Human sperm decondensation in vitro is related to cleavage rate and embryo quality in IVF.

PURPOSE: To investigate whether the ability of human spermatozoa to decondense in vitro in the presence of heparin (Hep) and glutathione (GSH) is related to assisted reproduction (ART) success. METHODS: Cross-sectional pilot study involving male partners of 129 infertile couples undergoing ICSI with (45) or without (84) donor oocytes at two infertility clinics in CABA, Argentina, between October 2012 and December 2013. In vitro decondensation kinetics with Hep and GSH and DNA fragmentation (TUNEL) were determined on the same sample used for ICSI. The possible relationship of decondensation parameters (maximum decondensation and decondensation velocity) and TUNEL values with ART success was evaluated. RESULTS: Embryo quality correlated positively with decondensation velocity (D60/D30) (Spearman's correlation, p < 0.05). According to D60/D30 values, patients were classified as slow decondensers (SlowD) (n = 68) or fast decondensers (FastD) (n = 61). Embryo quality was better in FastD (unpaired t test, p < 0.05). FastD and SlowD were subdivided according to use of donor oocytes. Among SlowD, biochemical and clinical pregnancy rates per transfer were significantly higher in donor (n = 19) vs. in non-donor (n = 31) cycles (Fisher's exact test, p < 0.05). TUNEL values were not related to embryo quality, but no clinical pregnancies or live births were achieved in TUNEL+ SlowD (n = 7). CONCLUSION: Decondensation kinetics of human spermatozoa in vitro with Hep and GSH could be related to embryo quality and ART success.

N-acetylcysteine inhibits lipid accumulation in mouse embryonic adipocytes.

Oxidative stress plays critical roles in the pathogenesis of diabetes, hypertension, and atherosclerosis; some authors reported that fat accumulation correlates to systemic oxidative stress in human and mice, but cellular redox environment effect on lipid accumulation is still unclear. In our laboratory we used mouse embryonic fibroblasts (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC) and accumulate lipids, as obesity model. Here we analyzed the role of the well-known antioxidant and glutathione precursor N-acetylcysteine (NAC) in cellular MAPK modulation and lipid accumulation. We evaluated the effect of NAC on the adipogenic differentiation pathway using different doses: 0.01, 0.1, 1 and 5mM; no toxic doses in these cells. A dose of 5mM NAC [DCN-5] provoked a significant decrease in triglyceride accumulation (72±10 [DCN-5] vs 169±15 [DC], p<0.01), as well in Oil Red O stained neutral lipid content (120±2 [DCN-5] vs 139±12 [DC], p<0.01). Molecular mechanisms responsible for adipogenic differentiation involve increase of the expression of phosphoERK½ and phosphoJNK, 5mM NAC treatment inhibited both pERK½ and pJNK protein levels. We also evaluated the mitotic clonal expansion (MCE) which takes place during adipogenesis and observed an increase in DC at a rate of 1.5 cells number compared to CC at day 2, whereas the highest doses of NAC significantly inhibited MCE. Our results suggest that NAC inhibits lipid accumulation and the MAPK phosphorylation in mouse embryonic fibroblasts during adipogenic differentiation and further contribute to probe the importance of cellular redox environment in adipogenesis.

GAL3ST2 from mammary gland epithelial cells affects differentiation of 3T3-L1 preadipocytes.

INTRODUCTION: In the mammary gland, the involution that occurs when lactation ends is an important period for cancer development. We have previously demonstrated stromal-epithelium interactions evaluating conditioned medium of adipose tissue on breast epithelial metalloproteases activity (Creydt et al., Clin Transl Oncol 15:124-131, 2013). Here, we evaluated the effects of conditioned medium of breast epithelial mammary cells on stromal cells. MATERIALS AND METHODS: Conditioned medium from normal murine mammary gland cell line (NMuMG) and conditioned medium proteins were obtained. Then, they were evaluated on modulation of adipocyte differentiation, using 3T3-L1 cell line. RESULTS: We described, for the first time, that breast epithelial mammary cells could produce the enzyme galactose 3-O-sulfotransferase 2 (GAL3ST2). Importantly, GAL3ST2 is present in NMMuMG and two human breast cancer cell lines, and it is more strongly expressed in more metastatic tumors. When 3T3-L1 preadipocyte differentiation was triggered in the presence of conditioned medium from NMuMG or GAL3ST2, triglyceride accumulation was decreased by 40 % and C/EBPß expression by 80 % in adipocytes. In addition, the expression of FABP4 (aP2), another marker of adipocyte differentiation, was inhibited by 40 % in GAL3ST2-treated cells. CONCLUSIONS: Taken together, these results suggest that GAL3ST2 would interfere with normal differentiation of 3T3-L1 preadipocytes; raising the possibility that it may affect normal differentiation of stromal preadipocytes and be a link to tumor metastatic capacity.

N-Acetylcysteine affects obesity-related protein expression in 3T3-L1 adipocytes.

OBJECTIVES: Oxidative stress plays critical roles in the pathogeneses of diabetes, hypertension, and atherosclerosis, but its effect on fat accumulation is still unclear. In this study, we analyzed the role of the well-known antioxidant and a glutathione (GSH) precursor N-acetylcysteine (NAC) in fat accumulation and the expression of obesity-associated proteins. METHODS: We studied the effects of 10 µM NAC on obesity-related protein expression in cultured 3T3-L1 preadipocytes, which are able to differentiate into mature adipocytes and accumulate lipids. RESULTS: NAC treatment inhibited fat accumulation and reduced the expression of obesity-related proteins, including monoamine oxidase A, heat shock protein 70 (HSP70), aminoacylase -1 (ACY-1), and transketolase. DISCUSSION: Our results suggest that the effects of NAC on triglycerides (Tgs) and protein expression are correlated. In support of this, we showed that NAC treatment affected both the Tg synthesis pathway and the expression levels of proteins implicated in human obesity.

Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa.

BACKGROUND: Human sperm nuclear decondensation in vivo involves protamine disulfide bond reduction by glutathione (GSH) and protamine/histone exchange, presumably with heparan sulfate (HS) as the protamine acceptor. The aim of the present study was to test the hypothesis that these two events occur simultaneously rather than sequentially, as has been hitherto accepted, and to test for the presence of HS in the human oocyte. METHODS: Spermatozoa and isolated sperm nuclei obtained from normal volunteers were exposed in vitro to heparin, the functional analogue of HS and either GSH or dithiothreitol (DTT) as the disulfide reducing agent. Decondensing reagents were added either simultaneously or sequentially. Percentage sperm nuclear decondensation was assayed by phase contrast microscopy. Thiol reduced status of isolated sperm nuclei was evaluated both indirectly [acridine orange (AO) staining of acid-denatured DNA] and directly [monobromobimane (mBBr) staining of protamine-free thiols]. The presence of HS in mature metaphase II (MII) human oocytes was analyzed by immunocytochemistry. RESULTS: Sequential addition of reagents always resulted in significantly lower decondensation if GSH was used as the disulfide bond reducer (P < 0.05 for sperm and P < 0.001 for nuclei), but only when heparin was used first, when DTT was the disulfide reducing agent (P < 0.05 for sperm and P < 0.01 for nuclei). Both AO staining of DNA and mBBr staining of protamines revealed that the addition of heparin to GSH but not to DTT significantly increased the thiol reduced status of sperm chromatin. HS was detected in the ooplasm of zona-free MII human oocytes. CONCLUSIONS: The results presented in this paper clearly show that heparin enhances the sperm chromatin thiol reducing activity of GSH in vitro, suggesting that in vivo thiol reduction and protamine/histone exchange could occur as simultaneous, rather than sequential, events. We also demonstrate for the first time the presence of HS in the human oocyte.

Elsevier Trophoblast Research Award lecture: Molecular mechanisms underlying estrogen functions in trophoblastic cells--focus on leptin expression.

The steroid hormone 17ß-estradiol is an estrogen that influences multiple aspects of placental function and fetal development in humans. During early pregnancy it plays a role in the regulation of blastocyst implantation, trophoblast differentiation and invasiveness, remodeling of uterine arteries, immunology and trophoblast production of hormones such as leptin. Estradiol exerts some effects through the action of classical estrogen receptors ERα and ERß, which act as ligand-activated transcription factors and regulate gene expression. In addition, estradiol can elicit rapid responses from membrane-associated receptors, like activation of protein-kinase pathways. Thus, the cellular effects of estradiol will depend on the specific receptors expressed and the integration of their signaling events. Leptin, the 16,000MW protein product of the obese gene, was originally considered an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblastic cells. Expression of leptin in placenta is highly regulated by key pregnancy molecules as hCG and estradiol. The aim of this paper is to review the molecular mechanisms underlying estrogen functions in trophoblastic cells; focusing on mechanisms involved in estradiol regulation of placental leptin expression.

Review: Leptin gene expression in the placenta--regulation of a key hormone in trophoblast proliferation and survival.

Leptin is a 16000 MW protein originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblast cells. Study of the major signaling pathways known to be triggered by leptin receptor has revealed that leptin stimulates JAK/STAT, MAPK and PI3K pathways in placental cells. Leptin also exerts an antiapoptotic action in placenta and this effect is mediated by the MAPK pathway. Moreover, leptin stimulates protein synthesis by activating the translational machinery via both PI3K and MAPK pathways. Expression of leptin in placenta is highly regulated, suggesting that certain key pregnancy molecules participate in such regulation. An important hormone in reproduction, hCG, induces leptin expression in trophoblast cells and this effect involves the MAPK signal transduction pathway. Moreover, the cyclic nucleotide cAMP, which has profound actions upon human trophoblast function, also stimulates leptin expression and this effect seems to be mediated by crosstalk between the PKA and MAPK signaling pathways. Estrogens play a central role in reproduction. 17ß-estradiol upregulates leptin expression in placental cells through genomic and non-genomic actions, probably via crosstalk between estrogen receptor-α and the MAPK and PI3K signal transduction pathways. Taken together these findings give a better understanding of the function of leptin and the regulatory mechanisms of leptin expression in human placental trophoblast and further support the importance of leptin in the biology of reproduction.

The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo.

BACKGROUND: Previous results from our laboratory have led us to propose heparan sulfate (HS) as a putative protamine acceptor during human sperm decondensation in vivo. The aim of this paper was to investigate the presence of glycosaminoglycans in the mammalian oocyte in an effort to better support this contention. METHODS: Two experimental approaches are used: oocyte labeling to identify the presence of HS and analysis of sperm decondensing ability of fresh oocytes in the presence or absence of specific glycosidases. RESULTS: Staining of mouse zona-intact oocytes with the fluorescent cationic dye, Rubipy, at pH 1.5 allowed for the detection of sulfate residues in the ooplasm by confocal microscopy. HS was detected in the ooplasm by immunocytochemistry. A sperm decondensation microassay using heparin and glutathione was successfully developed. The same level of sperm decondensation could be attained when heparin was replaced by mouse zona-free oocytes. Addition of heparinase to the oocyte/glutathione mixture significantly reduced sperm decondensation (P = 0.0159), while there was no effect following addition of either chondroitinase ABC or hyaluronidase. CONCLUSIONS: The results presented in this paper demonstrate for the first time that HS is present in the mammalian oocyte and show that HS is necessary for fresh oocytes to express their sperm decondensing ability in vitro.

Nuclear translocation of haeme oxygenase-1 is associated to prostate cancer.

The role of oxidative stress in prostate cancer has been increasingly recognised. Acute and chronic inflammations generate reactive oxygen species that result in damage to cellular structures. Haeme oxygenase-1 (HO-1) has cytoprotective effects against oxidative damage. We hypothesise that modulation of HO-1 expression may be involved in the process of prostate carcinogenesis and prostate cancer progression. We thus studied HO-1 expression and localisation in 85 samples of organ-confined primary prostate cancer obtained via radical prostatectomy (Gleason grades 4-9) and in 39 specimens of benign prostatic hyperplasia (BPH). We assessed HO-1 expression by immunohistochemical staining. No significant difference was observed in the cytoplasmic positive reactivity among tumours (84%), non-neoplastic surrounding parenchyma (89%), or BPH samples (87%) (P=0.53). Haeme oxygenase-1 immunostaining was detected in the nuclei of prostate cancer cells in 55 of 85 (65%) patients but less often in non-neoplastic surrounding parenchyma (30 of 85, 35%) or in BPH (9 of 39, 23%) (P<0.0001). Immunocytochemical and western blot analysis showed HO-1 only in the cytoplasmic compartment of PC3 and LNCaP prostate cancer cell lines. Treatment with hemin, a well-known specific inducer of HO-1, led to clear nuclear localisation of HO-1 in both cell lines and highly induced HO-1 expression in both cellular compartments. These findings have demonstrated, for the first time, that HO-1 expression and nuclear localisation can define a new subgroup of prostate cancer primary tumours and that the modulation of HO-1 expression and its nuclear translocation could represent new avenues for therapy.

Cell-cell communication between mouse mammary epithelial cells and 3T3-L1 preadipocytes: effect on triglyceride accumulation and cell proliferation

Interaction between parenchyma and stroma is essential for organogenesis, morphogenesis, and differentiation. Mammary gland has being the chosen model for developmental biologist because the most striking changes in morphology and function take place after birth. We have demonstrated a regulation of triglyceride accumulation by protein factors synthesized by normal mouse mammary gland epithelial cells (NMMG), acting on a cell line, 3T3-L1, long used as a model for adipogenesis. In this paper, we demonstrate that this inhibitory effect seems to be shared by other cells of epithelial origin but not by other cell types. We found a regulation of cell proliferation when NMMG cells are cultured in the presence of conditioned media from Swiss 3T3 or 3T3-L1 cells. We found a possible point of regulation for the mammary factor on a key enzyme of the lipid metabolic pathway, the glycerol-3-phosphate dehydrogenase. The inhibitory factor seems to have an effect on this enzyme's activity and reduces it. The results presented herein contribute to the understanding of cell-cell communication in a model of a normal mammary gland.

Cell-cell communication between mouse mammary epithelial cells and 3T3-L1 preadipocytes: effect on triglyceride accumulation and cell proliferation

Interaction between parenchyma and stroma is essential for organogenesis, morphogenesis, and differentiation. Mammary gland has being the chosen model for developmental biologist because the most striking changes in morphology and function take place after birth. We have demonstrated a regulation of triglyceride accumulation by protein factors synthesized by normal mouse mammary gland epithelial cells (NMMG), acting on a cell line, 3T3-L1, long used as a model for adipogenesis. In this paper, we demonstrate that this inhibitory effect seems to be shared by other cells of epithelial origin but not by other cell types. We found a regulation of cell proliferation when NMMG cells are cultured in the presence of conditioned media from Swiss 3T3 or 3T3-L1 cells. We found a possible point of regulation for the mammary factor on a key enzyme of the lipid metabolic pathway, the glycerol-3-phosphate dehydrogenase. The inhibitory factor seems to have an effect on this enzymes activity and reduces it. The results presented herein contribute to the understanding of cell-cell communication in a model of a normal mammary gland.(AU)

MDCK cells express serotonin-regulable 11beta-hydroxysteroiddehydrogenase type 2

Prior to this work, we found that adrenal as well as extra-adrenal factors activate the response of renal 11beta-hydroxysteroid dehydrogenase 2 to stressful situations. These results -showing ways through which the organism hinders the pathological occupation of mineralocorticoid receptors by glucocorticoids leading to sodium retention and hypertension- prompted the present study on the nature of the above-mentioned extra-adrenal factors. Serotonin was chosen because of its properties as a widely distributed neurohormone, known to interact with glucocorticoids at many sites, also exhibiting increased levels and effects under stressful situations. We studied serotonin effects on 11beta-hydroxysteroiddehydrogenase 2 activity in a cell line derived from distal nephronpolarized-epithelium, employing 3H-corticosterone as substrate. The end-product, 3H- 11 -dehydrocorticosterone was separated from the substrate by HPLC and quantified. Serotonin stimulated 1I beta-hydroxysteroiddehydrogenase 2 activity only at 2nM and 25pM, the magnitude of the responsedepending also on substrate concentration. The stimulation was blocked by thespecific inhibitors methiothepin and ketanserin. We postulate that the organism partially prevents renal mineralocorticoid receptor occupancy by glucocorticoids, circulating at enhanced levels under stressful situations, through serotonin-mediated catabolic regulation of the 11beta-hydroxysteroid dehydrogenase 2 activity. Given many, mostly positive, interactions between both hormones, this might eventually pave the way to studies on a new regulatory axis.

MDCK cells express serotonin-regulable 11beta-hydroxysteroiddehydrogenase type 2

Prior to this work, we found that adrenal as well as extra-adrenal factors activate the response of renal 11beta-hydroxysteroid dehydrogenase 2 to stressful situations. These results -showing ways through which the organism hinders the pathological occupation of mineralocorticoid receptors by glucocorticoids leading to sodium retention and hypertension- prompted the present study on the nature of the above-mentioned extra-adrenal factors. Serotonin was chosen because of its properties as a widely distributed neurohormone, known to interact with glucocorticoids at many sites, also exhibiting increased levels and effects under stressful situations. We studied serotonin effects on 11beta-hydroxysteroiddehydrogenase 2 activity in a cell line derived from distal nephronpolarized-epithelium, employing 3H-corticosterone as substrate. The end-product, 3H- 11 -dehydrocorticosterone was separated from the substrate by HPLC and quantified. Serotonin stimulated 1I beta-hydroxysteroiddehydrogenase 2 activity only at 2nM and 25pM, the magnitude of the responsedepending also on substrate concentration. The stimulation was blocked by thespecific inhibitors methiothepin and ketanserin. We postulate that the organism partially prevents renal mineralocorticoid receptor occupancy by glucocorticoids, circulating at enhanced levels under stressful situations, through serotonin-mediated catabolic regulation of the 11beta-hydroxysteroid dehydrogenase 2 activity. Given many, mostly positive, interactions between both hormones, this might eventually pave the way to studies on a new regulatory axis.(AU)

Heparin increases the adhesion of murine mammary adenocarcinoma cells (LM3). Correlation with the presence of heparin receptors on cell surface.

Mastocytosis is a common feature around solid tumors. Due to mast cell (MC) degranulation, heparin and other chemical mediators are released to surrounding tissues. The aim of this paper is to investigate the role of heparin and chemically modified heparins, on a murine mammary adenocarcinoma cell line adhesion properties, and the relationship with the presence of heparin binding sites in tumor cells. We show that heparin increases tumor cell adhesion in a dose-dependent manner. When the number of heparin binding sites was regulated, by culturing the cells with different FCS concentration for 24 hours, a correlation between binding capacity and heparin effect on cell adhesion was observed. The increment on cell adhesion by heparin was lower on cells with less heparin binding sites. Moreover, only heparin and a chemically modified heparin (partially N-desulfated N-acetylated), which bound to heparin-receptor, retained the ability to stimulate cell adhesion, while other modified heparins lost both effects. The increase in cell adhesion was observed on plastic dishes, albumin, as well as on fibronectin pre-coated ones suggesting that heparin effect is substratum independent. Our results show a direct relation between heparin binding to specific cell receptors and increase in cell attachment.

Synthesis, isolation and characterization of Plasmodium falciparum antigenic tetrabranched peptide dendrimers obtained by thiazolidine linkages.

Different chemical alternatives were evaluated for obtaining immunogenic polypeptidic macromolecules which could then be used as vaccines. These were based on the ligation reaction between an unprotected immunogenic peptide and an unprotected multifunctional core peptide; polyantigens, designated dendrimers because their form resembles that of dendritic cells, were thus obtained. The antigen-core ligation alternatives, studied by indirect synthesis, were the formation of oxime, hydrazone and thiazolidine linkages, making use of the reaction between a weak base (acting as nucleophile) and an alkyl aldehyde. The other alternative was the formation of a thioether linkage between a sulfydryl and an alkyl halide. Finally, a multiple antigen peptide (MAP) was synthesized by direct synthesis. All reactions were monitored by SEC-HPLC and SDS-PAGE. Dendrimer molecular mass obtained was confirmed by MS MALDI-TOF. Dendrimer purification was first carried out by concentrating crude reaction products with CP-5000 centricons and (using SEC-HPLC) pure tetramers were then obtained. A 20-residue 9376 immunogenic sequence, from Plasmodium falciparum apical merozoite antigen protein (AMA-1), was used to study the best alternative for chemical ligation. It was observed that thiazolidine formation proceeded with greater yield and in less time than the others. A tetramer has been simultaneously synthesized via thiazolidine with the SPf-66 antimalarial vaccine 45-residue monomer, proving the technique's versatility. The 9376 peptide disulfide bound polymer and SPf-66 (as well as their tetrameric thiazolidine dendrimers) were inoculated in rabbits to evaluate their antibody response. It was observed that titers for tetrameric thiazolidine dendrimers were not just greater but were also sustained over time. Western blot for pre-immune and immune sera showed that dendrimer sera recognized specific Plasmodium falciparum proteins as well as disulfide-bound polymers.

Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II).

The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.

Kinetic properties of microsomal 3beta hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H.

3beta-hydroxysteroid dehydrogenase 5-ene isomerase (3betaHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3betaHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K(m)=0.17+/-0. 03 and 1.02 microM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V(max) while K(m) was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K(i) was calculated from replot of Dixon's slope for each substrate concentration. K(i) from the intercept and the slope of this replot were similar (0.276+/-0.01 and 0.263+/-0.02 microM) and higher than the K(m) for DHEA. The K(m) and K(i) values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V(max) while K(m) values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.

Plasmodium falciparum AMA-1 erythrocyte binding peptides implicate AMA-1 as erythrocyte binding protein.

The role of AMA-1 during merozoite invasion has not yet been determined. However, reported experimental evidence suggests that this protein can be used, in particular as erythrocyte-binding protein, since, Fab fragments against this protein are able to block merozoite invasion. Using a previously described methodology, eight peptides with high binding activity to human erythrocyte, scattered along the different domains and having around 130 nM affinity constants, were identified in the Plasmodium falciparum AMA-1 protein. Their binding activity was sialic acid independent. Some of these peptides showed homology with the erythrocyte binding domains of one of the apical organelle protein family, MAEBL, identified in rodent malarial parasites. One of these peptides shares amino acid sequence with a previously reported B-cell epitope which induces antibodies to block parasite growth. The critical residues were identified for erythrocyte binding conserved peptides 4313 (DAEVAGTQYRLPSGKCPVFG), 4321 (VVDNWEKVCPRKNLQNAKFG), 4325 (MIKSAFLPTGAFKADRYKSH) and 4337 (WGEEKRASHTTPVLMEKPYY). All conserved peptides were able to block merozoite invasion of new RBC and development, suggesting that these peptides are involved in P. falciparum invasion.

Activity of renal 11betahydroxysteroid dehydrogenase 2 (11betaHSD2) in stressed animals.

The enzyme 11betaHSD2 protects the non-selective mineralocorticoid receptor from occupation by glucocorticoids in aldosterone target tissues. We studied the effect of stress elicited by intubation with a rubber catheter and administration of 10 ml of 0.45% NaCl (G3), of 10 ml of 200 mM HCl (G4) or intubation alone (G2) on the kinetics of the renal enzyme compared with untreated rats (G1). Microsomes were incubated with increasing masses of 3H corticosterone and 400 microM NAD at pH=7.4 during 5 minutes. Samples were extracted with ethyl acetate and analyzed by TLC. Results for n=4: Vmax for G1, 4.82 +/- 0.67. G2, 10.04 +/- 0.16***. G3, 9.16 +/- 0.74**. G4, 10.19 +/- 0.79*** pmoles/min/mg prot. Km for G1, 22.37 +/- 2.42. G2, 50.72 +/- 7.05*. G3, 55.25 +/- 8.37**. G4, 27.40 +/- 3.20 nM. (***p<0.001, **p<0.01 and *p<0.05 vs G1). All treatments increased Vmax. Intubation alone and gavage with 0.45% NaCl, but not with 200 mM HCl, increased Km. Taking together, the results could reflect a way to prevent occupation of type I receptors by increased levels of circulating glucocorticoids due to stressful situations. This protection seems more efficient under acidotic conditions causing--in addition to an increased Vmax--a low Km for the enzyme.

Dose-dependence of the effects of corticosterone and the non-glucocorticoid, 18-hydroxycorticosterone, on the brush-border Na+/K+ exchanger.

Up to now, only glucocorticoids were thought to act on the renal proximal Na+/H+ exchanger. Using fluorimetric techniques we studied the kinetics of Na+/H+ exchange in brush border vesicles from ADX rats treated with increasing doses of corticosterone (B) and 18-hydroxycorticosterone (18OHB). Significant linear correlations were obtained when the Vmax of each treatment were plotted against log doses. 18OHB exhibits a slightly higher sensitivity than B and log-dose responses were steeper for 18OHB than for B treated rats. Differences between both treatments were highly significant at the 4.8 microg/100 g level, corresponding to the physiological blood level of 18OHB. Physiological doses of both steroids elicited equal Na+/H+ exchange-responses. 18OHB is not a glucocorticoid since even 88 microg/100 g did not promote hepatic glycogen deposition while the same dose of B increases glycogen deposits 3.5-fold. These results demonstrate the importance of the Na+/H+ exchanger as a mediator between corticoid action and H+ transport and that of the non-glucocorticoid 18OHB in this process.