cerevisiae.

Incorporation of membrane and secretory proteins into COPII vesicles are facilitated either by the direct interaction of cargo proteins with COPII coat proteins, or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in the yeast Saccharomyces cerevisiae. The ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that the efficient incorporation of Mnn4, a type II membrane protein required for mannosyl phosphate transfer to glycoprotein-linked oligosaccharides, into COPII vesicles is also dependent on the function of Svp26. We show that Mnn4 is localized to the Golgi. In addition to Mnn4, Mnn6 is known to be also required for the transfer of mannosyl phosphate to the glycans. We show, by indirect immunofluorescence, that Mnn6 localizes to the ER. As in the case with Svp26, deletion of the MNN6 gene results in the accumulation of Mnn4 in ER. In vitro COPII vesicle budding assays show that Svp26 and Mnn6 facilitate the incorporation of Mnn4 into COPII vesicles. In contrast to Svp26, which is itself efficiently captured into the COPII vesicles, Mnn6 was not incorporated into the COPII vesicles. Mnn4 and Mnn6 have the DXD motif which is often found in the many glycosyltransferases and functions to coordinate a divalent cation essential for the reaction. Alcian blue dye binding assay shows that substitution of the first D in this motif present in Mnn4 by A impairs the Mnn4 function. In contrast, amino acid substitutions in DXD motifs present in Mnn6 did not affect the function of Mnn6. These results suggest that Mnn4 may be directly involved in the mannosyl phosphate transfer reaction.

Svp26 facilitates ER exit of mannosyltransferases Mnt2 and Mnt3 in Saccharomyces cerevisiae.

After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these cargo proteins into COPII vesicles are facilitated either by direct interaction of cargo proteins with COPII coat proteins or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in yeast Saccharomyces cerevisiae. ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that efficient incorporation of Mnt2 and Mnt3 into COPII vesicles is also dependent on the function of Svp26. Mnt2 and Mnt3 are Golgi-localized α-1,3-mannosyltransferases with type II membrane topology involved in protein O-glycosylation. Immunoisolation of the yeast Golgi subcompartments quantitatively showed that Mnt2 and Mnt3 are more abundant in the early Golgi fraction than in the late Golgi fraction. Subcellular fractionation and fluorescence microscopy showed that deletion of the SVP26 gene results in the accumulation of Mnt2 and Mnt3 in ER. Using an in vitro COPII vesicle formation assay, we further demonstrate that Svp26 facilitates incorporation of Mnt2 and Mnt3 into COPII vesicles. Finally, we showed that Mnt2 and Mnt3 were co-immunoprecipitated with Svp26 from digitonin-solubilized membranes. These results indicate that Svp26 functions as an ER exit adaptor protein of Mnt2 and Mnt3.

A novel ER membrane protein Ehg1/May24 plays a critical role in maintaining multiple nutrient permeases in yeast under high-pressure perturbation.

Previously, we isolated 84 deletion mutants in Saccharomyces cerevisiae auxotrophic background that exhibited hypersensitive growth under high hydrostatic pressure and/or low temperature. Here, we observed that 24 deletion mutants were rescued by the introduction of four plasmids (LEU2, HIS3, LYS2, and URA3) together to grow at 25 MPa, thereby suggesting close links between the genes and nutrient uptake. Most of the highly ranked genes were poorly characterized, including MAY24/YPR153W. May24 appeared to be localized in the endoplasmic reticulum (ER) membrane. Therefore, we designated this gene as EHG (ER-associated high-pressure growth gene) 1. Deletion of EHG1 led to reduced nutrient transport rates and decreases in the nutrient permease levels at 25 MPa. These results suggest that Ehg1 is required for the stability and functionality of the permeases under high pressure. Ehg1 physically interacted with nutrient permeases Hip1, Bap2, and Fur4; however, alanine substitutions for Pro17, Phe19, and Pro20, which were highly conserved among Ehg1 homologues in various yeast species, eliminated interactions with the permeases as well as the high-pressure growth ability. By functioning as a novel chaperone that facilitated coping with high-pressure-induced perturbations, Ehg1 could exert a stabilizing effect on nutrient permeases when they are present in the ER.

Immunoisolaton of the yeast Golgi subcompartments and characterization of a novel membrane protein, Svp26, discovered in the Sed5-containing compartments.

The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.

Saccharomyces cerevisiae Bor1p is a boron exporter and a key determinant of boron tolerance.

Boron is toxic to living organisms when present in excess. Saccharomyces cerevisiae Bor1p is a plasma membrane protein that decreases the intracellular concentration of boron and confers boron tolerance in yeasts. We investigated the detailed characteristics of boron transport by Bor1p and its roles in boron tolerance. Boron transport assays showed that the bor1 deletion mutant (bor1Delta) accumulates higher intracellular concentrations of boron and has a lower rate of boron export. The bor1Delta showed greater susceptibility to high concentrations of boron than the wild-type strain, and the growth rates of both strains were negatively correlated with the intracellular concentrations of boron. With normal to toxic levels of external boron, green fluorescent protein (GFP)-tagged Bor1p localized to the plasma membrane irrespective of the concentration of boron in the medium. Taken together, these results establish Bor1p as a plasma membrane boron exporter and a key determinant of boron tolerance.

Molecular cloning and characterization of a Pichia pastoris ortholog of the yeast Golgi GDP-mannose transporter gene.

There are two structural profiles in the yeast Golgi. The Golgi of Saccharomyces cerevisiae is composed of a number of vesicular compartments dispersed in the cytoplasm as recognized by a large number of Golgi marker proteins. In contrast, the Golgi of Pichia pastoris was reported to be organized in a small number of stacked cisternae located near the transitional endoplasmic reticulum (tER) sites by electron microscopy and immunofluorescent staining of a few marker proteins. The guanosine diphosphate (GDP)-mannose transporter (GMT) is an essential component in the yeast Golgi apparatus. We isolated an ortholog of the GMT gene of P. pastoris and visualized the gene product by epitope tagging to verify the structural characteristics of the Golgi. The tagged product in P. pastoris cell was observed in rod-like compartments in which Och1 mannosyltransferase was also found and the tER marker Sec12 and Sec13 proteins localized very close to them. The present results add further evidence of the restricted localization of the Golgi in P. pastoris cell.

Effectiveness of two-step (consecutive) embryo transfer in patients who have two embryos on day 2: comparison with cleavage-stage embryo transfer.

OBJECTIVE: To evaluate the effectiveness, for patients who have only two embryos on day 2, of a two-step (consecutive) embryo transfer (ET) procedure in which a cleaved embryo is transferred on day 2 and a single blastocyst is transferred on day 5. DESIGN: Observational comparative study. SETTING: Private IVF clinic. PATIENT(S): Ninety two-step ET cycles were performed in patients who had two embryos on day 2 (two-step group). Ninety day-2 ET cycles were performed in age- and infertility-matched patients who had two embryos on day 2 (control group). INTERVENTION(S): Cleaved-ET, extended culture of one embryo, and a second transfer of a blastocyst. MAIN OUTCOME MEASURE(S): Implantation and pregnancy rates. RESULT(S): The pregnancy and implantation rates in the two-step group (respectively 33.3% and 17.2%) were significantly higher than those in the control group (18.9% and 9.4%). Thirty-nine of the patients in the two-step group (43.3%) could not proceed to the second step of ET because no viable blastocyst could be obtained, but four of them conceived anyway. CONCLUSION(S): Taking advantage of both day-2 ET and blastocyst transfer, two-step ET may be an effective option for ET in patients who have an insufficient number of embryos.

Effectiveness of antiadhesion barriers in preventing adhesion after myomectomy in patients with uterine leiomyoma.

BACKGROUND: Myomectomy often causes adhesion formation and decreases subsequent fertility. The purpose of the present study was to evaluate the effectiveness of several antiadhesion barrier materials in preventing adhesion after myomectomy. METHODS: We prospectively classified 63 women undergoing myomectomy alone into four groups according to the type of antiadhesion material used: Hyaluronic acid-carboxymethylcellulose film (Seprafilm) (n = 21, Group 1), Dextran 40 (10% Dextran 40 Low Injection) (n = 17, Group 2), factor 13 with fibrinogen (Beriplast) (n = 12, Group 3) and control (n = 13, Group 4). We performed early second-look laparoscopy after the seventh post-operative day in all patients and examined adhesion formation in the abdominal cavity. The incidence of adnexal adhesions was evaluated according to the American Fertility Association (AFS) adhesion score. RESULTS: The incidence of uterine adhesion was 14.3% in Group 1, 70.6% in Group 2, 75.0% in Group 3 and 76.9% in Group 4. Adhesion formation in Group 1 was significantly less than that in Group 2 (p = 0.0004), Group 3 (p = 0.0005) and Group 4 (p = 0.0003). The incidence of peritoneal adhesion was 14.3% in Group 1, 29.4% in Group 2, 41.6% in Group 3 and 69.2% in Group 4. Adhesion formation in Group 1 was significantly less than that in Group 4 (p = 0.001). AFS scores in Groups 1-4 were 0.38+/-1.02, 4.58 +/- 7.02, 0.83 +/- 1.99 and 8.53 +/- 8.79 (mean +/- S.D.), respectively. Group 1 had the lowest AFS score and the difference between Group 1 and Group 4 was significant (p < 0.0001). The AFS score in Group 3 was also significantly less than that of Group 4 (p = 0.0009). CONCLUSION: Seprafilm was highly effective and was superior to the other antiadhesion materials tested in preventing uterine adhesions after myomectomy.

Follistatin-related gene (FLRG) expression in human endometrium: sex steroid hormones regulate the expression of FLRG in cultured human endometrial stromal cells.

Follistatin-related gene (FLRG) encodes a novel secreted glycoprotein that is highly homologous to follistatin and binds activins and bone morphogenetic proteins, members of the TGF beta superfamily of growth/differentiation factors. FLRG protein inhibits activin-induced and bone morphogenetic protein-2-induced transcriptional responses in a dose-dependent manner, and its mRNA is abundantly expressed in human placenta, heart, aorta, testis, and adrenal gland. In this study we showed that FLRG mRNA was expressed in human endometrium across the menstrual cycle and in decidua of early pregnancy. In the proliferative phase of the menstrual cycle, FLRG protein was detected predominantly in the cytoplasm of endometrial epithelium. In the secretory phase and in early pregnancy, it was also detected in the nuclei of endometrial stromal cells. Using in vitro decidualization model, we demonstrated that 17 beta-estradiol plus progesterone, but not 17 beta-estradiol or progesterone alone, induced FLRG expression significantly. These results suggest that FLRG expression in endometrial stromal cells is regulated by the concerted action of ovarian steroid hormones via decidualization, and FLRG protein may participate in the regulation of stromal cell decidualization as a binding protein for members of TGF beta superfamily.