Characterization of an intracellular aspartic protease (PsAPA) from Penicillium sp. XT7 and its application in collagen extraction.

An intracellular aspartic protease, PsAPA, was identified from Penicillium sp. XT7. This protease was belonged to penicillopepsin and was expressed in Pichia pastoris GS115. The recombinant PsAPA had a specific activity of 4289.7 ± 261.7 U/mg. The pH and temperature maxima of the enzyme were 3.0 and 30 °C, respectively. The PsAPA was stable in the pH range from 3.0 to 6.0 and was completely inactivated after incubation at 50 °C for 15 min. Presence of Mn2+ and Cu2+ increased the proteolytic activity and ß-mercaptoethanol and SDS showed inhibitory effects, whereas 0.05 M pepstatin A strongly inhibited it. PsAPA could effectively hydrolyze animal proteins, including myoglobin, and hemoglobin but not collagens. PsAPA increased the yield of collagen extraction compared to the acid extraction method. The above properties show that the novel low-temperature acidic protease, PsAPA, is comparable to commercial proteases (porcine pepsin) and has great potential for collagen extraction.

Apoplastic Proteases: Powerful Weapons against Pathogen Infection in Plants.

Plants associate with diverse microbes that exert beneficial, neutral, or pathogenic effects inside the host. During the initial stages of invasion, the plant apoplast constitutes a hospitable environment for invading microbes, providing both water and nutrients. In response to microbial infection, a number of secreted proteins from host cells accumulate in the apoplastic space, which is related to microbial association or colonization processes. However, the molecular mechanisms underlying plant modulation of the apoplast environment and how plant-secreted proteases are involved in pathogen resistance are still poorly understood. Recently, several studies have reported the roles of apoplastic proteases in plant resistance against bacteria, fungi, and oomycetes. On the other hand, microbe-secreted proteins directly and/or indirectly inhibit host-derived apoplastic proteases to promote infection. These findings illustrate the importance of apoplastic proteases in plant-microbe interactions. Therefore, understanding the protease-mediated apoplastic battle between hosts and pathogens is of fundamental importance for understanding plant-pathogen interactions. Here, we provide an overview of plant-microbe interactions in the apoplastic space. We define the apoplast, summarize the physical and chemical properties of these structures, and discuss the roles of plant apoplastic proteases and pathogen protease inhibitors in host-microbe interactions. Challenges and future perspectives for research into protease-mediated apoplastic interactions are discussed, which may facilitate the engineering of resistant crops.

Chinese Yellow Rice Wine Processing with Reduced Ethyl Carbamate Formation by Deleting Transcriptional Regulator Dal80p in Saccharomyces cerevisiae.

Ethyl carbamate (EC) is a potential carcinogen that forms spontaneously during Chinese rice wine fermentation. The primary precursor for EC formation is urea, which originates from both external sources and arginine degradation. Urea degradation is suppressed by nitrogen catabolite repression (NCR) in Saccharomyces cerevisiae. The regulation of NCR is mediated by two positive regulators (Gln3p, Gat1p/Nil1p) and two negative regulators (Dal80p/Uga43p, Deh1p/Nil2p/GZF3p). DAL80 revealed higher transcriptional level when yeast cells were cultivated under nitrogen-limited conditions. In this study, when DAL80-deleted yeast cells were compared to wild-type BY4741 cells, less urea was accumulated, and genes involved in urea utilization were up-regulated. Furthermore, Chinese rice wine fermentation was conducted using dal80Δ cells; the concentrations of urea and EC were both reduced when compared to the BY4741 and traditional fermentation starter. The findings of this work indicated Dal80p is involved in EC formation possibly through regulating urea metabolism and may be used as the potential target for EC reduction.

The impact of intracellular aminopeptidase on C2C12 myoblast proliferation and differentiation.

The ubiquitin-proteasome pathway is essential for skeletal muscle growth and development. Proteasomes generate oligopeptides in the cytoplasm, and these peptides are considered to be rapidly degraded to amino acids by several intracellular aminopeptidases. However, the role of intracellular aminopeptidases in muscle growth remains unknown. In this study, therefore, we investigated the role of intracellular aminopeptidases in C2C12 myoblast proliferation and differentiation. Inhibition of intracellular aminopeptidases by Bestatin methyl ester (Bes-ME) decreased leucine and alanine aminopeptidase activity, and impaired proliferation and differentiation of C2C12 myoblasts. Furthermore, we observed that the inhibition of intracellular aminopeptidases reduced intracellular levels of amino acid and ATP level, and suppressed the phosphorylation of the mTOR pathway. These results suggested that intracellular aminopeptidases affect C2C12 myoblast proliferation and differentiation via mTOR pathway; however, further studies are required to clarify the role of aminopeptidase in skeletal muscle.

Interplay between compartmentalized NAD+ synthesis and consumption: a focus on the PARP family.

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor for redox enzymes, but also moonlights as a substrate for signaling enzymes. When used as a substrate by signaling enzymes, it is consumed, necessitating the recycling of NAD+ consumption products (i.e., nicotinamide) via a salvage pathway in order to maintain NAD+ homeostasis. A major family of NAD+ consumers in mammalian cells are poly-ADP-ribose-polymerases (PARPs). PARPs comprise a family of 17 enzymes in humans, 16 of which catalyze the transfer of ADP-ribose from NAD+ to macromolecular targets (namely, proteins, but also DNA and RNA). Because PARPs and the NAD+ biosynthetic enzymes are subcellularly localized, an emerging concept is that the activity of PARPs and other NAD+ consumers are regulated in a compartmentalized manner. In this review, I discuss NAD+ metabolism, how different subcellular pools of NAD+ are established and regulated, and how free NAD+ levels can control signaling by PARPs and redox metabolism.

ß-Cyclodextrin modified silver nanoclusters for highly sensitive fluorescence sensing and bioimaging of intracellular alkaline phosphatase.

In this paper, silver nanoclusters (AgNCs) were synthesized by one facile, low-cost and green method with histidine acting as the stabilizer and ascorbic acid performing as reducing agent. AgNCs displayed excellent fluorescence property and good biocompatibility and were further functionalized by ß-cyclodextrin (CD-AgNCs) for the highly sensitive detection of alkaline phosphatase (ALP) activity. ALP catalyzed the dephosphorylation of p-nitrophenylphosphate (NPP) to produce p-nitrophenol (NP), and NP can effectively quench the fluorescence of CD-AgNCs via the inner filter effect (IFE) due to the UV-vis absorption spectrum of NP had considerable overlap with the excitation spectrum of CD-AgNCs. Benefiting from the IFE and the host-guest interaction between CD and NP, this CD-AgNCs based system for ALP activity analysis displayed a good linear range of 0.02-10.0 U L-1 and the detection limit as low as 0.0046 U L-1. In addition, this proposed strategy also showed satisfactory performance for ALP analysis in human serum samples and bioimaging of intracellular ALP in living cells, indicating the great application prospect of this system for ALP activity analysis in medical related researches.

Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis.

Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicity. Here, we show that a unique domain within the MLCK splice variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identify a domain-binding small molecule (divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, tumor necrosis factor (TNF)-induced MLCK1 recruitment as well as downstream myosin light chain (MLC) phosphorylation, barrier loss, and diarrhea in vitro and in vivo. Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.

A novel ß-xylosidase from Anoxybacillus sp. 3M towards an improved agro-industrial residues saccharification.

An intracellular ß-xylosidase (AbXyl), from the thermoalkaline Anoxybacillus sp. 3M, was purified and characterized. The homodimeric enzyme (140 kDa) was optimally active at 65 °C and pH 5.5, exhibited half life of 10 h at 60 °C, 78 and 88% residual activity after 24 h, at pH 4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+ inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by ß-mercaptoethanol. In the presence of p-nitrophenyl-ß-d-xylopyranoside, AbXyl exhibited Km of 0.19 mM, Kcat of 453.29 s-1, Kcat Km-1 of 2322 s-1 mM and was moderately influenced by xylose (Ki 21.25 mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars.

Subcellular localization of sterol biosynthesis enzymes.

Cholesterol synthesis is a complex, coordinated process involving a series of enzymes. As of today, our understanding of subcellular localization of cholesterol biosynthesis enzymes is far from complete. Considering the complexity and intricacies of this pathway and the importance of functions of DHCR7, DHCR24 and EBP enzymes for human health, we undertook a study to determine their subcellular localization and co-localization. Using expression constructs and antibody staining in cell cultures and transgenic mice, we found that all three enzymes are expressed in ER and nuclear envelope. However, their co-localization was considerably different across the cellular compartments. Furthermore, we observed that in the absence of DHCR7 protein, DHCR24 shows a compensatory upregulation in a Dhcr7-/- transgenic mouse model. The overall findings suggest that the sterol biosynthesis enzymes might not always work in a same functional complex, but that they potentially have different, multifunctional roles that go beyond the sterol biosynthesis pathway. Furthermore, the newly uncovered compensatory mechanism between DHCR7 and DHCR24 could be of importance for designing medications that would improve cholesterol production in patients with desmosterolosis and Smith-Lemli-Opitz syndrome.

Molecular imaging of telomerase and the enzyme activity-triggered drug release by using a conformation-switchable nanoprobe in cancerous cells.

So far, the development of a unique strategy for specific biomolecules activity monitoring and precise drugs release in cancerous cells is still challenging. Here, we designed a conformation-switchable smart nanoprobe to monitor telomerase activity and to enable activity-triggered drug release in cancerous cells. The straightforward nanoprobe contained a gold nanoparticle (AuNP) core and a dense layer of 5-carboxyfluorescein (FAM)-labeled hairpin DNA shell. The 3' region of hairpin DNA sequence could function as the telomerase primer to be elongated in the presence of telomerase, resulting in the conformational switch of hairpin DNA. As a result, the FAM fluorescence was activated and the anticancer drug doxorubicin (Dox) molecules which intercalated into the stem region of the hairpin DNA sequence were released into cancerous cells simultaneously. The smart method could specifically distinguish cancerous cells from normal cells based on telomerase activity. It also showed a good performance for monitoring telomerase activity in the cytoplasm by molecular imaging and precise release of Dox triggered by telomerase activity in cancerous cells. These advantages may offer a great potential of this method for monitoring telomerase activity in cancer progression and estimating therapeutic effect.

Tissue-specific and intracellular localization of indican synthase from Polygonum tinctorium.

The plant Polygonum tinctorium produces the secondary metabolite indican (indoxyl-ß-D-glucoside), a precursor of the blue dye indigo. P. tinctorium synthesizes indican through the actions of the UDP-glucosyltransferase (UGT), indican synthase. Herein, we partially purified an indican synthase from the leaves and subsequently performed peptide mass fingerprinting analysis. Consequently, we identified a fragment that was homologous to a UDP-glucosyltransferase 72B (UGT72B) family member. We named it PtIgs (P. tinctoriumindoxyl-ß-D-glucoside synthase) and obtained the full-length cDNA using rapid amplification of the cDNA ends. The primary structure of PtIGS, which PtIgs encoded, showed high identity with indican synthases (ItUGT1 and ItUGT2) from Indigofera tinctoria (Inoue et al., 2017). Moreover, in expression analyses of P. tinctorium, PtIGS mRNA was virtually found only in the leaves, was most highly expressed in the 1st leaves, and decreased with leaf age. Because PtIGS expression tended to reflect indican contents and synthesis activities, we concluded that PtIGS functions as an indican synthase in plant cells. To examine intracellular localization of PtIGS, crude leaf extracts were separated into cytosol and microsome fractions, and found PtIGS in the cytosol and in microsome fractions. Furthermore, microsomal PtIGS was soluble in the presence of detergents and urea and was strongly associated with membranes. Finally, we confirmed endoplasmic reticulum (ER) membrane localization of PtIGS using ultracentrifugation with a sucrose density gradient. These data suggest that PtIGS interacts with some kind of proteins on ER membranes to certainly carry out a delivery of substrate.

Peptide-templated multifunctional nanoprobe for feasible electrochemical assay of intracellular kinase.

Protein kinases play a critical role in regulation of intracellular signal transduction, whose aberrant expression is closely associated with various dangerous human diseases. In this paper, we propose a feasible electrochemical assay of intracellular kinase by incorporating peptide nanoprobe-assisted signal labeling and signal amplification. Protein kinase A (PKA)-specific peptide P1 is self-assembled on the surface of a gold electrode, serine of which could be phosphorylated with catalysis of PKA in the presence of adenosine-5'-triphosphate (ATP). Another artificial peptide P2 contains a short template for preparation of copper nanoparticles-based nanoprobe (P2-CuNPs) and provides arginine residues for specific recognition of phosphorylation site. After PKA-catalyzed phosphorylation, phosphorylated P1 specially binds with P2-CuNPs through ultra-stable phosphate-guanidine interaction, and thus results in amplified electrochemical response from surface-attached CuNPs. Our method demonstrates a satisfactory sensitivity toward PKA detection with a detection limit of 0.0019 U/mL, which is also successfully applied in intracellular PKA assay and inhibitory study with high specificity comparable to ELISA. Therefore, the facile method suggests a promising potential use in kinase-related biochemical fundamental research, disease diagnosis and drug discovery in the future.

Teicoplanin-functionalized magnetic beads for detection of Staphylococcus aureus via inhibition of the luminol chemiluminescence by intracellular catalase.

An affinity-based protocol is described for the detection of Staphylococcus aureus (S. aureus). It is utilizing teicoplanin-functionalized magnetic beads as carriers. Teicoplanin, which binds to the walls of cells of S. aureus via five hydrogen bonds, acts as the recognition agent. Captured S. aureus is magnetically separated from the sample matrix and then specifically lysed by lysostaphin which cleaves the cross-linking pentaglycine bridges of peptidoglycan in the cell wall. Lastly, S. aureus is quantified via the inhibitory effect of released intracellular catalase on a chemiluminescent (CL) system composed of peroxidase, luminol, H2O2 and p-iodophenol because catalase decomposes H2O2. S. aureus can be detected with CL response in the 140 to 1.4 × 107 CFU·mL-1 concentration range and a detection limit as low as 47 CFU·mL-1 at a signal-to-noise ratio of 3. The method was evaluated by analyzing spiked samples including milk, human urine and saline injection solutions. The reliability was demonstrated by a recovery test and by comparison with a conventional plate counting method. Graphical abstract An antibiotic-affinity protocol is developed to detect Staphylococcus aureus (S. aureus) by utilizing teicoplanin-functionalized magnetic beads (Teic-MBs) as carriers. S. aureus can be quantified by measuring the inhibition of luminol chemiluminescence (CL) signal by intracellular catalase.

Subcellular localization defines modification and production of Δ9-tetrahydrocannabinolic acid synthase in transiently transformed Nicotiana benthamiana.

OBJECTIVE: Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis. RESULTS: Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 ± 12 fkat g FW-1 activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat g FW-1 activity towards CBCA production). CONCLUSION: Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.

Human hyaluronic acid synthase-1 promotes malignant transformation via epithelial-to-mesenchymal transition, micronucleation and centrosome abnormalities.

BACKGROUND: Human hyaluronic acid (HA) molecules are synthesized by three membrane spanning Hyaluronic Acid Synthases (HAS1, HAS2 and HAS3). Of the three, HAS1 is found to be localized more into the cytoplasmic space where it synthesizes intracellular HA. HA is a ubiquitous glycosaminoglycan, mainly present in the extracellular matrix (ECM) and on the cell surface, but are also detected intracellularly. Accumulation of HA in cancer cells, the cancer-surrounding stroma, and ECM is generally considered an independent prognostic factors for patients. Higher HA production also correlates with higher tumor grade and more genetic heterogeneity in multiple cancer types which is known to contribute to drug resistance and results in treatment failure. Tumor heterogeneity and intra-tumor clonal diversity are major challenges for diagnosis and treatment. Identification of the driver pathway(s) that initiate genomic instability, tumor heterogeneity and subsequent phenotypic/clinical manifestations, are fundamental for the diagnosis and treatment of cancer. Thus far, no evidence was shown to correlate intracellular HA status (produced by HAS1) and the generation of genetic diversity in tumors. METHODS: We tested different cell lines engineered to induce HAS1 expression. We measured the epithelial traits, centrosomal abnormalities, micronucleation and polynucleation of those HAS1-expressing cells. We performed real-time PCR, 3D cell culture assay, confocal microscopy, immunoblots and HA-capture methods. RESULTS: Our results demonstrate that overexpression of HAS1 induces loss of epithelial traits, increases centrosomal abnormalities, micronucleation and polynucleation, which together indicate manifestation of malignant transformation, intratumoral genetic heterogeneity, and possibly create suitable niche for cancer stem cells generation. CONCLUSIONS: The intracellular HA produced by HAS1 can aggravate genomic instability and intratumor heterogeneity, pointing to a fundamental role of intracellular HA in cancer initiation and progression.

Lipoquality control by phospholipase A2 enzymes.

The phospholipase A2 (PLA2) family comprises a group of lipolytic enzymes that typically hydrolyze the sn-2 position of glycerophospholipids to give rise to fatty acids and lysophospholipids. The mammalian genome encodes more than 50 PLA2s or related enzymes, which are classified into several subfamilies on the basis of their structures and functions. From a general viewpoint, the PLA2 family has mainly been implicated in signal transduction, producing bioactive lipid mediators derived from fatty acids and lysophospholipids. Recent evidence indicates that PLA2s also contribute to phospholipid remodeling for membrane homeostasis or energy production for fatty acid ß-oxidation. Accordingly, PLA2 enzymes can be regarded as one of the key regulators of the quality of lipids, which I herein refer to as lipoquality. Disturbance of PLA2-regulated lipoquality hampers tissue and cellular homeostasis and can be linked to various diseases. Here I overview the current state of understanding of the classification, enzymatic properties, and physiological functions of the PLA2 family.

Enzymatic mechanisms of biological magnetic sensitivity.

Primary biological magnetoreceptors in living organisms is one of the main research problems in magnetobiology. Intracellular enzymatic reactions accompanied by electron transfer have been shown to be receptors of magnetic fields, and spin-dependent ion-radical processes can be a universal mechanism of biological magnetosensitivity. Magnetic interactions in intermediate ion-radical pairs, such as Zeeman and hyperfine (HFI) interactions, in accordance with proposed strict quantum mechanical theory, can determine magnetic-field dependencies of reactions that produce biologically important molecules needed for cell growth. Hyperfine interactions of electrons with nuclear magnetic moments of magnetic isotopes can explain the most important part of biomagnetic sensitivities in a weak magnetic field comparable to the Earth's magnetic field. The theoretical results mean that magnetic-field dependencies of enzymatic reaction rates in a weak magnetic field that can be independent of HFI constant a, if H << a, and are determined by the rate constant of chemical transformations in the enzyme active site. Both Zeeman and HFI interactions predict strong magnetic-field dependence in weak magnetic fields and magnetic-field independence of enzymatic reaction rate constants in strong magnetic fields. The theoretical results can explain the magnetic sensitivity of E. coli cell and demonstrate that intracellular enzymatic reactions are primary magnetoreceptors in living organisms. Bioelectromagnetics. 38:511-521, 2017. © 2017 Wiley Periodicals, Inc.

Enzyme activity and expression pattern of intra- and extracellular chitinase and ß-1,3-glucanase of Wickerhamomyces anomalus EG2 using glycol chitin and glucan-containing high polymer complex.

We investigated cell growth and activity of intra- and extracellular chitinase, ß-1,3-glucanase, and chitin deacetylase with SDS-PAGE by incubating W. anomalus EG2 in PDB and YPD media for 24h in presence of different concentrations (0%, 0.1%, 0.3%, and 0.5%) of colloidal chitin. Maximum cell growth was observed in both PDB and YPD media without colloidal chitin. In the absence of colloidal chitin, maximum extracellular ß-1,3-glucanase activity of 32.96 and 47.28 units/mL was reported at 18h in PDB medium and 6h in YPD medium, respectively. In addition, extracellular chitinase was unaffected by various concentrations of carboxymethyl chitin in both PDB and YPD media. In the absence of colloidal chitin, maximum intracellular chitinase activity was indicated to be 9.82 and 9.86 units/mg protein in PDB and YPD media, respectively. Maximum intracellular ß-1,3-glucanase activity reported was 17.34 units/mg protein in PDB medium containing 0.5% colloidal chitin and 15.0 units/mg protein in YPD medium containing 0.3% colloidal chitin. Five major isozymes, GN1, GN2, GN3, GN4, and GN5, of intracellular ß-1,3-glucanase were detected with glucan-containing high polymer complex as a substrate with or without colloidal chitin.

Science of intracrinology in postmenopausal women.

OBJECTIVE: To illustrate the marked differences between classical endocrinology that distributes hormones to all tissues of the body through the bloodstream and the science of intracrinology, whereby each cell of each peripheral tissue makes a small and appropriate amount of estrogens and androgens from the inactive precursor dehydroepiandrosterone (DHEA), DHEA being mainly of adrenal origin. Because only the inactivated sex steroids are released in the blood, influence in the other tissues is avoided. METHODS: Molecular biology has been used for the identification/characterization of the steroid-forming and steroid-inactivating enzymes, whereas steroids have been measured by mass spectrometry-based assays validated according to the US Food and Drug Administration guidelines. RESULTS: Evolution over 500 million years has engineered the expression of about 30 steroid-forming enzymes specific for each peripheral tissue. These tissue-specific enzymes transform DHEA into the appropriate small amounts of estrogens and androgens for a strictly intracellular and local action. Humans, contrary to species below primates, also possess intracellular steroid-inactivating enzymes, especially glucuronyl transferases and sulfotransferases, which inactivate the estrogens and androgens at their local site of formation, thus preventing the release of a biologically significant amount of estradiol (E2) and testosterone in the circulation. Since DHEA becomes the unique source of sex steroids after menopause, serum E2 and testosterone are thus maintained at low biologically inactive concentrations with no activity outside the cells of origin. DHEA secretion, unfortunately, starts decreasing at about the age of 30 at various rates in different women. Moreover, there is no feedback mechanism to increase DHEA secretion when the concentration of serum DHEA decreases. Considering this mechanism is unique to the human, it seems logical to replace DHEA locally in women suffering from vulvovaginal atrophy (genitourinary syndrome of menopause). The clinical data obtained using a small dose of intravaginal DHEA (prasterone) confirm the mechanisms of intracrinology mentioned above which avoid biologically significant changes in serum E2 and testosterone. CONCLUSIONS: The symptoms and signs of vulvovaginal atrophy (genitourinary syndrome of menopause) can be successfully treated by the intravaginal administration of DHEA without safety concerns. This strategy exclusively replaces in the vagina the missing cell-specific intracellular estrogens and androgens. This approach avoids systemic exposure and the potential risks of estrogen exposure for the tissues other than the vagina.