Enrique Werner

Expression of GM‐CSF receptors in male germ cells and their role in signaling for increased glucose and vitamin C transport

We studied the expression and function of the granulocyte‐macrophage colony stimulating factor (GM‐CSF) receptor in male germ cells. RT‐PCR showed expression of mRNAs encoding the α‐ and β‐subunits of the GM‐CSF receptor in human testis, and the presence of the α‐ and β‐proteins was confirmed by immunoblotting with anti‐α and anti‐β‐antibodies. Immunolocalization studies showed the level of expression of GM‐CSF α‐ and β‐subunits in the germ line in the testis and in ejaculated spermatozoa. Receptor binding studies using radiolabeled GM‐CSF revealed that bull spermatozoa have about 105 high‐affinity sites with a Kd of 222 pM and ≈1100 low‐affinity sites with a Kd of 10 nM. GM‐CSF signaled, in a time‐ and dose‐dependent manner, for an increased uptake of glucose and vitamin C. J. Cell. Biochem. 80:625–634, 2001.

Comparative study on the larvicidal activity of drimane sesquiterpenes and nordrimane compounds against Drosophila melanogaster til-til.

Natural compounds from Drimys winteri Forst and derivatives exhibited larvicidal effects against Drosophila melanogaster til-til. The most active compound was isodrimenin (4). The highest lethal concentration to the larvae of D. melanogaster was 4.5 ± 0.8 mg/L. At very low concentrations drimenol (1), confertifolin (3), and drimanol (5) displayed antifeedant and larvae growth regulatory activity. The antifeedant results of nordrimanic and drimanic compounds were better in first instar larvae. The EC50 value of polygodial (2) was 60.0 ± 4.2 mg/L; of diol 15 45.0 ± 2.8 mg/L, and of diol 17 36.9 ± 3.7 mg/L, while the new nordrimane compound 12 presented a value of 83.2 ± 3.5 mg/L.

Expression of granulocyte–macrophage colony stimulating factor (GM-CSF) in male germ cells: GM-CSF enhances sperm motility

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine capable of stimulating proliferation, maturation and function of hematopoietic cells. Receptors for this cytokine are composed of two subunits, alpha and beta, and are expressed on myeloid progenitors and mature mononuclear phagocytes, monocytes, eosinophils and neutrophils, as well as in other nonhematopietic cells. We have recently demonstrated that bull spermatozoa express functional GM-CSF receptors that signal for increased glucose and Vitamin C uptake. In this study, we analyzed the expression of GM-CSF in bovine and human germ cells and its influence in bovine sperm motility. Reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunoblotting analysis demonstrated that adult bovine and human testes expressed GM-CSF. In addition, immunolocalization studies confirmed the presence of GM-CSF in the germ cell line in bovine and human testes. Computer-assisted evaluation of patterns of sperm motility demonstrated that the addition of GM-CSF enhances several parameters of sperm motility in the presence of glucose or fructose substrates.

Expression of key substrate cycle enzymes in rat spermatogenic cells: Fructose 1,6 bisphosphatase and 6 phosphofructose 1-kinase

A substrate cycle composed of phosphofructo 1‐kinase I (PFK) and fructose 1,6 bisphosphatase I (FBPase) has been proposed in rat spermatids. This substrate cycle can explain the ability of glucose to induce a decrease in intracellular ATP, a phenomenon that was related to regulation of [Ca2+]i in these cells. In spite of the importance of this metabolic cycle, the expression and activities of the enzymes that compose such cycle have not been systematically studied in spermatogenic cells. Here, we show that PFK and FBPase activities were present in pachytene spermatocytes and round spermatids extracts. Expression of PFK at the mRNA and protein levels showed a relatively similar expression in spermatogenic cells, but a stronger expression in Sertoli cells. Instead, expression of FBPase at the mRNA and protein levels was stronger in round and elongating spermatids as compared to other spermatogenic cells. A similar pattern was observed when evidencing FBPase activity by a NADPH‐nitroblue tetrazolium‐linked cytochemical assay in isolated pachytene spermatocytes and round spermatids. Rat spermatids also showed the ability to convert lactate to fructose‐ and glucose‐6‐P, indicating that both glycolytic and gluconeogenic fluxes are present in these cells. Our results indicate that a coordinated expression of key substrate cycle enzymes, at the level of PFK/FBPase, appear in the last stages of spermatogenic cell differentiation, suggesting that the co‐regulation of these enzymes are required for the ability of these cells to respond to glucose and induce metabolic and Ca2+ signals that can be important for sperm development and function. J. Cell. Physiol. 212:807–816, 2007.

Synthesis and Evaluation of Novel Oxyalkylated Derivatives of 2′,4′-Dihydroxychalcone as Anti-Oomycete Agents against Bronopol Resistant Strains of Saprolegnia sp.

A series of novel oxyalkylchalcones substituted with alkyl groups were designed and synthesized, and the antioomycete activity of the series was evaluated in vitro against Saprolegnia strains. All tested O-alkylchalcones were synthesized by means of nucleophilic substitution from the natural compound 2′,4′-dihydroxychalcone (1) and the respective alkyl bromide. The natural chalcone (1) and 10 synthetic oxyalkylchalcones (2–11) were tested against Saprolegnia parasitica and Saprolegnia australis. Among synthetic analogs, 2-hydroxy,4-farnesyloxychalcone (11) showed the most potent activity against Saprolegnia sp., with MIC and MOC values of 125 µg/mL (similar to bronopol at 150 µg/mL) and 175 µg/mL, respectively; however, 2′,4′-dihydroxychalcone (1) was the strongest and most active molecule, with MIC and MOC values of 6.25 µg/mL and 12.5 µg/mL.

Chemical Characterization and Anti-Oomycete Activity of Laureliopsis philippianna Essential Oils against Saprolegnia parasitica and S. australis

Laureliopsis philippiana (Looser) R. Schodde (Monimiaceae) is a native tree widespread in the forest areas in the south of Chile and Argentina, known for its medicinal properties and excellent wood. The aim of this study was to evaluate the chemical composition of L. philippiana leaf and bark essential oils (EOs) using gas chromatography-mass spectrometry (GC-MS), and to quantify its anti-oomycete activity, specifically against Saprolegnia parasitica and S. australis. Only six components were identified in leaf EO, 96.92% of which are phenylpropanoids and 3.08% are terpenes. As for bark EO, 29 components were identified, representing 67.61% for phenylpropanoids and 32.39% for terpenes. Leaf EO was characterized mainly by safrole (96.92%) and β-phellandrene (1.80%). Bark EO was characterized mainly by isosafrole (30.07%), safrole (24.41%), eucalyptol (13.89%), methyleugenol (7.12%), and eugenol (6.01%). Bark EO has the most promising anti-Saprolegnia activity, with a minimum inhibition concentration (MIC) value of 30.0 µg/mL against mycelia growth and a minimum fungicidal concentration (MFC) value of 50.0 μg/mL against spores; for leaf EO, the MIC and MFC values are 100 and 125 µg/mL, respectively. These findings demonstrate that bark EO has potential to be developed as a remedy for the control of Saprolegnia spp. in aquaculture.

SYNTHESIS AND CONFORMATIONAL ANALYSIS OF LEPTOCARPIN DERIVATIVES: INFLUENCE OF MODIFICATION OF THE OXIRANE RINGON LEPTOCARPIN'S CYTOTOXIC ACTIVITY

The reaction in acidconditions of Leptocarpin 1, a compound with antitumor activity, formed two new isomeric products, 8b-angeloyl-1b,3b-dihydroxy-4,10-dimethyl,-D11(13) methylen-4Z,9Z-dieneheliangol-6,12-olide 2 and 8b-angeloyl-1b,3b-dihydroxy-4-methyl-D11(13),D11(14)-dimethylen-4Z-eneheliangol-6,12-olide 3, whose structures reported in this study were established by spectroscopy (1H-NMR, 13C-NMR, MS and IR) and confirmedthrough ROESY experiments and theoretical studies by molecular mechanics. The in vitro cytotoxicity of these isomeric compounds was less active than leptocarpin, showing the importance of the oxirane ring in the biological activity. Cytotoxic activity was measured in six cancer cell lines

Hemi-Synthesis and Anti-Oomycete Activity of Analogues of Isocordoin

An efficient synthesis of a series of 4′-oxyalkyl-isocordoin analogues (2–8) is reported for the first time. Their structures were confirmed by 1H-NMR, 13C-NMR, and HRMS. Their anti-oomycete activity was evaluated by mycelium and spores inhibition assay against two selected pathogenic oomycetes strains: Saprolegnia parasitica and Saprolegnia australis. The entire series of isocordoin derivatives (except compound 7) showed high inhibitory activity against these oomycete strains. Among them, compound 2 exhibited strong activity, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values of 50 µg/mL and 75 µg/mL, respectively. The results showed that 4′-oxyalkylated analogues of isocordoin could be potential anti-oomycete agents.

PRELIMINARY ANTIPROLIFERATIVE EVALUATION OF NATURAL,-SYNTHETIC BENZALDEHYDES AND BENZYL ALCOHOLS

Vanillin, o-vanillin, natural and synthetic benzaldehydes and benzyl alcohols were assessed for antiproliferative effects using different human cell lines. Benzyl alcohols were synthesized from benzaldehydes reduced with NaBH4 in methanol solution. A new method for deprotection of ether compounds with TiCl4 solution was achieved with better performance, than previously reported. Twenty four compounds were tested. The in vitro growth inhibition assay was based on sulphorhodamine dye to quantify cell viability. Catechol 9 derived from piperonal as well as compounds 4 and 12 showed higher cytotoxicity on breast and prostate cancer cell lines (MDA-MB-231 and PC-3 respectively). o-Vanillin 5 has the highest cytotoxicity for all cell lines. IC50 values of 35.40 ± 4.2 μM Breast MDA-MB231; 47.10 ± 3.8 μM Prostate PC-3; 72.50 + 5.4 μM Prostate DU-145; 85.10 + 6.5 μM and Colon HT-29, were obtained without toxicity towards dermal human fibroblast (DHF cells).

Structure-Activity Relationship of Dialkoxychalcones to Combat Fish Pathogen Saprolegnia australis

To investigate the anti-Saprolegnia activities of chalconic compounds, nine dialkoxychalcones 2–10, along with their key building block 2′,4′-dihydroxychalcone 1, were evaluated for their potential oomycide activities against Saprolegnia australis strains. The synthesis afforded a series of O-alkylated derivatives with typical chalcone skeletons. Compounds 4–10 were reported for the first time. Interestingly, analogue 8 with the new scaffold demonstrated remarkable in vitro growth-inhibitory activities against Saprolegnia strains, displaying greater anti-oomycete potency than the standard drugs used in the assay, namely fluconazole and bronopol. In contrast, a dramatic loss of activity was observed for O-alkylated derivatives 2, 3, 6, and 7. These findings have highlighted the therapeutic potential of the natural compound 1 scaffold to be exploitable as a drug lead with specific activity against various Saprolegnia strains.