Recent Advances in the Use of the Dimerization Strategy as a Means to Increase the Biological Potential of Natural or Synthetic Molecules.

The design of C2-symmetric biologically active molecules is a subject of interest to the scientific community. It provides the possibility of discovering medicine with higher biological potential than the parent drugs. Such molecules are generally produced by classic chemistry, considering the shortness of reaction sequence and the efficacy for each step. This review describes and analyzes recent advances in the field and emphasizes selected C2-symmetric molecules (or axial symmetric molecules) made during the last 10 years. However, the description of the dimers is contextualized by prior work allowing its development, and they are categorized by their structure and/or by their properties. Hence, this review presents dimers composed of steroids, sugars, and nucleosides; known and synthetic anticancer agents; polyphenol compounds; terpenes, known and synthetic antibacterial agents; and natural products. A special focus on the anticancer potential of the dimers transpires throughout the review, notwithstanding their structure and/or primary biological properties.

Oncostatin M and STAT3 Signaling Pathways Support Human Trophoblast Differentiation by Inhibiting Inflammatory Stress in Response to IFNγ and GM-CSF.

Interleukin-6 (IL-6) superfamily cytokines play critical roles during human pregnancy by promoting trophoblast differentiation, invasion, and endocrine function, and maintaining embryo immunotolerance and protection. In contrast, the unbalanced activity of pro-inflammatory factors such as interferon gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at the maternal-fetal interface have detrimental effects on trophoblast function and differentiation. This study demonstrates how the IL-6 cytokine family member oncostatin M (OSM) and STAT3 activation regulate trophoblast fusion and endocrine function in response to pro-inflammatory stress induced by IFNγ and GM-CSF. Using human cytotrophoblast-like BeWo (CT/BW) cells, differentiated in villous syncytiotrophoblast (VST/BW) cells, we show that beta-human chorionic gonadotrophin (ßhCG) production and cell fusion process are affected in response to IFNγ or GM-CSF. However, those effects are abrogated with OSM by modulating the activation of IFNγ-STAT1 and GM-CSF-STAT5 signaling pathways. OSM stimulation enhances the expression of STAT3, the phosphorylation of STAT3 and SMAD2, and the induction of negative regulators of inflammation (e.g., IL-10 and TGFß1) and cytokine signaling (e.g., SOCS1 and SOCS3). Using STAT3-deficient VST/BW cells, we show that STAT3 expression is required for OSM to regulate the effects of IFNγ in ßhCG and E-cadherin expression. In contrast, OSM retains its modulatory effect on GM-CSF-STAT5 pathway activation even in STAT3-deficient VST/BW cells, suggesting that OSM uses STAT3-dependent and -independent mechanisms to modulate the activation of pro-inflammatory pathways IFNγ-STAT1 and GM-CSF-STAT5. Moreover, STAT3 deficiency in VST/BW cells leads to the production of both a large amount of ßhCG and an enhanced expression of activated STAT5 induced by GM-CSF, independently of OSM, suggesting a key role for STAT3 in ßhCG production and trophoblast differentiation through STAT5 modulation. In conclusion, our study describes for the first time the critical role played by OSM and STAT3 signaling pathways to preserve and regulate trophoblast biological functions during inflammatory stress.

The V86M mutation in HIV-1 capsid confers resistance to TRIM5α by abrogation of cyclophilin A-dependent restriction and enhancement of viral nuclear import.

BACKGROUND: HIV-1 is inhibited early after entry into cells expressing some simian orthologues of the tripartite motif protein family member TRIM5α. Mutants of the human orthologue (TRIM5αhu) can also provide protection against HIV-1. The host protein cyclophilin A (CypA) binds incoming HIV-1 capsid (CA) proteins and enhances early stages of HIV-1 replication by unknown mechanisms. On the other hand, the CA-CypA interaction is known to increase HIV-1 susceptibility to restriction by TRIM5α. Previously, the mutation V86M in the CypA-binding loop of HIV-1 CA was found to be selected upon serial passaging of HIV-1 in cells expressing Rhesus macaque TRIM5α (TRIM5αrh). The objectives of this study were (i) to analyze whether V86M CA allows HIV-1 to escape mutants of TRIM5αhu, and (ii) to characterize the role of CypA in the resistance to TRIM5α conferred by V86M. RESULTS: We find that in single-cycle HIV-1 vector transduction experiments, V86M confers partial resistance against R332G-R335G TRIM5αhu and other TRIM5αhu variable 1 region mutants previously isolated in mutagenic screens. However, V86M HIV-1 does not seem to be resistant to R332G-R335G TRIM5αhu in a spreading infection context. Strikingly, restriction of V86M HIV-1 vectors by TRIM5αhu mutants is mostly insensitive to the presence of CypA in infected cells. NMR experiments reveal that V86M alters CypA interactions with, and isomerisation of CA. On the other hand, V86M does not affect the CypA-mediated enhancement of HIV-1 replication in permissive human cells. Finally, qPCR experiments show that V86M increases HIV-1 transport to the nucleus of cells expressing restrictive TRIM5α. CONCLUSIONS: Our study shows that V86M de-couples the two functions associated with CA-CypA binding, i.e. the enhancement of restriction by TRIM5α and the enhancement of HIV-1 replication in permissive human cells. V86M enhances the early stages of HIV-1 replication in restrictive cells by improving nuclear import. In summary, our data suggest that HIV-1 escapes restriction by TRIM5α through the selective disruption of CypA-dependent, TRIM5α-mediated inhibition of nuclear import. However, V86M does not seem to relieve restriction of a spreading HIV-1 infection by TRIM5αhu mutants, underscoring context-specific restriction mechanisms.

CD40 pathway activation reveals dual function for macrophages in human endometrial cancer cell survival and invasion.

Reproductive malignancies are a major cause of cancer death in women worldwide. CD40 is a TNF receptor family member, which upon activation may mediate tumor regression. However, despite the great potential of CD40 agonists, their use as a therapeutic option for reproductive cancers has never been investigated. Because CD40 ligation is a potent pathway of macrophage activation, an in vitro model of pro-inflammatory type-1 (Mϕ-1) and anti-inflammatory type-2 (Mϕ-2) macrophages was developed to determine whether and how macrophage CD40 pathway activation might influence endometrial tumor cell behavior. Analysis of tumor growth kinetic in the endometrial cancer xenograft model indicates that, when injected once into the growing tumors, CD40-activated Mϕ-1 greatly reduced, while CD40-activated Mϕ-2 increased tumor size when compared to control isotype-activated Mϕ-1 and Mϕ-2, respectively. In vitro assays indicated that CD40-activated Mϕ-2 increased cell viability but failed to promote cell invasion. CD40-activated Mϕ-1, in contrast, decreased cell survival but greatly increased cell invasion in tumor cells less susceptible to cell death by apoptosis; they also induced the expression of some pro-inflammatory genes, such as IL-6, LIF, and TNF-α, known to be involved in tumor promotion and metastasis. The presence of IFN-γ is minimally required for CD40-activated Mϕ-1 to promote tumor cell invasion, a process that is mediated in part through the activation of the PI3K/Akt2 signaling pathway in tumor cells. From these results, we speculate that some functions of CD40 in tumor-associated Mϕs might limit the therapeutic development of CD40 agonists in endometrial cancer malignancies.

Investigating a new C2-symmetric testosterone dimer and its dihydrotestosterone analog: Synthesis, antiproliferative activity on prostate cancer cell lines and interaction with CYP3A4.

The synthesis of a 17α-linked C2-symmetric testosterone dimer and its dihydrotestosterone analog is reported. The dimers were synthesized using a short five-step reaction sequence with 28% and 38% overall yield for the testosterone and dihydrotestosterone dimer, respectively. The dimerization reaction was achieved by an olefin metathesis reaction with 2nd generation Hoveyda-Grubbs catalyst. The dimers and their corresponding 17α-allyl precursors were tested for the antiproliferative activity on androgen-dependent (LNCaP) and androgen-independent (PC3) prostate cancer cell lines. The effects on cells were compared with that of the antiandrogen cyproterone acetate (CPA). The results showed that the dimers were active on both cell lines, with an increased activity towards androgen-dependent LNCaP cells. However, the testosterone dimer (11) was fivefold more active than the dihydrotestosterone dimer (15), with an IC50 of 11.7 µM vs. 60.9 µM against LNCaP cells, respectively, and more than threefold more active than the reference drug CPA (IC50 of 40.7 µM). Likewise, studies on the interaction of new compounds with drug-metabolizing cytochrome P450 3A4 (CYP3A4) showed that 11 was a fourfold stronger inhibitor than 15 (IC50 of 3 µM and 12 µM, respectively). This suggests that changes in the chemical structure of sterol moieties and the manner of their linkage could largely affect both the antiproliferative activity of androgen dimers and their crossreactivity with CYP3A4.

CD40 translocation to lipid rafts: signaling requirements and downstream biological events.

CD40, a member of the TNF receptor family, is expressed on a variety of immune and non-immune cells. Its interaction with its ligand, CD154, plays a pivotal role in humoral and cell-mediated immunity. A low level of CD40 is constitutively associated within membrane lipid rafts and, upon engagement, this level is significantly enhanced. In this study, our objective is to evaluate the process of CD40/lipid raft association in terms of the signals required for its initiation and the resulting biological outcomes. Here, we show the CD40/lipid raft association to be independent of PI-3-kinase, Src family kinases and p38 MAPK pathways. Moreover, CD40 lacking its intracellular domain, which is usually required for CD40-mediated signaling, still localizes to lipid rafts upon engagement, confirming that the CD40/lipid raft association is independent of signaling events. As to the biological outcomes of the CD40/lipid raft association, we show that disrupting lipid raft integrity selectively abolishes CD40-mediated Akt phosphorylation. In addition, replacing the transmembrane domain of CD40 with that of CD45 (a protein excluded from lipid rafts) dramatically reduced CD40-mediated Akt phosphorylation and B7.1 upregulation, while not influencing p38, ERK and JNK activation. Together, these findings clarify the requirements for CD40/lipid raft association and the signals triggered upon CD40 engagement by CD154.

Leukemia inhibitory factor regulates differentiation of trophoblastlike BeWo cells through the activation of JAK/STAT and MAPK3/1 MAP kinase-signaling pathways.

It is well established that syncytium formation involves the fusion of mononucleated trophoblasts into a multinucleated structure and the secretion of hormonal factors, such as human chorionic gonadotropin (hCG). These morphological and biochemical changes are regulated by a plethora of ligands, which upon binding to specific receptors trigger the activation of many signaling pathways, such as janus kinase/signal transducer and activator of transcription (JAK/STAT) and the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinases 1 and 2 (MAPK3/1). We used the forskolin-induced syncytialization of trophoblastlike BeWo cells to characterize at the cellular level the effect mediated by leukemia inhibitory factor (LIF) on trophoblast differentiation and to describe its action at the molecular level. Forskolin induces both hCG secretion and BeWo cell syncytial fusion. Although LIF had no effect on the undifferentiated state of the cells, the cytokine generated a strong reduction in forskolin-induced hCG release. In contrast to its effect on hCG secretion, LIF exerts a synergistic effect toward forskolin-induced fusion. LIF reduced hormonal production through a STAT1- and STAT3-dependent mechanism, whereas MAPK3/1 was not involved in this process. However, both types of signaling molecules were required to mediate the action of LIF in forskolin-induced cell fusion. These data provide novel insights into the regulation of trophoblast cell differentiation by LIF and describe for the first time the molecular mechanism underlying the effect of the cytokine.

Locating the binding sites of two aminobenzoic acid derivatives on tRNA: drug binding efficacy and RNA structure.

The binding of tRNA to aminobenzoic acid derivatives DAB-0 (N'-[4-(2,5-dioxo-pyrrolidin-1-yl)-benzoyl]-hydrazine carboxylic acid tert-butyl ester) and DAB-1 (N'-[4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-benzoyl]-hydrazine carboxylic acid tert-butyl ester) was investigated in aqueous solution at physiological pH. Thermodynamic parameters ΔH0 -4.8 to -4.30 (kJ mol-1), ΔS0 24.20 to 22 (J mol-1K-1) and ΔG0 -12 to -11.40 (kJ mol-1) showed that DAB-0 and DAB-1 readily bind tRNA via ionic interactions with DAB-1 forming stronger tRNA adducts. Similar binding sites to A-T and G-C bases were located with DAB-0 and DAB-1. The binding efficacy ranged from 40% to 50%. No alteration of tRNA conformation was detected upon drug complexation. Communicated by Ramaswamy H. Sarma.

Innovative C2-symmetric testosterone and androstenedione dimers: Design, synthesis, biological evaluation on prostate cancer cell lines and binding study to recombinant CYP3A4.

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17ß-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17ß-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17ß-OH (14α and 14ß) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14ß) with an average IC50 of 13.3 µM. In LNCaP cells, 14ß was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC50 of 12.0 µM vs. 59.6 µM, respectively). At low concentrations (0.25-0.5 µM), 14α and 14ß were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein.

Leukemia inhibitory factor regulates the activation of inflammatory signals in macrophages and trophoblast cells.

The pleiotropic cytokine leukemia inhibitory factor (LIF) is a key gestational factor known to establish dynamic cellular and molecular cross talk at the feto-maternal interface. Previously, we described the regulatory role of the LIF-trophoblast-IL10 axis in the process of macrophage deactivation in response to pro-inflammatory cytokines. However, the direct regulatory effects of LIF in macrophage and trophoblast cell function remains elusive. In this study, we aimed to examine whether and how LIF regulates the behavior of macrophages and trophoblast cells in response to pro-inflammatory stress factors. We found that LIF modulated the activating effects of interferon-gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in macrophages and trophoblast cells by reducing the phosphorylation levels of signal transducer and activator of transcription-1 (Stat1) and -5 (Stat5). Cell activation with IFNγ inhibited cell invasion and migration but this immobilizing effect was abrogated when macrophages and trophoblast cells were deactivated with LIF; macrophage cell motility restitution could in part be explained by the positive effects of LIF in Stat3 activation and matrix metalloproteinase 9 (MMP-9) expression. Pharmacological inhibition of Stat1 and Stat3 indicated that IFNγ-induced Stat1 activation mediated macrophage motility inhibition, and that cell motility in IFNγ-activated macrophages is restored via LIF-induced Stat3 activation and Stat1 inhibition. Moreover, IFNγ-induced TNFα gene expression was also abrogated by LIF through Stat1 inhibition and Stat3 activation. Finally, we have found that cell invasion of trophoblast cells is inhibited when they were cocultured with GM-CSF-differentiated, IFNγ-stimulated macrophages. This effect, however, was inhibited when macrophages were exposed to LIF. Overall, this in vitro study reveals for the first time the anti-inflammatory and pro-gestational activities of LIF by acting directly on macrophages and trophoblast cells.

Relevance of iNOS expression in tumor growth and maintenance of cancer stem cells in a bladder cancer model.

The expression of inducible nitric oxide (NO) synthase (iNOS) in human bladder cancer (BC) is a poor prognostic factor associated with invasion and tumor recurrence. Here, we evaluated the relevance of iNOS expression in BC progression and in cancer stem cell (CSC) maintenance in a murine BC model. Also, iNOS expression and CSC markers were analyzed in human BC samples. iNOS inhibitors (L-NAME or 1400W) or shRNA were used on murine BC model with different iNOS expressions and invasiveness grades: MB49 (iNOS+, non-muscle invasive (NMI)) and MB49-I (iNOS++, muscle invasive (MI)), in order to analyzed cell proliferation, tumor growth, angiogenesis, number of CSC, and pluripotential marker expression. iNOS, SOX2, Oct4, and Nanog expressions were also analyzed in human BC samples by qPCR and immunohistochemistry. iNOS inhibtion reduced parameters associated with tumor progression and reduced the number of CSC, wich resulted higher in MB49-I than in MB49, in concordance with the higher expression of SOX2, Oct4, and Nanog. The expression of SOX2 was notoriously diminished, when iNOS was inhibited only in the MI cell line. Similar results were observed in human samples, where MI tumors expressed higher levels of iNOS and pluripotential genes, in comparison to NMI tumors with a positive correlation between those and iNOS, suggesting that iNOS expression is associated with CSC. iNOS plays an important role in BC progression and CSC maintenance. Its inhibition could be a potential therapeutic target to eradicate CSC, responsible for tumor recurrences. KEY MESSAGES: • iNOS expression is involved in bladder tumor development, growth, and angiogenesis. • iNOS expression is involved in bladder cancer stem cell generation and maintenance, playing an important role regulating their self-renewal capacity, especially in muscle invasive murine bladder cancer cells. • iNOS expression is higher in human muscle invasive tumors, in association with a high expression of pluripotential genes, especially of SOX2.

Molecular therapy with derivatives of amino benzoic acid inhibits tumor growth and metastasis in murine models of bladder cancer through inhibition of TNFα/NFΚB and iNOS/NO pathways.

Muscle-invasive bladder cancer (MIBC) is an aggressive form of urothelial bladder carcinoma (UBC) with poorer outcomes compared to the non-muscle invasive form (NMIBC). Higher recurrent rates and rapid progression after relapse in UBC is known to be linked with chronic inflammation. Here, the preclinical murine models of NMIBC (MB49) and MIBC (MB49-I) were used to assess the antitumor effects of DAB-1, an anti-inflammatory aminobenzoic acid derivative we have developed in order to target cancer-related inflammation. A subchronic toxicity study on cancer-free mice shown that DAB-1 treatment did not affect normal mouse development or normal function of vital organs. In mice bearing MB49-I tumors, whole body accumulation of the radioconjugate [131I]DAB-1 was higher than in control mice, the main sites of [131I]DAB-1 accumulation being the liver (34%), the intestines (21%), and the tumors (18%). In vivo molecular therapy of ectopic and orthotopic tumors indicated that treatment with DAB-1 efficiently inhibited tumor growth, metastasis formation, and mortality rate. The antitumor efficacy of DAB-1 was associated with strong decreased tumor cell proliferation and iNOS expression in tumor tissues and deactivation of macrophages from tumor-bearing mice. Mechanistic investigations revealed that DAB-1 efficiently inhibited i) TNFα/NFΚB and IL6/STAT3 signaling pathways activation; ii) TNFα-induced NO production by decreasing NFΚB transcriptional activation and functional iNOS expression; and iii) cellular proliferation with minimal or no effects on cell mortality or apoptosis. In conclusion, this study provides preclinical and biological/mechanistic data highlighting the potential of DAB-1 as a safe and efficient therapeutic agent for the treatment of patients with NMIBC and MIBC.

Spermatozoa modulate epididymal cell proliferation and protein secretion in vitro.

Normal epididymal function, such as protein expression and secretion, is primarily regulated by testicular androgens and temperature. However, the role of spermatozoa in this critical process has never been studied. In order to determine whether sperm itself could regulate epididymal function, we have developed a cell culture system of bovine epididymal cells to study the interactions between spermatozoa and the epididymal epithelium. Primary cells from caput, corpus, and cauda epididymal tissues were cultured in the presence of androgens at 32 degrees C (scrotal) and 37 degrees C (abdominal). Newly synthesized proteins were metabolically labeled with (35)S-methionine after sperm co-incubation and the pattern of secreted proteins was analyzed by two-dimensional polyacrylamide gel electrophoresis. Proliferation rate, protein secretion rate and electrophoretic patterns of secreted proteins were evaluated 48 hr post-co-incubation. Incubation at 32 degrees C indicated that spermatozoa stimulation increases the level of protein secretion of cultured cells from all epididymal sections while it slightly decreases proliferation of corpus cells. At 37 degrees C, spermatozoa co-incubation significantly decreases the protein secretion rate of cultured cells from all epididymal sections. Independently of cell incubation temperature, spermatozoa stimulation induces both an increase in the intensity of radiolabeled proteins and the appearance of new secreted proteins of caput cells without affecting the protein pattern of corpus or cauda cells. Incubation at 37 degrees C, however, greatly modifies the pattern of proteins expressed at 32 degrees C by cauda cells. Taken together, these results support the hypothesis that spermatozoa themselves affect epididymal cell function, most importantly for caput epididymides.

Requirement of the extracellular cysteine at position six for CD40/CD40 dimer formation and CD40-induced IL-8 expression.

We recently showed that oligomerization of CD40 molecules on cell surface leads to disulfide-linked CD40/CD40 dimer formation, an event that is necessary for CD40-induced B7-2 expression in human B cells. Here, we demonstrate that CD40/CD40 dimers formation also occurs in different cell types such as T24 bladder cancer cells and CD40-transfected HEK 293 cells. Disulfide bonds mediate the formation of CD40/CD40 homodimers in CD40-activated cells. To determine the potential residue(s) involved in disulfide bonds formation and subsequent CD40-induced IL-8 expression, we generated a CD40 mutant in which the extracellular cysteine 6 was replaced by a glutamine (CD40-C6Q). CD40-induced IL-8 mRNA expression and protein synthesis were studied in stably transfected HEK 293 cells that were sorted out along with similar levels of expression of wild type (CD40-WT) and CD40-C6Q molecules. In contrast to cells expressing CD40-WT protein, disulfide-linked CD40/CD40 dimer formation was completely abolished in HEK 293 cells expressing CD40-C6Q proteins. Abolishment of disulfide-linked CD40/CD40 dimers in these transfected cells was sufficient to inhibit CD40-induced mRNA expression and secretion of IL-8. This study identifies the extracellular cysteine 6 of CD40 molecules as a potential molecular target to disrupt the expression of CD40-induced pro-inflammatory cytokines by epithelial cells.

The proacrosin binding protein, sp32, is tyrosine phosphorylated during capacitation of pig sperm.

Mammalian sperm must undergo capacitation, a preparation period in the female reproductive tract or in vitro, in order to fertilize. We have previously described a Mr 32 000 tyrosine phosphorylated protein, "p32," that appears in pig sperm during capacitation. The identity of p32 remains unknown; if and how it is involved during capacitation is not understood. The objective of the present study was to identify p32 by proteomic techniques. Western blotting of proteins separated successively under nonreducing and then reducing conditions showed the appearance of the tyrosine phosphorylated p32 only when sperm were incubated in capacitating conditions. The spot was sequenced by mass spectrometry/mass spectrometry and identified as "sp32," a protein implicated in proacrosin maturation. The same membranes probed with anti-sp32 antibody demonstrated that sp32 is present in both noncapacitating and capacitating conditions and revealed exactly the same spot as p32. Immunoprecipitation with either anti-phosphotyrosine or anti-sp32 antibody corroborated these results. Indirect immunofluorescence with anti-phosphotyrosine antibody or anti-sp32 antibody show similar labeling of capacitated sperm, supporting the hypothesis that p32 is a tyrosine phosphorylated form of sp32. After ionophore treatment to induce the acrosome reaction, anti-sp32 and anti-phosphotyrosine labeling on the acrosome disappeared. These results demonstrate that sp32, a (pro)acrosin binding protein, is the p32, a tyrosine phosphorylated protein related to capacitation. We will now focus on the significance of tyrosine phosphorylation on sp32 function during fertilization-related events.

Use of phosphoproteomics to study tyrosine kinase activity in capacitating boar sperm. Kinase activity and capacitation.

It is generally accepted that sperm capacitation is associated with the protein kinase A-mediated appearance of tyrosine phosphoproteins, although the substrates and kinase(s) involved have not been identified. We described a Mr 32,000 tyrosine phosphoprotein, "p32", appearing in porcine sperm coincident with capacitation. We also discovered a tyrosine kinase-like enzyme in boar sperm of Mr 32,000 ("TK-32") with enhanced activity during capacitation. The present work was conducted to further characterize and to identify these capacitation-related protein(s). Fresh porcine sperm were incubated to induce capacitation then immunoprecipitation, immunoblotting and proteomic analysis revealed seven tyrosine-phosphorylated proteins aligned in the range of Mr 30,000 with different isoelectric pH values (pI). Therefore, p32 may be composed of several tyrosine phosphoproteins. Three were identified as acrosin-binding sp32 (pI 6.5), and two triosephosphate isomerase isoforms (pI 7.1 and 7.9). At present, however, proteonomic analysis has not revealed any kinase at Mr 32,000. Immunoprecipitation experiments show that p32 and TK-32 are different molecules, as TK-32 activity remains in the supernatant of the antiphosphotyrosine precipitates. Finally, in-gel renaturation and immunoblotting suggest that TK-32 is a mitogen-activated protein kinase (MAPK). The discovery of p32 and the MAPK-like TK-32 provides new insight regarding the mechanisms underlying capacitation in the pig.

Identification of an anti-inflammatory derivative with anti-cancer potential: The impact of each of its structural components on inflammatory responses in macrophages and bladder cancer cells.

Inflammation plays a crucial role in many types of cancer and is known to be involved in their initiation and promotion. As such, it is presently recognized as an important risk factor for several types of cancers such as bladder, prostate and breast cancers. The discovery of novel anti-inflammatory compounds can have a huge implication not only for the treatment of cancer but also as preventive and protective treatment modalities. We have recently identified a new compound (1) that presents interesting anti-inflammatory activity. In order to better understand its biological action, we have divided the molecule in its basic components and verified their respective contribution towards the anti-inflammatory response of the whole molecule. We have discovered that only the combination of the maleimide function together with the tert-butyloxycarbonylhydrazinamide function lead to important anti-inflammatory properties. The main derivative 1 can decrease the activating effects of INFγ or IL6 on human (hMϕs) macrophages by 38% or by 64% at a concentration of 10 µM as indicated by a decrease of STAT1 or STAT3 activation. The expression of pro-inflammatory markers CD40 and MHCII in INFγ stimulated hMϕs were reduced by 87% and 49%, respectively with a 3 h pretreatment of 1 at 10 µM. The cell motility assay revealed that 1 at 10 µM can reduce relative cell motility induced by IL6 by 92% in comparison with the untreated control hMϕ monolayers. Compound 1 reduced by 91% the inflammatory response induced by the cytokines (INFγ + TNFα) in the macrophage-like J774A.1 cells at a concentration of 25 µM, as measured by the detection of NO production with the Griess reagent. Furthermore, upon removal of the tert-butyloxycarbonyl protective group the unprotected derivative as a hydrochloride salt (1A) retains interesting anti-inflammatory activity and was found to be less toxic than the parent compound (1).

The activating effect of IFN-γ on monocytes/macrophages is regulated by the LIF-trophoblast-IL-10 axis via Stat1 inhibition and Stat3 activation.

Interferon gamma (IFN-γ) and leukemia inhibitory factor (LIF) are key gestational factors that may differentially affect leukocyte function during gestation. Because IFN-γ induces a pro-inflammatory phenotype in macrophages and because trophoblast cells are principal targets of LIF in the placenta, we investigated whether and how soluble factors from trophoblast cells regulate the effects of IFN-γ on macrophage activation. IFN-γ reduces macrophage motility, but enhances Stat1 activation, pro-inflammatory gene expression and cytotoxic functions. Soluble factors from villous cytotrophoblasts (vCT+LIF cells) and BeWo cells (BW/ST+LIF cells) that were differentiated in the presence of LIF inhibit macrophage Stat1 activation but inversely sustain Stat3 activation in response to IFN-γ. vCT+LIF cells produce soluble factors that induce Stat3 activation; this effect is partially abrogated in the presence of neutralizing anti-interleukin 10 (IL-10) antibodies. Moreover, soluble factors from BW/ST+LIF cells reduce cell proliferation but enhance the migratory responses of monocytes. In addition, these factors reverse the inhibitory effect of IFN-γ on monocyte/macrophage motility. BW/ST+LIF cells also generate IFN-γ-activated macrophages with enhanced IL-10 expression, but reduced tumor-necrosis factor alpha (TNF-α), CD14 and CD40 expression as well as impaired cytotoxic function. Additional assays performed in the presence of neutralizing anti-IL-10 antibodies and exogenous IL-10 demonstrate that reduced macrophage cytotoxicity and proliferation, but increased cell motility result from the ability of trophoblast IL-10 to sustain Stat3 activation and suppress IFN-γ-induced Stat1 activation. These in vitro studies are the first to describe the regulatory role of the LIF-trophoblast-IL-10 axis in the process of macrophage activation in response to pro-inflammatory cytokines.

Boar sperm storage capacity of BTS and Androhep Plus: viability, motility, capacitation, and tyrosine phosphorylation.

Androhep Plus, a long-term extender (up to 7 days) and Beltsville Thawing Solution (BTS), a short-term extender (up to 3 days), are commonly used for liquid storage of porcine semen. To test the hypothesis that modifications in sperm viability, motility, chlortetracycline (CTC) fluorescence patterns, and protein tyrosine phosphorylation occur during semen storage in extenders, we compared these end points at different periods of storage in either Androhep Plus or BTS. Sperm from five boars were assessed daily over 12 days of storage (n = 5 ejaculates from different boars). Viability was not different (P < 0.05 between extenders, except on Day 2, when Androhep Plus maintained better viability. Differences in the percentage of motile (total) sperm due to extender were evident on Days 2, 4, 5, and 6, when Androhep Plus was superior to BTS (P < 0.05). The percentages of progressively motile sperm also differed, with Androhep Plus supporting higher rates on Days 2, 4, 5, 7, 8, 9, 10, and 11 (P < 0.05). The CTC fluorescence pattern distribution differed due to extender as early as Day 2; storage in Androhep Plus induced higher levels of pattern B sperm (P < 0.05) than storage in BTS. A tyrosine-phosphorylated protein of Mr 21,000 appeared after 10 days in sperm incubated in BTS, and was identified as a phospholipid hydroperoxide glutathione peroxidase. Therefore, modifications in viability, motility, CTC fluorescence patterns, and sperm protein tyrosine phosphorylation were apparent during sperm storage in extenders; these may affect the fertilizing capacity of the semen.

Lantana macrophylla Schauer (Verbenaceae) ethanolic extract induces activation of ERK1/2 and p38 MAPKs pathway and Ca2+ imbalance in human trophoblasts derived cell lines.

Lantana macrophylla Schauer (Verbenaceae) a medicinal plant used to treat menstrual and respiratory disorders was investigated. The ethanolic extract from leaves was subjected to phytochemical and biological analysis. BeWo and JEG-3 cells were used to evaluate human chorionic gonadotropin hormone (hCG) production, syncytial formation, Ca2+ uptake and Ca2+ handling protein expression. The cAMP production and the mitogen-activated protein kinases (MAPKs) phosphorylation were also investigated. Phytochemical analysis yield three triterpenes: oleanolic, ursolic and latonolic acid. Viability assay showed no significant cytotoxic effect. A significant decrease in hCG production but not a disturbance on BeWo cell fusion were observed. The cAMP pathway was not affected by L. macrophylla extract alone; although the cAMP production inducted by forskolin was diminished. Both ERK1/2 and p38 MAPKs pathways were activated. Increased intracellular Ca2+ concentration ([Ca2+]i) was observed after 24 h treatment in a time and dose dependent manner; however only L. macrophylla at 10 µg/mL induced increased [Ca2+]i after 10 min treatment. CaBP28K and PMCA1/4 were modulated at protein and mRNA levels, respectively. This study showed for the first time the effect of triterpenoids from L. macrophylla leaves on trophoblasts-like cells and indicates a potential toxic effect of this plant in the placental development and fetal growth.