Small GTP-binding protein Rap1 mediates EGF and HB-EGF signaling and modulates EGF receptor expression in HTR-8/SVneo extravillous trophoblast cells.

Purpose: Extravillous trophoblasts (EVTs) invade the endometrium to establish a fetomaternal interaction during pregnancy. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) stimulate EVT invasion by binding to the EGF receptor (EGFR). We examined the role of the small GTP-binding protein Rap1 in EGF- and HB-EGF-stimulated EVT invasion. Methods: Expression of Rap1 in the first-trimester placenta was examined by immunohistochemistry. Effect of EGF or HB-EGF on Rap1 activation (GTP-Rap1) and Rap1 knockdown on invasion was assessed in EVT cell line (HTR-8/SVneo). In addition, effect of Rap1 knockdown and Rap1GAP (a Rap1 inactivator) overexpression on the activation of EGF signaling and EGFR expression were examined. Results: Rap1 was expressed by EVTs, villous cytotrophoblasts, and syncytiotrophoblasts in the placenta. EGF and HB-EGF activated Rap1 and promoted invasion of HTR-8/SVneo, and these effects were inhibited by Rap1 knockdown. The EGF- and HB-EGF-induced phosphorylation of AKT, ERK1/2, p38MAPK, and Src was inhibited by Rap1 knockdown. Furthermore, the knockdown of Rap1 reduced the EGFR protein level. Overexpression of Rap1GAP repressed EGF- and HB-EGF-induced Rap1 activation and reduced EGFR expression. Conclusion: Rap1 may function as a mediator of EGF and HB-EGF signaling pathways and can modulate EGFR expression in EVTs during placental development.

Alpha 1 Antitrypsin Regulates Trophoblast Syncytialization and Inflammatory Factor Expression.

The serine protease inhibitor alpha1-antitrypsin (A1AT) may possess protective functions of impaired organs in a manner independent of its protease inhibitor activity. A1AT expression has been shown to fluctuate in patients with pregnancy-induced hypertension, which suggests that A1AT may play a role in the syncytialization of villous trophoblasts. A1AT expression was knocked down in primary trophoblasts. RNA was extracted from these cells and subjected to RNA-sequencing analysis to determine the levels of expression of markers of syncytialization and inflammation. In addition, A1AT protein was localized in trophoblastic cells in placental tissues. Knockdown of A1AT upregulated the expression of FOSL1 and markers of syncytialization, as well as cell fusion, whereas overexpression of A1AT had the opposite effects. FOSL1 overexpression stimulated syncytialization, similar to the effects of A1AT knock down. Inhibitors of p38MAPK and JNK reduce the expression of inflammatory factors, whereas a p38MAPK inhibitor suppressed FOSL1 expression. Collectively, these findings indicated A1AT may negatively regulate inflammatory responses by controlling the activation of p38MAPK and JNK, and that p38MAPK mediates trophoblast syncytialization by altering FOSL1 expression. Therefore, a dysfunction in A1AT could be responsible for abnormal placental formation and pregnancy-associated disorders.

Beyond BRCA Status: Clinical Biomarkers May Predict Therapeutic Effects of Olaparib in Platinum-Sensitive Ovarian Cancer Recurrence.

The purpose of this study was to investigate the predictors of the effect of olaparib on platinum-sensitive recurrent ovarian cancer with unknown germline BRCA mutations. We retrospectively examined 20 patients with platinum-sensitive ovarian cancer who were treated at the Nippon Medical School Chiba Hokusoh Hospital, Japan, from 2018 to 2020. We found that the median progression-free survival was 11.4 months (95% Confidence interval (CI): 3.8-Not Available (NA)) in the group with NLPN score [recurrent neutrophil-lymphocyte ratio (rNLR) × number of previous regimens] >7.51, and median progression-free survival was not reached in the group with NLPN score <7.51 (95% CI: 21.8-NA) (p = 0.0185). There was a clear correlation between the degree of dose reduction of olaparib and recurrence (p = 0.00249). Our results show that NLPN scores lower than 7.51 are associated with a favorable outcome of olaparib treatment for platinum-sensitive recurrent ovarian cancer. In cases with a high rNLR, it may be necessary to start olaparib treatment as early as possible to obtain low NLPN scores. Our results imply that the effectiveness of olaparib can be determined after recurrence and before platinum treatment begins. As newer drugs for ovarian cancer are developed, the measurement of biomarker levels at the start of treatment for recurrent ovarian cancer, as shown in our study, may provide strong support for cancer treatment protocols.

Statistical Evaluation of the First Year of a Neonatal Intensive Care Unit Established in a Medical School Hospital.

BACKGROUND: There has been significant progress in reducing perinatal mortality in Japan. However, due to changes in social conditions, the total fertility rate and the number of births are decreasing, whereas the number of low birth weight infants is increasing along with the number of newborn babies that require intensive care. Further, although the number of high-level perinatal medical centers has increased, so has that of infants who need long-term hospitalization. Conversely, the number of regular obstetric facilities has decreased, thus resulting in insufficient beds for neonatal care. To fill this gap, we established a neonatal intensive care unit (NICU) at our hospital. This study aimed to evaluate our new type by comparing the data from ours with that from other facilities. METHODS: The other facilities assessed were two high-level NICU facilities and two regular obstetric facilities. Data, including sex, gestational age, birth weight, Apgar scores at 1 and 5 min, delivery method, and presence of breathing disorders, were extracted from medical records. RESULTS: The birth weight and gestational age distributions were significantly different in the institutions, except in one facility without a NICU. The new NICU saw more infants with low birth weight and respiratory disorders than the regular obstetric facilities. CONCLUSION: The comparison of birth weight and gestational age distributions, cases of respiratory disorders, and delivery methods indicate that our new NICU is positioned as an intermediate facility between a high-level NICU and a regular obstetrics facility.

Clinical Tips of Cesarean Section in Case of Breech, Transverse Presentation, and Incarcerated Uterus.

Cesarean section in breech or transverse presentation involves more complicated procedures than cesarean section in cephalic presentation because the former requires additional manipulations for guiding the presenting part of the fetus, liberation of the arms, and the after-coming head delivery; therefore, those cesarean sections are likely to be more invasive. Making a rather wide uterine incision to prevent uterine injury during delivery of the fetus facilitates smooth delivery of the fetus. Furthermore, in cases of breech or transverse presentation, it is important to initially identify the presenting part of the fetus and guide it to the incision opening in the lower uterine segment, because delivering the presenting part of the fetus first is a basic rule of delivery of the fetus. Smooth delivery of the fetus by means of breech extraction can prevent excessive stress or injury to the fetus. Therefore, it is important to acquire the knowledge and skills necessary to perform these techniques, including the internal version. Smooth delivery of the fetus is also less invasive for the mother because an extension of the uterine excision or injury to arteries and veins in the uterus and parametrium can be avoided. Incarcerated uterus occurring in cases of pregnancy with intrapelvic adhesion, endometriosis, cervical myoma, or extended cervix may result in excessive uterine and cervical injury when a transverse incision of the lower uterine segment is performed without caution. These conditions may result in difficulty in fetal delivery. Therefore, it is important to identify risks in advance and to choose the incision line with great care. Countermeasures for difficult delivery of the fetus need to be mastered by all practitioners of obstetrics. If the transverse incision fails to reach the uterine cavity, an inverted T-shaped or J-shaped incision should be made. Risks of complications such as injury to the cervical canal, the vagina, the bladder or ureter, and massive hemorrhage must be kept in mind.

Alternating electric field application induced non-contact and enzyme-free cell detachment.

Low intensity (< 2 Vpp/cm (peak to peak voltage/cm)), high frequency (10-30 MHz), and 10 min alternating electric fields (sine wave with no DC component) induce non-contact and enzyme-free cell detachment of anchorage-dependent cells directly from commercially available cell culture flasks and stack plates. 0.25 Vpp/cm, 20 MHz alternating electric field for 10 min at room temperature (RT) induced maximum detachment and separated 99.5 ± 0.1% (mean ± SEM, n = 6) of CHO-K1 and 99.8 ± 0.2% of BALB/3T3 cells from the culture flasks. Both vertical and lateral alternating electric field applications for 10 min at RT detach the CHO-K1 cells from 25 cm2 culture flasks. The alternating electric field application induced cell detachment is almost noncytotoxic, and over 90% of the detached cells remained alive. The alternating electric field applied CHO-K1 cells for 90 min showed little or no lag phase and immediately enter exponential phase in cell growth. Combination of the 20 MHz alternating electric field and enzymatic treatment for 4 min at 37 °C showed synergetic effect and quickly detached human induced pluripotent stem cells from a laminin-coated culture flask compared with the only enzymatic treatment. These results indicate that the rapid cell detachment with both the electric field application and the enzymatic treatment could be applied to subcultures of cells that are susceptible to prolonged enzymatic digestion damage for mass culture of sustainable clinical use.

Valproate-Induced Polycystic Ovary Syndrome in a Girl with Epilepsy: A Case Study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common ovulatory disorder that can be induced by sodium valproate (VPA). PATIENT: We report a case of PCOS that developed in a 15-year-old girl with idiopathic epilepsy after she took VPA. VPA administration stopped her seizures, but it also led to weight gain and amenorrhea, and the patient was diagnosed with PCOS on the basis of diagnostic imaging and serological examination results. Cessation of VPA administration led to reduced weight gain and restored menstruation. CONCLUSIONS: The risk of PCOS developing in patients with epilepsy is known to be high, and the association of VPA with PCOS is well established, so if physicians feel this is the best drug to prescribe for female patients with epilepsy, they should carefully monitor the patients' weight and menstruation, and immediately perform ovarian imaging and hormonal examinations if any abnormalities are observed.

Glibenclamide inhibits NLRP3 inflammasome-mediated IL-1ß secretion in human trophoblasts.

Infection-associated pregnancy complications cause premature delivery. Caspase-1 is involved in the maturation of interleukin (IL)-1ß, which is activated by the NLRP3 inflammasome. To characterize the significance of the NLRP3 inflammasome pathway in the placenta, the effects of activators and inhibitors on NLRP3-related molecules were examined using isolated primary trophoblasts. Caspase-1 and IL-1ß mRNA expression was markedly increased in response to lipopolysaccharide (LPS), a toll-like receptor (TLR)4 ligand. Treatment with the potassium ionophore nigericin significantly increased the level of activated caspase-1. Treatment with either LPS or nigericin stimulated IL-1ß secretion, whereas pretreatment with the ATP-sensitive K+ channel inhibitor glibenclamide, the Rho-associated coiled-coil kinase inhibitor Y-27632, or a caspase-1 inhibitor significantly decreased nigericin-induced IL-1ß secretion. In addition, dibutyryl-cAMP, which induces trophoblast differentiation, decreased expression of NLRP3, caspase-1, and IL-1ß. These findings suggest that trophoblasts can secrete IL-1ß through the NLRP3/caspase-1 pathway, which is suppressed by glibenclamide, and that the TLR4-mediated NLRP3 inflammasome pathway is more likely to be stimulated in undifferentiated than differentiated trophoblasts. Our data support the hypothesis that inhibition of the NLRP3 inflammasome can suppress placental inflammation-associated disorders.

Statistical hypothesis testing of factor loading in principal component analysis and its application to metabolite set enrichment analysis.

BACKGROUND: Principal component analysis (PCA) has been widely used to visualize high-dimensional metabolomic data in a two- or three-dimensional subspace. In metabolomics, some metabolites (e.g., the top 10 metabolites) have been subjectively selected when using factor loading in PCA, and biological inferences are made for these metabolites. However, this approach may lead to biased biological inferences because these metabolites are not objectively selected with statistical criteria. RESULTS: We propose a statistical procedure that selects metabolites with statistical hypothesis testing of the factor loading in PCA and makes biological inferences about these significant metabolites with a metabolite set enrichment analysis (MSEA). This procedure depends on the fact that the eigenvector in PCA for autoscaled data is proportional to the correlation coefficient between the PC score and each metabolite level. We applied this approach to two sets of metabolomic data from mouse liver samples: 136 of 282 metabolites in the first case study and 66 of 275 metabolites in the second case study were statistically significant. This result suggests that to set the number of metabolites before the analysis is inappropriate because the number of significant metabolites differs in each study when factor loading is used in PCA. Moreover, when an MSEA of these significant metabolites was performed, significant metabolic pathways were detected, which were acceptable in terms of previous biological knowledge. CONCLUSIONS: It is essential to select metabolites statistically to make unbiased biological inferences from metabolomic data when using factor loading in PCA. We propose a statistical procedure to select metabolites with statistical hypothesis testing of the factor loading in PCA, and to draw biological inferences about these significant metabolites with MSEA. We have developed an R package "mseapca" to facilitate this approach. The "mseapca" package is publicly available at the CRAN website.

Hydroxysteroid (17-ß) dehydrogenase 1 is dysregulated by miR-210 and miR-518c that are aberrantly expressed in preeclamptic placentas: a novel marker for predicting preeclampsia.

In this study, to search for novel preeclampsia (PE) biomarkers, we focused on microRNA expression and function in the human placenta complicated with PE. By comprehensive analyses of microRNA expression, we identified 22 microRNAs significantly upregulated in preeclamptic placentas, 5 of which were predicted in silico to commonly target the mRNA encoding hydroxysteroid (17-ß) dehydrogenase 1 (HSD17B1), a steroidogenetic enzyme expressed predominantly in the placenta. In vivo HSD17B1 expression, at both the mRNA and protein levels, was significantly decreased in preeclamptic placentas. Of these microRNAs, miR-210 and miR-518c were experimentally validated to target HSD17B1 by luciferase assay, real-time PCR, and ELISA. Furthermore, we found that plasma HSD17B1 protein levels in preeclamptic pregnant women reflected the decrease of its placental expression. Moreover, a prospective cohort study of plasma HSD17B1 revealed a significant reduction of plasma HSD17B1 levels in pregnant women at 20 to 23 and 27 to 30 weeks of gestation before PE onset compared with those with normal pregnancies. The sensitivities/specificities for predicting PE at 20 to 23 and 27 to 30 weeks of gestation were 0.75/0.67 (cutoff value=21.9 ng/mL) and 0.88/0.51 (cutoff value=30.5 ng/mL), and the odds ratios were 6.09 (95% CI: 2.35-15.77) and 7.83 (95% CI: 1.70-36.14), respectively. We conclude that HSD17B1 is dysregulated by miR-210 and miR-518c that are aberrantly expressed in preeclamptic placenta and that reducing plasma level of HSD17B1 precedes the onset of PE and is a potential prognostic factor for PE.

A case report of complete chorioamniotic membrane separation.

We describe a patient with complete chorioamniotic membrane separation (CMS). During embryologic development, the chorionic and amniotic membranes each arise from their own germ layers and form a celomic cavity in the first trimester of pregnancy. By the early second trimester, the cavity has shrunk and the membranes become conjugated. However, the membranes may separate spontaneously or because of an invasive intrauterine procedure. This pathologic condition is referred to as CMS. Extensive CMS can lead to miscarriage, fetal death, neonatal death, amniotic band syndrome, umbilical cord complications, and preterm delivery. In this case, CMS was detected in the 29th week of pregnancy with a routine ultrasonographic examination in the absence of a distinct non-reassuring fetal status or an abnormality of the intrauterine environment. The patient had undergone amniocentesis at 16 weeks of pregnancy for chromosomal analysis. Ultrasonography showed a floating membranous structure in almost every view of the intra-amniotic cavity. Thus, complete CMS was believed to have occurred. Therefore, precautionary checkups and examinations were periodically performed. Childbirth took place uneventfully by means of elective cesarean section in the 37th week of pregnancy. Because pathological examination showed complete CMS, the validity of the prenatal diagnosis was confirmed.

A training session in a clinical simulation laboratory for the acquisition of clinical skills by newly recruited medical interns.

In organized orientation programs for newly recruited medical interns of the Nippon Medical School Hospital, the working committee of the clinical simulation laboratory introduced a laboratory training session that was designed to improve the clinical skills of the medical interns. The session consisted of 6 training courses, comprising internal examination, tracheal intubation, auscultation of heart sounds, bandaging and the collection of samples of venous and arterial blood. Medical interns rotated to a new course every 30 minutes and did practical trainings in each of the 6 skills. A total of 36 newly recruited medical interns participated in the training session. The majority of medical interns took part in the practical training actively and positively. The session was efficiently carried out from the standpoints of human resources and the teaching hours involved. A post training questionnaire survey, completed by the medical interns, revealed that many of them valued the sessions for comprehensibility of the instructions, the descriptions in the manual and the content of the training; however, only 21% thought that they had successfully acquired the clinical skills. Medical interns must continually engage in self-training to steadily acquire basic clinical skills. The convenience of a clinical simulation laboratory, together with the reinforcement of the education of clinical skills during internship, is necessary to strengthen the educational benefits of the training session.

[Countermeasure for obstetric hemorrhage].

Although the number of maternal death in Japan has decreased especially since 1990's and its total number has reached the level of developed countries, obstetric hemorrhage is still equally important as obstetric embolism and hypertension in pregnancy as a cause of maternal death. Intrapartum abnormal bleeding is defined as hemorrhage which amounts to more than 500 ml during intrapartum period by Japan Society of Obstetrics and Gynecology (JSOG). However, according to the official register of peripartum data in Japan, the upper normal limit (mean + 1.5 SD) of the amount of hemorrhage during intrapartum period was 900 g at vaginal singleton delivery, 1600 g at singleton cesarean delivery, 1900 g at multifetal vaginal delivery and 2600 g at multifetal cesarean delivery. Thus, upper normal limit is varied depending on mode of deliveries and fetal number. The character of obstetric DIC is marked consumptive coagulopathy and increased fibrinolytic system. Principal strategy for treatment of obstetric DIC is compensation of expended coagulating factors. Thus, the most important strategy is to administer FFP However, it is necessary to be unfreezed for administration of FFP, and it is time-consuming. Prompt and firm decision for administration of FFP is important. Priority of administration of platelet concentrate is not high. Rather the use of antithrombin should be considered. Guideline of response to obstetric critical hemorrhage has been tentatively constructed by Japanese Society of Anesthesiologists, JSOG and other related academic societies. According to the guideline, recommended medical intervention depends on the shock index. At the extremely severe clinical state like placenta previa with accreta and placental abruption, multidisciplinary and prompt approach fulfills very important role to bring through. Strategy of treatment for obstetric DIC and the guideline of response to obstetric critical hemorrhage are also important at such state. Level 1 and other medical devices for critical care also become strong tools for severe cases. However, the most important is adequate and prompt estimation and direction by workforce.

Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.

In this study, we performed small RNA library sequencing using human placental tissues to identify placenta-specific miRNAs. We also tested the hypothesis that human chorionic villi could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation. By small RNA library sequencing, most placenta-specific miRNAs (e.g., MIR517A) were linked to a miRNA cluster on chromosome 19. The miRNA cluster genes were differentially expressed in placental development. Subsequent validation by real-time PCR and in situ hybridization revealed that villous trophoblasts express placenta-specific miRNAs. The analysis of small RNA libraries from the blood plasma showed that the placenta-specific miRNAs are abundant in the plasma of pregnant women. By real-time PCR, we confirmed the rapid clearance of the placenta-specific miRNAs from the plasma after delivery, indicating that such miRNAs enter into maternal circulation. By using the trophoblast cell line BeWo in culture, we demonstrated that miRNAs are indeed extracellularly released via exosomes. Taken together, our findings suggest that miRNAs are exported from the human placental syncytiotrophoblast into maternal circulation, where they could target maternal tissues. Finally, to address the biological functions of placenta-specific miRNAs, we performed a proteome analysis of BeWo cells transfected with MIR517A. Bioinformatic analysis suggests that this miRNA is possibly involved in tumor necrosis factor-mediated signaling. Our data provide important insights into miRNA biology of the human placenta.

DmGEN shows a flap endonuclease activity, cleaving the blocked-flap structure and model replication fork.

Drosophila melanogaster XPG-like endonuclease (DmGEN) is a new category of nuclease belonging to the RAD2/XPG family. The DmGEN protein has two nuclease domains (N and I domains) similar to XPG/class I nucleases; however, unlike class I nucleases, in DmGEN these two nuclease domains are positioned close to each other as in FEN-1/class II and EXO-1/class III nucleases. To confirm the properties of DmGEN, we characterized the active-site mutant protein (E143A E145A) and found that DmGEN had flap endonuclease activity. DmGEN possessed weak nick-dependent 5'-3' exonuclease activity. Unlike XPG, DmGEN could not incise the bubble structure. Interestingly, based on characterization of flap endonuclease activity, DmGEN preferred the blocked-flap structure as a substrate. This feature is distinctly different from FEN-1. Furthermore, DmGEN cleaved the lagging strand of the model replication fork. Immunostaining revealed that DmGEN was present in the nucleus of actively proliferating Drosophila embryos. Thus, our studies revealed that DmGEN belongs to a new class (class IV) of the RAD2/XPG nuclease family. The biochemical properties of DmGEN and its possible role are also discussed.

[Autistic psychopathy or pervasive developmental disorder: how has Asperger's syndrome changed in the past sixty years?].

Bosch (1970) was the first author who used "Asperger's syndrome" in English literature. In those days, "Kanner's syndrome" i.e. autism, which had been under schizophrenic-versus-undeveloped arguments from the 1960's, was always contrasted with Asperger's "autistic psychopathy in children". From then on there have been vicissitudes over the notion of "Asperger's syndrome" and its clinical presentation. Nowadays, the restricted notion of "Asperger's syndrome" is dominant and used in both DSM-IV-TR and ICD-10. However, debates concerning the aspect of Asperger s "psychopathy" in clinical study and practice have long disappeared. In daily life, when we describe someone as "like Asperger's", it means a personality deviation that is to the degree of Asperger's "psychopathy". The history of Asperger's "psychopathy" is still developing in our culture.

The cytotrophoblast layer of human chorionic villi becomes thinner but maintains its structural integrity during gestation.

Chorionic villi in the human placenta serve as essential structures in fetomaternal exchanges. According to the embryology and placentology literature, during the first trimester, the cytotrophoblast (CTB) layer that is subjacent to the syncytiotrophoblast (STB) and supported by a basal lamina is nearly complete, but later, it becomes discontinuous. In the present study, we investigated the structural integrity of the CTB layer in the normal villous tree by advanced microscopy techniques using an antibody to hepatocyte growth factor (HGF) activator inhibitor type 1 (SPINT1), a potent inhibitor of HGF activators expressed exclusively on villous CTB. In full-term placenta, the cell surface of the CTB layer was spread over the basal lamina but was not interrupted. Morphometric analysis showed that throughout the villous tree, 80% of the continuity of the CTB layer of full-term placenta and 90% of that of first-trimester placenta were preserved. Gestation was accompanied by unique structural change in the basal domain of the trophoblast layer. The initially cuboidal-shaped CTB cells were transformed to flat cells with many cellular processes that, together with those of the adjacent STB, eventually covered the trophoblast basal lamina in a complex network of interdigitations. In addition, the expression levels of SPINT1, ST14, HGF, and MET mRNAs in the villous tree increased over the course of gestation. These results suggest that the structural integrity of the SPINT1-positive CTB layer may play an important role in villous differentiation and in maintenance of the villous tree via the HGF signaling system during gestation.

Ultrahigh-resolution immunofluorescence microscopy using ultrathin cryosections: subcellular distribution of caveolin-1alpha and CD31 in human placental endothelial cells.

We compared the z-axis resolutions achieved by immunofluorescence (IF) microscopic imaging of tissue sections of different thicknesses (ultrathin cryosections, optical sections of cryostat sections and conventional cryostat sections). We used these images to determine the distribution of caveolin-1alpha (CAV-1alpha) and CD31 in endothelial cells of full-term, human placenta. Anti-CAV-1alpha antibody was used to visualize caveolae, which are among the smallest organelles. By using ultrathin cryosections as substrates for IF microscopy, we were able to resolve discrete caveolae that were primarily present immediately beneath the endothelial cell surface. In contrast, neither conventional nor confocal images from cryostat sections were able to resolve individual caveolae, despite dramatic reductions in the confocal image degradation that arises from out-of-focus fluorescence signals. Anti-CD31 antibody labeled the endothelial cell surface exclusively. Quantitative analysis of ultrathin cryosections showed that about 2.5 times more CD31 was expressed on the luminal surface of cells than on the abluminal surface. Our results demonstrate that ultrathin cryosections can serve as excellent substrates for ultrahigh-resolution IF microscopy.

Drosophila damaged DNA binding protein 1 contributes to genome stability in somatic cells.

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.

DmGEN, a novel RAD2 family endo-exonuclease from Drosophila melanogaster.

A novel endo-exonuclease, DmGEN (Drosophila Melanogaster XPG-like endonuclease), was identified in D.melanogaster. DmGEN is composed of five exons and four introns, and the open reading frame encodes a predicted product of 726 amino acid residues with a molecular weight of 82.5 kDa and a pI of 5.36. The gene locus on Drosophila polytene chromosomes was detected at 64C9 on the left arm of chromosome 3 as a single site. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nucleases, especially XPG. Although the XPG-N- and XPG-I-domains are highly conserved in sequence, locations of the domains are similar to those of FEN-1 and EXO-1, and the molecular weight of the protein is close to that of EXO-1. In vitro, DmGEN showed endonuclease and 3'-5' exonuclease activities with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), but the endonuclease action with dsDNA was quite specific: 5'-3' exonuclease activity was found to occur with nicked DNA, while dsDNA was endonucleolytically cut at 3-4 bp from the 5' end. Homologs are widely found in mammals and higher plants. The data suggest that DmGEN belongs to a new class of RAD2 nuclease.