Redox regulation of apurinic/apyrimidinic endonuclease 1 activity in Long-Evans Cinnamon rats during spontaneous hepatitis.

The Long-Evans Cinnamon (LEC) rat is an animal model for Wilson's disease. This animal is genetically predisposed to copper accumulation in the liver, increased oxidative stress, accumulation of DNA damage, and the spontaneous development of hepatocellular carcinoma. Thus, this animal model is useful for studying the relationship of endogenous DNA damage to spontaneous carcinogenesis. In this study, we have investigated the apurinic/apyrimidinic endonuclease 1 (APE1)-mediated excision repair of endogenous DNA damage, apurinic/apyrimidinic (AP)-sites, which is highly mutagenic and implicated in human cancer. We found that the activity was reduced in the liver extracts from the acute hepatitis period of LEC rats as compared with extracts from the age-matched Long-Evans Agouti rats. The acute hepatitis period had also a heightened oxidative stress condition as assessed by an increase in oxidized glutathione level and loss of enzyme activity of glyceraldehyde 3-phosphate dehydrogenase, a key redox-sensitive protein in cells. Interestingly, the activity reduction was not due to changes in protein expression but apparently by reversible protein oxidation as the addition of reducing agents to extracts of the liver from acute hepatitis period reactivated APE1 activity and thus, confirmed the oxidation-mediated loss of APE1 activity under increased oxidative stress. These findings show for the first time in an animal model that the repair mechanism of AP-sites is impaired by increased oxidative stress in acute hepatitis via redox regulation which contributed to the increased accumulation of mutagenic AP-sites in liver DNA.

Robust predictive control strategy of SCIG-based drive system.

This article presents a robust finite control set predictive scheme for a stand-alone squirrel cage induction generator (SCIG) drive. This technique is considered an alternative to the drive system due to the inclusion of system nonlinearities and fast dynamic response. The control objective in the distributed generation environment is to fix the output voltage to follow the stand-alone requirement. The strategy establishes optimized switching instants for cost function minimization for both source and load converter control and diminished cross-coupling amid active and reactive power during transient scenarios. The scheme is designed to achieve the minimal effect caused by the parameter uncertainties. During source and load changes, this work will also address the maintenance of dc-link voltage, machine, and load variables at the set value, supported by machine and load-end converter control to achieve stand-alone load objectives. In addition, the presented scheme is also tested with random variation of speed to check the efficacy of the control configuration. The drive performance is evaluated by simulation using MATLAB/Simulink environment. Comprehensive real-time findings obtained from a scaled laboratory test bench using dSPACE-1104 are provided to verify the feasibility of the predictive solution.

Regulating the licensing of DNA replication origins in metazoa.

Eukaryotic DNA replication is a highly conserved process; the proteins and sequence of events that replicate animal genomes are remarkably similar to those that replicate yeast genomes. Moreover, the assembly of prereplication complexes at DNA replication origins ('DNA licensing') is regulated in all eukaryotes so that no origin fires more than once in a single cell cycle. And yet there are significant differences between species both in the selection of replication origins and in the way in which these origins are licensed to operate. Moreover, these differences impart advantages to multicellular animals and plants that facilitate their development, such as better control over endoreduplication, flexibility in origin selection, and discrimination between quiescent and proliferative states.

Multilevel hysteresis modulation (MHM) for sliding mode controlled dual inverter PV systems.

In this article, proposes an adaptive multiband hysteresis modulation and its characterization based on a robust nonlinear sliding-mode control (RNSMC) strategy of a dual two-level inverter topologies for photovoltaic (PV) system subject to robust and fast-tracking characteristics and external disturbance rejection. Using the RNSMC based novel, vector control schemes are designed and implemented to improve its robustness and against the rejection of external disturbances. The control scheme ensures the active power is stable across the whole operation of the PV systems (standalone and grid-connected). The reachability and stability analysis are obtaining the robustness of the controller. Tsypkin's method is used to realize the switching frequency characterization. The development and implementation approach of the proposed control strategy is thoroughly detailed, and its effectiveness is validated on the simulation and real-time laboratory tests. Moreover, the proposed scheme is superior in keeping the enhanced efficiency, multilevel voltage waveforms, and total harmonic distortion (THD) reduction in current within the necessary standard limitations even under different operating conditions.

Regulated phosphorylation of a major UDP-glucuronosyltransferase isozyme by tyrosine kinases dictates endogenous substrate selection for detoxification.

Whereas UDP-glucuronosyltransferase-2B7 is widely distributed in different tissues, it preferentially detoxifies genotoxic 4-OH-estradiol and 4-OH-estrone (4-OHE(1)) with barely detectable 17ß-estradiol (E(2)) conversion following expression in COS-1 cells. Consistent with the UDP-glucuronosyltransferase requirement for regulated phosphorylation, we discovered that 2B7 requires Src-dependent tyrosine phosphorylation. Y236F-2B7 and Y438F-2B7 mutants were null and 90% inactive, respectively, when expressed in COS-1. We demonstrated that 2B7 incorporated immunoprecipitable [(33)P]orthophosphate and that 2B7His, previously expressed in SYF-(Src,Yes,Fyn)(-/-) cells, was Src-supported or phosphorylated under in vitro conditions. Unexpectedly, 2B7 expressed in SYF(-/-) and SYF(+/-) cells metabolized 4-OHE(1) at 10- and 3-fold higher rates, respectively, than that expressed in COS-1, and similar analysis showed that E(2) metabolism was 16- and 9-fold higher than in COS-1. Because anti-Tyr(P)-438-2B7 detected Tyr(P)-438-2B7 in each cell line, results indicated that unidentified tyrosine kinase(s) (TKs) phosphorylated 2B7 in SYF(-/-). 2B7-transfected COS-1 treated with increasing concentrations of the Src-specific inhibitor PP2 down-regulated 4-OHE(1) glucuronidation reaching 60% maximum while simultaneously increasing E(2) metabolism linearly. This finding indicated that increasing PP2 inhibition of Src allows increasing E(2) metabolism caused by 2B7 phosphorylation by unidentified TK(s). Importantly, 2B7 expressed in SYF(-/-) is more competent at metabolizing E(2) in cellulo than 2B7 expressed in COS-1. To confirm Src-controlled 2B7 prevents toxicity, we showed that 2B7-transfected COS-1 efficiently protected against 4-OH-E(1)-mediated depurination. Finally, our results indicate that Src-dependent phosphorylation of 2B7 allows metabolism of 4-OHE(1), but not E(2), in COS-1, whereas non-Src-phosphorylated 2B7 metabolizes both chemicals. Importantly, we determined that 2B7 substrate selection is not fixed but varies depending upon the TK(s) that carry out its required phosphorylation.

BRCA1-mediated signaling pathways in ovarian carcinogenesis.

The link between loss or defect in functional BRCA1 and predisposition for development of ovarian and breast cancer is well established. Germ-line mutations in BRCA1 are responsible for both hereditary breast and ovarian cancer, which is around 5-10% for all breast and 10-15% of all ovarian cancer cases. However, majority of cases of ovarian cancer are sporadic in nature. The inactivation of cellular BRCA1 due to mutations or loss of heterozygosity is one of the most commonly observed events in such cases. Complement-resistant retroviral BRCA1 vector, MFG-BRCA1, is the only approved gene therapy for ovarian cancer patients by the Federal and Drug Administration. Given the limited available information, there is a need to evaluate the effects of BRCA1 on the global gene expression pattern for better understanding the etiology of the disease. Here, we use Ingenuity Pathway Knowledge Base to examine the differential pattern of global gene expression due to stable expression of BRCA1 in the ovarian cancer cell line, SKOV3. The functional analysis detected at least five major pathways that were significantly (p < 0.05) altered. These include: cell to cell signaling and interaction, cellular function and maintenance, cellular growth and proliferation, cell cycle and DNA replication, and recombination repair. In addition, we were able to detect several biologically relevant genes that are central for various signaling networks involved in cellular homeostasis; TGF-ß1, TP53, c-MYC, NF-κB and TNF-α. This report provides a comprehensive rationale for tumor suppressor function(s) of BRCA1 in ovarian carcinogenesis.

Antioxidative effects of the spice cardamom against non-melanoma skin cancer by modulating nuclear factor erythroid-2-related factor 2 and NF-κB signalling pathways.

The role of dietary factors in inhibiting or delaying the development of non-melanoma skin cancer (NMSC) has been investigated for many years. Cardamom, which is a dietary phytoproduct, has been commonly used in cuisines for flavour and has numerous health benefits, such as improving digestion and stimulating metabolism and having antitumorigenic effects. We have investigated the efficacy of dietary cardamom against 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin papillomatogenesis in Swiss albino mice that closely resembles human NMSC. Mice were grouped into normal wild type (untreated), vehicle-treated (acetone), carcinogen-treated (DMBA), and DMBA and cardamom-treated (DMBA+CARD) to delineate the role of cardamom against DMBA-induced papillomatogenesis. Oral administration of cardamom to DMBA-treated mice up-regulated the phase II detoxification enzymes, such as glutathione-S-transferase and glutathione peroxidase, probably via activation of nuclear factor erythroid-2-related factor 2 transcription factor in 'DMBA+CARD' mice. Furthermore, reduced glutathione, glutathione reductase, superoxide dismutase and catalase were also up-regulated by cardamom in the same 'DMBA+CARD' group of mice compared with DMBA-treated mice. Cardamom ingestion in DMBA-treated mice blocked NF-κB activation and down-regulated cyclo-oxygenase-2 expression. As a consequence, both the size and the number of skin papillomas generated on the skin due to the DMBA treatment were reduced in the 'DMBA+CARD' group. Thus, the results from the present study suggest that cardamom has a potential to become a pivotal chemopreventive agent to prevent papillomagenesis on the skin.

Transcriptional regulation of the base excision repair pathway by BRCA1.

Inactivation of the breast cancer susceptibility gene BRCA1 plays a significant role in the development of a subset of breast cancers, although the major tumor suppressor function of this gene remains unclear. Previously, we showed that BRCA1 induces antioxidant-response gene expression and protects cells against oxidative stress. We now report that BRCA1 stimulates the base excision repair pathway, a major mechanism for the repair of oxidized DNA, by stimulating the activity of key base excision repair (BER) enzymes, including 8-oxoguanine DNA glycosylase (OGG1), the DNA glycosylase NTH1, and the apurinic endonuclease redox factor 1/apurinic endonuclease 1 (REF1/APE1), in human breast carcinoma cells. The increase in BER enzyme activity appears to be due, primarily, to an increase in enzyme expression. The ability of BRCA1 to stimulate the expression of the three BER enzymes and to enhance NTH1 promoter activity was dependent upon the octamer-binding transcription factor OCT1. Finally, we found that OGG1, NTH1, and REF1/APE1 each contribute to the BRCA1 protection against oxidative stress due to hydrogen peroxide and that hydrogen peroxide stimulates the expression of BRCA1 and the three BER enzymes. These findings identify a novel mechanism through which BRCA1 may regulate the repair of oxidative DNA damage.

BRCA1 localization to the telomere and its loss from the telomere in response to DNA damage.

BRCA1, a tumor suppressor, participates in DNA damage signaling and repair. Previously, we showed that BRCA1 overexpression caused inhibition of telomerase activity and telomere shortening in breast and prostate cancer cells. We now report that BRCA1 knockdown causes increased telomerase reverse transcriptase expression, telomerase activity, and telomere length; but studies utilizing a combination of BRCA1 and telomerase reverse transcriptase small interfering RNAs suggest that BRCA1 also regulates telomere length independently of telomerase. Using telomeric chromatin immunoprecipitation assays, we detected BRCA1 at the telomere and demonstrated time-dependent loss of BRCA1 from the telomere following DNA damage. Further studies suggest that BRCA1 interacts with TRF1 and TRF2 in a DNA-dependent manner and that some of the nuclear BRCA1 colocalizes with TRF1/2. Our findings further suggest that Rad50 is required to localize BRCA1 at the telomere and that the association of BRCA1 with Rad50 does not require DNA. Finally, we found that BRCA1 regulates the length of the 3' G-rich overhang in a manner that is dependent upon Rad50. Our findings suggest that BRCA1 is recruited to the telomere in a Rad50-dependent manner and that BRCA1 may regulate telomere length and stability, in part through its presence at the telomere.

Effect of garlic on lipid peroxidation and antioxidation enzymes in DMBA-induced skin carcinoma.

OBJECTIVE: Naturally occurring phytochemicals display an active cancer preventive strategy to inhibit, delay, or reverse human carcinogenesis. Studies have indicated that certain daily-consumed dietary phytochemicals have cancer protective effects mediated by carcinogens. Lipid peroxide plays a detrimental role in all cancers including skin carcinogenesis. Garlic, a phytochemical, has acquired a special position in the folklore of many cultures as a formidable prophylactic and therapeutic medicinal agent. In this report, we pursue the chemopreventive effect of aqueous garlic on skin carcinogenesis. METHODS: "Swiss albino mice" were divided into five groups depending on the combination of skin cancer-inducing 7,12-dimethylbenz[a]anthracene and garlic treatments. Histology of the affected skin and biochemical assays for lipid peroxide, catalase, superoxide dismutase, glutathione-S-transferase, and glutathione peroxidase were performed to demonstrate the effect of garlic in mice. Immunoblotting was performed with cyclo-oxygenase-2, p53, and caspase-3 to demonstrate expressions of the respective proteins in skin lysates. RESULTS: Garlic extracts inhibited the oxidative modification of lipids, thus protecting cells from injury by the oxidized molecules. The best chemopreventive action of garlic was observed in mice in which garlic treatment was performed before and after the induction of skin carcinogenesis. Garlic ingestion delayed formation of skin papillomas in animals and simultaneously decreased the size and number of papillomas, which was also reflected in the skin histology of the mice treated. CONCLUSION: The protective effects against skin cancer elicited by garlic in mice are believed to be due at least in part to the induction cellular defense systems.

Tgf-Beta signaling in development.

The transforming growth factor-beta (TGF-beta) superfamily comprises nearly 30 growth and differentiation factors that include TGF-betas, activins, inhibins, and bone morphogenetic proteins (BMPs). Multiple members of the TGF-beta superfamily serve key roles in stem cell fate commitment. The various members of the family can exhibit disparate roles in regulating the biology of embryonic stem (ES) cells and tumor suppression. For example, TGF-beta inhibits proliferation of multipotent hematopoietic progenitors, promotes lineage commitment of neural precursors, and suppresses epithelial tumors. BMPs block neural differentiation of mouse and human ES cells, contribute to self-renewal of mouse ES cells, and also suppress tumorigenesis. ES cells and tumors may be exposed to multiple TGF-beta members, and it is likely that the combination of growth factors and cross-talk among the intracellular signaling pathways is what precisely defines stem cell fate commitment. This Connections Map Pathway in the Database of Cell Signaling integrates signaling not only from TGF-beta and BMP but also from the ligands nodal and activin, and describes the role of the signaling pathways activated by these ligands in mammalian development. Much of the evidence for the connections shown comes from studies on mouse and human ES cells or mouse knockouts. This pathway is important for understanding not only stem cell biology, but also the molecular effectors of TGF-beta and BMP signaling that may contribute to cancer suppression or progression and thus are potential targets for therapeutic intervention.

Regional FMRP deficits and large repeat expansions into the full mutation range in a new Fragile X premutation mouse model.

Carriers of FMR1 alleles with 55-200 repeats in the 5' UTR are at risk for Fragile X associated tremor and ataxia syndrome. The cause of the neuropathology is unknown but is thought to be RNA-mediated. Maternally transmitted premutation alleles are also at risk of expansion of the repeat tract into the "full mutation" range (>200 repeats). The mechanism responsible for expansion is unknown. Full mutation alleles produce reduced amounts of the FMR1 gene product, FMRP, which leads to Fragile X mental retardation syndrome. We have developed a murine model for Fragile X premutation carriers that recapitulates key features seen in humans including a direct relationship between repeat number and Fmr1 mRNA levels, an inverse relationship with FMRP levels and Purkinje cell dropout that have not been seen in a previously described knock-in mouse model. In addition, these mice also show a differential deficit of FMRP in different parts of the brain that might account for symptoms of the full mutation that are seen in premutation carriers. As in humans, repeat instability is high with expansions predominating and, for the first time in a mouse model, large expansions into the full mutation range are seen that occur within a single generation. Thus, contrary to what was previously thought, mice may be good models not only for the symptoms seen in human carriers of FMR1 premutation alleles but also for understanding the mechanism responsible for repeat expansion, a phenomenon that is responsible for a number of neurological and neurodevelopmental disorders.

The fragile X syndrome repeats form RNA hairpins that do not activate the interferon-inducible protein kinase, PKR, but are cut by Dicer.

We show here that under physiologically reasonable conditions, CGG repeats in RNA readily form hairpins. In contrast to its DNA counterpart that forms a complex mixture of hairpins and tetraplexes, r(CGG)22 forms a single stable hairpin with no evidence for any other folded structure even at low pH. RNA with the sequence (CGG)9AGG (CGG)12AGG(CGG)97, found in a fragile X syndrome pre-mutation allele, forms a number of different hairpins. The most prominent hairpin forms in the 3' part of the repeat and involves the 97 uninterrupted CGG repeats. In contrast to the CUG-RNA hairpins formed by myotonic dystrophy type 1 repeats, we found no evidence that CGG-RNA hairpins activate PKR, the interferon-inducible protein kinase that is activated by a wide range of double-stranded RNAs. However, we do show that the CGG-RNA is digested, albeit inefficiently, by the human Dicer enzyme, a step central to the RNA interference effect on gene expression. These data provide clues to the basis of the toxic effect of CGG-RNA that is thought to occur in fragile X pre-mutation carriers. In addition, RNA hairpins may also account for the stalling of the 40S ribosomal subunit that is thought to contribute to the translation deficit in fragile X pre-mutation and full mutation alleles.

A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells.

DNA-protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polß, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA-protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.

Photolysis of fluchloralin in aqueous methanol.

The photodegradation of fluchloralin by UV irradiation or sunlight in aqueous methanolic solution has been examined. In the presence of titanium dioxide five photoproducts were obtained, but only four in its absence. One photoproduct, 2, 2'-azoxy-bis(alpha,alpha,alpha-trifluoro-6-nitro-p-toluidine) is reported for the first time as a metabolite of fluchloralin. In natural sunlight the rate of degradation was higher than in UV light and titanium dioxide had almost no effect on the rate of degradation.

Dodecamer d-AGATCTAGATCT and a homologous hairpin form triplex in the presence of peptide REWER.

We have designed a dodecamer d-AGATCTAGATCT (RY12) with alternate oligopurines and oligopyrimidines tracts and its homologous 28 bp hairpin oligomer (RY28) that forms a triple helix only in the presence of a pentapeptide REWER. An intermolecular triplex is formed by the single strand invasion of the RY28 duplex by RY12 in the presence of REWER. 5'- oligopurine end of RY12 binds to oligopurine sequence of RY28 in a parallel orientation and its oligopyrimidine stretch then changes strand and adopts an antiparallel orientation with the other strand of the duplex. Evidence for the formation of the triplex come from our studies of the UV melting curves, UV mixing curves, gel retardation assay, and chemical sequencing of 1∶1 mixture of dodecamer and hairpin oligonucleotides in the presence and absence of the peptide REWER. RY12 exists as a duplex that melts at 35°C. The hairpin (RY28) melts at 68°C. 1∶1 mixture of RY12 and RY28 in the absence of REWER gives a biphasic transition curve with thermodynamic properties corresponding to those of the melting of the duplex of RY12 and the hairpin RY28. However, the melting curve of this mixture is triphasic in the presence of the REWER; the thermodynamic parameters associated with the first phase (melting of the duplex of RY12), second phase (melting of the triplex) and the third phase (melting of the hairpin) show dependence on the molar ratio of peptide to oligonucleotides. Under appropriate conditions, gel retardation assay showed a shifted band that corresponds to a possible triplex. Chemical sequencing of KMnO4 and DEPC treated mixture of RY12, RY28 and REWER revealed the footprint of triplex.

LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy.

Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.

Central Role of Ubiquitination in Genome Maintenance: DNA Replication and Damage Repair.

Faithful transmission of genetic information through generations ensures genomic stability and integrity. However, genetic alterations occur every now and then during the course of genome duplication. In order to repair these genetic defects and lesions, nature has devised several repair pathways which function promptly to prevent the cell from accumulating permanent mutations. These repair mechanisms seem to be significantly impacted by posttranslational modifications of proteins like phosphorylation and ubiquitination. Protein ubiquitination is emerging as a critical regulatory mechanism of DNA damage response. Non-proteolytic, proteasome-independent functions of ubiquitin involving monoubiquitination and polyubiquitination of DNA repair proteins contribute significantly to the signaling of DNA repair pathways. In this paper, we will particularly highlight the work on ubiquitin-mediated signaling in the repair processes involving the Fanconi anemia pathway, translesional synthesis, nucleotide excision repair, and repair of double-strand breaks. We will also discuss the role of ubiquitin ligases in regulating checkpoint mechanisms, the role of deubiquitinating enzymes, and the growing possibilities of therapeutic intervention in this ubiquitin-conjugation system.

BRCA1 regulates follistatin function in ovarian cancer and human ovarian surface epithelial cells.

Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells.

Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction.

BACKGROUND: Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details. RESULTS: In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg2+/Mn2+, and that neither Zn2+ or nor Ca2+ can activate hTDP2. Mg2+ also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg2+ controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg2+ concentration above which it is inhibitory for hTDP2 activity. CONCLUSION: These results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.