Effect of 17β-estradiol on adhesion of Mytilus galloprovincialis hemocytes to selected substrates. Role of alpha2 integrin subunit

The process of hemocyte adhesion to extracellular matrix (ECM) proteins plays a crucial role in cell immunity. In most of these interactions between ECM proteins and cells, integrins are involved. The results of the present study showed that incubation of Mytilus galloprovincialis hemocytes with 17β-estradiol caused significant increased adhesion of hemocytes to ECM proteins and specifically to laminin-1, collagen IV and oxidized collagen IV, in relation to control cells. The adhesion of hemocytes to oxidized collagen was significantly higher than to either collagen IV or to laminin-1. In accordance with this, inhibition of either NADPH oxidase or nitric oxide (NO) synthase attenuated 17β-estradiol effect on hemocyte adhesion, suggesting that the high levels of free radicals, produced after 17β-estradiol effect, could contribute to the high adhesion of hemocytes to laminin-1 and collagen IV. The implication of ROS was further confirmed by the use of the oxidant rotenone, which caused elevation of cell adhesion in relation to control and by the antioxidant NAC which attenuated 17β-estradiol effect. The mechanism of 17β-estradiol induced adhesion to laminin-1, collagen IV and oxidized collagen IV involves a large number of intracellular components, as Na+/H+ exchanger (NHE), all isoforms of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K) and c-jun N-terminal kinase (JNK) as well as alpha2 integrin subunit. Maintenance of high cyclic adenosine-3'-5'-monophosphate (cAMP) levels caused non significant higher adhesion of hemocytes to ECM proteins in relation to control cells. Our results showed that 17β-estradiol caused a significant increase in α? integrin subunit levels, which was reduced after inhibition of NHE, PI3K, PKC, NO synthase, NADPH oxidase and JNK. In addition, our results showed that apart from 17β-estradiol, high cAMP and high ROS levels caused significantly higher induction of α? integrin subunit levels in relation to control. Our results imply a potential involvement of cAMP in immune responses of Mytilus hemocytes, which needs further investigation.     

Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50?kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO₂ hydration reaction: k_cat =?4.1?×?10⁵ s^?1 and k_cat/K_m of 3.6?×?10⁷ M^?1 × s^?1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a K_I of 380?nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO₂.     

Effect of γ-hexachloran and sevin on the survival of the Black Sea mussel,Mytilus galloprovincialis Lam

Some observations on the effect of -hexachloran and sevin on the survival of two size groups of the Black Sea mussel, Mytilus galloprovincialis Lam. were made. The results of the present study are compared with those made earlier on the same species and on fishes.     

Effects of the “Aegean Sea” oil spill on biotransformation enzymes,oxidative stress and DNA-adducts in digestive gland of the mussel (Mytilus edulus L.)

Possible molecular biomarkers of impact by organic pollution on mussels were applied to samples from five sites along the Galician Coast, Spain, taken 6 months after the oil spill from the tanker “Aegean Sea.” Whole body aliphatic hydrocarbon concentrations were similar at all sites, but specific chemical ratios (resolved/unresolved hydrocarbons; carbon preference index; pristane/phytane) indicated a predominance of degraded petrogenic hydrocarbons nearer the oil spill. Levels of whole body polycyclic aromatic hydrocarbons (sum of 13 PAHs) increased steadily towards the oil spill, and were paralleled by increases in digestive gland levels of total cytochrome P-450, CYP1A-like protein and lipid peroxidation (corr. coeffs. with PAHs of 0.64–0.67). Differences were more marked in CYP1A-like protein than total cytochrome P450, indicating induction of specific P450 isoenzyme(s). No differences between sites were seen for benzo[a]pyrene hydroxylase, glutathione S-transferase, Superoxide dismutase and DT-diaphorase activities. Bulky, hydrophobic DNA-adducts were detected in digestive gland of mussels from industrial and urban sites, but not from the site nearest to the oil spill which had the highest tissue levels of PAHs. Overall the results indicate induction of cytochrome P450(s) and oxidative damage in mussel with oil exposure.     

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk—I. Purification and ion requirements

• 1.1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively.
• 2.2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn²⁺, Zn²⁺ and Mg²⁺. The K_m for Mg²⁺ is lower than the physiological concentration of cation in the tissue, whereas its K_m for Mn²⁺ and Zn²⁺ is greater than the respective in vivo concentrations.
• 3.3. The joint action of Mg²⁺ and Zn²⁺ increases the affinity of the enzyme for the substrate Fru-1,6-P₂, though V_max is reduced.
• 4.4. Na⁺ strongly inhibits the enzyme even at very low concentrations. K⁺ has no effect whatsoever.

     

Cloning,expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32?kDa. Moreover, the determined kinetic parameters for the CO₂ hydration reaction were k_cat?=??4.2?×?10⁵?s^?1 and k_cat/K_m of 3.5?×?10⁷?M^?1 ×s^?1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50?kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.     

Effect of 17β-estradiol on calcium response to phytohaemagglutinin in human lymphocytes

Pre-treatment of human lymphocytes with 17-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17-estradiol, since the isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5-androstan), have no effect. Since the effect of the 17-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.     

Effect of 17β-estradiol on cells stained for FSHβ and/or LHβ in the dog pituitary gland

Summary The effect of long-term treatment (52 weeks) with high doses of 17-estradiol (1.28 mg/kg/week intramuscularly) on gonadotrophs was studied in the pituitary gland of the beagle bitch. For immunochemical staining the immunoperoxidase technique and antisera to the specific beta () subunits of FSH and LH were employed. For control purposes antisera to the following hormones were also used: bovine TSH, canine GH, canine PRL and porcine ACTH¹. In the pars distalis and pars tuberalis of control bitches, in addition to the cells which react solely with antisera to either LH or FSH, most cells were reactive to both antisera. The cells stained for FSH were less numerous than those shown to contain LH. TSH, PRL, GH and ACTH/MSH were localized in distinctly different cell types in the pars distalis of all control animals. In the treated bitches, almost complete regression of cells classically identified as gonadotrophs and stained for LH was observed. On the other hand, using the antiserum to FSH, selective immunochemical staining was localized in cells fitting the morphological characteristics of TSH cells. All these cells were also stained for TSH. However, a few cells were also shown to react solely with the antiserum to TSH. These cells, which seem to contain both TSH and FSH, were further clearly differentiated from PRL, GH and ACTH/MSH cells on the basis of their cytological features, intraglandular distribution and by immunochemical double staining. These observations support the concept that the one cell-one hormone theory may not necessarily apply to the glycoprotein hormones of the dog pituitary gland.Abbreviations of Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin The authors are grateful to Mrs. K. Oertel for carrying out the experimental work on animals, to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

The study of core-shell molecularly imprinted polymers of 17β-estradiol on the surface of silica nanoparticles

In this paper, highly selective core-shell molecularly imprinted polymers of 17β-estradiol on the surface of silica nanoparticles (SiO(2)@E2-MIPs) were prepared. The SiO(2)@E2-MIPs were characterized by Fourier transform infrared spectrometer (FT-IR), transmission electron microscope (TEM), dynamic adsorption and static adsorption tests. The sorption capacity of the SiO(2)@E2-MIPs were nearly 5 times that of the non-imprinted polymers (NIPs), and it only took 25 min to achieve the sorption equilibrium. It indicated that the SiO(2)@E2-MIPs exhibited a high selectivity, large adsorption capacity and fast kinetics. When the SiO(2)@E2-MIPs were used as dispersive solid-phase extraction (dSPE) absorbents to selectively enrich and determine estrogens in duck feed, the average recoveries of E2 and estriol (E3) were higher than 96.74% and 72.07%, respectively, and the relative standard deviations (RSD) of E2 and E3 were less than 1.61% and 3.28%, respectively. The study provides an effective method for the separation and enrichment of estrogens in the complex matrix samples by the SiO(2)@E2-MIPs.     

Regucalcin is expressed in rat mammary gland and prostate and down-regulated by 17β-estradiol

Regucalcin is involved in maintenance of calcium homeostasis due to the activation of Ca²⁺ pumping enzymes in the plasma membrane. It has a suppressive effect in cell proliferation, DNA and RNA synthesis, and may be associated with the abnormal cell division on tumor tissues. On the other hand both estrogens and Ca²⁺ are implicated in breast and prostate cancer but there are no studies focused on the expression of regucalcin in rat mammary gland or prostate. Furthermore, it is known that the expression of regucalcin in rat liver and kidney is regulated by 17β-estradiol (E₂). The aim of this study is to analyze if regucalcin is expressed in rat mammary gland and prostate and if it is regulated by E₂ in these tissues. We demonstrated for the first time that regucalcin mRNA and protein are present in rat mammary gland and prostate by in situ hybridization and immunohistochemistry, respectively. Furthermore, we show by Real-time PCR that E₂ down-regulates regucalcin expression in rat mammary gland and prostate.     

Androgen dynamics in vitro in the human prostate gland. Effect of oestradiol-17β

Normal, hyperplastic and adenocarcinomatous human prostatic tissue was perfused in vitro with radioactively labelled androstenedione, testosterone and 5alpha-dihydrotestosterone with and without added oestradiol-17beta. Various parameters of tissue-steroid relationship were measured at the steady state. When oestradiol (0.11 or 0.22mumol/l) was added to the perfusing medium, the entry of the steroids into the tissue and their metabolism was increased in the majority of the glands studied. The ;uptake' of all the steroids varied, in response to the addition of oestradiol, in both normal and adenocarcinomatous glands in a way differing from the response of hyperplastic glands. As a consequence, the tissue clearance of the steroids, particularly of androstenedione and testosterone, increased in normal and adenocarcinomatous glands in the presence of oestradiol, and decreased in the hyperplastic tissues. At a concentration 0.33mumol/l, oestradiol decreased the entry of the steroids in all the tissues studied, while the clearance of steroids tended to decrease. The significance of these findings in terms of the regulation of androgen dynamics in vivo in the normal and diseased human prostate, with particular regard to the response to oestrogen treatment, is discussed.     

Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17β-estradiol at estrus

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17β-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17β-estradiol treatment. Expression of NF-κB and IκB proteins, and IκB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus.     

β-Glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are β-glucuronidase (β-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that β-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas β-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, β-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that β-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed. This study was funded by the University of the Basque Country through a grant to Consolidated Research Groups. U.I. is a recipient of a pre-doctoral fellowship from the Basque Government.     

Alterations in the subcellular distribution of 17β-estradiol dehydrogenase in porcine endometrial cells over the course of the estrous cycle

The uteri of German landrace gilts slaughtered at different days of the cycle were processed for immunocytochemistry and biochemical analyses. Plasma was collected for hormone assays. The monoclonal antibody F1 against the structure-bound 17-estradiol dehydrogenase of porcine endometrial epithelium was applied to rehydrated paraffin sections either as a direct, peroxidase-linked probe or in combination with a fluorescing secondary antibody. The oxidation of estradiol was measured in homogenates of tissue powdered in liquid nitrogen. Immunoreactivity was restricted to endometrial epithelium. In the glandular epithelium, faint dots of fluorescence became visible at day 4, which apparently coalesced to spherical structures of 2–4 m diameter at the cell basis between days 11 through 17 before disappearing by day 18. A similar distribution was observed for the oxidation products of diaminobenzidine beginning with a faint uniform staining and followed by the appearance of intensely stained basal bodies persisting until day 17. Essentially the same time course was seen in the luminal epithelium but with a different distribution. Immunoreactive material amassed in the apical region of the cells, but the conspicuous aggregations were absent. Time course and intensities of the immunological responses are matched by the enzymatic activity measured in parallel. Both correlate with the plasma progesterone levels, suggesting an induction of the enzyme by the hormone. An involvement of the cytoskeleton in the sequence of subcellular distribution patterns is discussed.     

Effects of 17β-estradiol and bisphenol A on the formation of reproductive organs in planarians

Planarians have a remarkable capacity for regeneration after ablation, and they reproduce asexually by fission. However, some planarians can also reproduce and maintain their sexual organs. During the regenerative process, their existing sexual organs degenerate and new ones develop. However, little is known about hormonal regulation during the development of reproductive organs in planarians. In this study, we investigated the effects of 17β-estradiol (a steroid) and bisphenol A (an endocrine disrupter) on the formation of sexual organs in the hermaphroditic planarian Dugesia ryukyuensis. Under control conditions, all worm tissues regenerated into sexual planarians with sexual organs within 4 weeks after ablation. However, in the presence of bisphenol A or 17β-estradiol, although they apparently regenerated into sexual planarians, the yolk glands, which are one of the female sexual organs, failed to regenerate even 7 weeks after ablation. These data suggest that planarians have a steroid hormone system, which plays a key role in the formation and maturation of sexual organs.     

The effect of 17β-estradiol treatment on the mass and the turnover of bone in ovariectomized rats taking a mild dose of thyroxin

We performed the dosing experiment to establish whether estrogen administration has any beneficial effects on the mass and the turnover of bone in ovariectomized rats taking a mild dose of thyroxin. Thirty-five Wistar rats, 28 weeks of age, received ovariectomies (OVX) or sham operations and were divided into five groups. Group I was the sham group, Groups 2–5 were ovariectomized. Group 2 was the OVX-control, Group 3 treated with thyroxin 30 μg/kg/day (T4), Group 4, 17β-estradiol 0.3 mg/kg/week (E2), and Group 5, the combination of T4 and E2. The duration of the experiment was 12 weeks. At the end of the experiment, serum chemistries were measured. Bone minerals in the femur were determined with single photon absorptiometry and bone turnover was assessed histomorphometrically. Alkaline-phosphatase increased in Group 3 (OVX-T4), but it reduced in Groups 4 (OVX-E2) and 5 (OVX-T4 + E2). Bone minerals decreased in Groups 2 (OVX) and 3. In Group 4, it was preserved at the same level as in Group 1. Group 5 showed a significant increase of bone mass compared with Group 1. Eroded surface and osteoid surface increased in Groups 2 and 3 and they were reduced in Groups 4 and 5. Bone volume and mineral apposition rate were at a maximum in Group 5. This study demonstrated that 17β-estradiol was capable of preventing the bone mass decrease by regulating the turnover in ovariectomized rats taking a mild dose of thyroxin. Osteoblast function appeared to be stimulated in combination with 17β-estradiol and thyroxin.