Potential immunomodulatory response associated with L-mimosine in male Wistar rats.

Leucaena leucocephala is a plant that is used as animal and human food worldwide. This plant contains the toxic compound namely L-mimosine. The main mechanism of action of this compound involves its ability to chelate metal ions, which may interfere with the proliferative activity of cells and being studied for the treatment of cancer. However, little is known about the effect of L-mimosine on immune responses. Thus, the aim of this study was to evaluate the effects of L-mimosine on immune responses in Wistar rats. Different doses of L-mimosine (25, 40 and 60 mg/kg body weight/day) were administered orally by gavage to adult rats for 28 days. No clinical signs of toxicity were observed in animals, but a decrease in the T-dependent response to sheep red blood cells (SRBC) in animals treated with 60 mg/kg L-mimosine and an increase in the intensity of S. aureus phagocytosis by macrophages in animals treated with 40 or 60 mg/kg L-mimosine were observed. Therefore, these findings suggest that L-mimosine did not compromise macrophage activity and inhibited T-dependent clonal expansion during the immune response.

Calcitriol potentially alters HeLa cell viability via inhibition of autophagy.

Objective: This study was conducted to evaluate the effect of Calcitriol on cellular death in HeLa cells via autophagy and turn over due to mitochondria homeostasis. Methods: HeLa cell lines were grown in 24-well plates and treated with Calcitriol at varying doses (0.013 µM-0.325 µM) for varying time periods (2, 6, 12, and 18 h). Cell proteins were extracted with scrapers and lysed using RIPA buffer. Western blots were performed for proteins involved with autophagy (Lc3, p62), signaling (mTOR, PI3K, HIF1α), mitochondria (PGC1α, COX4, and Tom 20), and apoptosis (Caspase 3, Caspase 9, and PARP). Protein carbonyl levels were determined by measuring the indirect ROS level. An inhibition study using L-mimosine was performed to analyze the significance of HIF1α. Results: Calcitriol treatment induced cytotoxicity in a dose- and time-dependent manner and caused growth arrest in HeLa cells. The PI3K-AKT-mTOR pathway was activated, leading to inhibition of autophagy and alterations in mitochondria biogenesis homeostasis. Treatment with Calcitriol produced protein carbonyl levels similar to those in the cisplatin-treated and control groups. Increased ROS levels may cause toxicity and induce cell death specifically in cancer cells but not in normal cells. The inhibition of HIF1α partially rescued the HeLa cells from the toxic effects of Calcitriol treatment. Conclusion: We suggest that Calcitriol may shut down mitochondrial homeostasis in HeLa cells by inducing the PI3K-AKT-mTOR pathway and inhibiting autophagy, which leads to cell death.

Methods of Mimosine Extraction from Leucaena leucocephala (Lam.) de Wit Leaves.

Mimosine is a nonprotein amino acid biosynthesized from OAS (O-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3,4-dihydroxypyridine). This amino acid constitutively occurs in all parts of Leucaena leucocephala (Lam.) de Wit plants and is found at higher concentrations in seeds and leaves. This metabolite has several useful activities, such as antioxidant, allelochemical, insecticidal, antimicrobial, metal chelating, and antitumor. Mimosine is well studied in biomedical research due its ability to inhibit cells in the late G1 phase and to induce cell apoptosis. Two simple methods of mimosine extraction from leucaena leaves, pulverized and whole maceration, are described herein in detail.

Influence of the Nonprotein Amino Acid Mimosine in Peptide Conformational Propensities from Novel Amber Force Field Parameters.

Mimosine is a nonprotein amino acid derived from plants known for its ability to bind to divalent and trivalent metal cations such as Zn2+, Ni2+, Fe2+, or Al3+. This results in interesting antimicrobial and anticancer properties, which make mimosine a promising candidate for therapeutic applications. One possibility is to incorporate mimosine into synthetic short peptide drugs. However, how this amino acid affects the peptide structure is not well understood, reducing our ability to design effective therapeutic compounds. In this work, we used computer simulations to understand this question. We first built parameters for the mimosine residue to be used in combination with two classical force fields of the Amber family. Then, we used atomistic molecular dynamics simulations with the resulting parameter sets to evaluate the influence of mimosine in the structural propensities for this amino acid. We compared the results of these simulations with homologous peptides, where mimosine is replaced by either phenylalanine or tyrosine. We found that the strong dipole in mimosine induces a preference for conformations where the amino acid rings are stacked over more extended conformations. We validated our results using quantum mechanical calculations, which provide a robust foundation for the outcome of our classical simulations.

Anthelmintic Effect of Leucaena leucocephala Extract and Its Active Compound, Mimosine, on Vital Behavioral Activities in Caenorhabditis elegans.

Helminth infections continue to be a neglected global threat in tropical regions, and there have been growing cases of anthelmintic resistance reported towards the existing anthelmintic drugs. Thus, the search for a novel anthelmintic agent has been increasing, especially those derived from plants. Leucaena leucocephala (LL) is a leguminous plant that is known to have several pharmacological activities, including anthelmintic activity. It is widely known to contain a toxic compound called mimosine, which we believed could be a potential lead candidate that could exert a potent anthelmintic effect. Hence, this study aimed to validate the presence of mimosine in LL extract and to investigate the anthelmintic effect of LL extract and mimosine on head thrashing, egg-laying, and pharyngeal pumping activities using the animal model Caenorhabditis elegans (C. elegans). Mimosine content in LL extract was confirmed through an HPLC analysis of spiking LL extract with different mimosine concentrations, whereby an increasing trend in peak heights was observed at a retention time of 0.9 min. LL extract and mimosine caused a significant dose-dependent increase in the percentage of worm mortality, which produced LC50s of 73 mg/mL and 6.39 mg/mL, respectively. Exposure of C. elegans to different concentrations of LL extract and mimosine significantly decreased the head thrashing, egg-laying, and mean pump amplitude of pharyngeal pumping activity. We speculated that these behavioral changes are due to the inhibitory effect of LL extract and mimosine on an L-type calcium channel called EGL-19. Our findings provide evidential support for the potential of LL extract and its active compound, mimosine, as novel anthelmintic candidates. However, the underlying mechanism of the anthelmintic action has yet to be elucidated.

A novel methylated analogue of L-Mimosine exerts its therapeutic potency through ROS production and ceramide-induced apoptosis in malignant melanoma.

Melanoma is an aggressive and highly metastatic type of skin cancer where the design of new therapies is of utmost importance for the clinical management of the disease. Thus, we have aimed to investigate the mode of action by which a novel methylated analogue of L-Mimosine (e.g., L-SK-4) exerts its therapeutic potency in an in vitro model of malignant melanoma. Cytotoxicity was assessed by the Alamar Blue assay, oxidative stress by commercially available kits, ROS generation, caspase 3/7 activation and mitochondrial membrane depolarisation by flow cytometry, expression of apoptosis-related proteins by western immunoblotting and profiling of lipid biosynthesis by a metabolomic approach. Overall, higher levels of ROS, sphingolipids and apoptosis were induced by L-SK-4 suggesting that the compound's therapeutic potency is mediated through elevated ROS levels which promote the upregulation of sphingolipid (ceramide) biosynthesis thus leading to the activation of both extrinsic and intrinsic apoptosis, in an experimental model of malignant melanoma.

The identification of dual protective agents against cisplatin-induced oto- and nephrotoxicity using the zebrafish model.

Dose-limiting toxicities for cisplatin administration, including ototoxicity and nephrotoxicity, impact the clinical utility of this effective chemotherapy agent and lead to lifelong complications, particularly in pediatric cancer survivors. Using a two-pronged drug screen employing the zebrafish lateral line as an in vivo readout for ototoxicity and kidney cell-based nephrotoxicity assay, we screened 1280 compounds and identified 22 that were both oto- and nephroprotective. Of these, dopamine and L-mimosine, a plant-based amino acid active in the dopamine pathway, were further investigated. Dopamine and L-mimosine protected the hair cells in the zebrafish otic vesicle from cisplatin-induced damage and preserved zebrafish larval glomerular filtration. Importantly, these compounds did not abrogate the cytotoxic effects of cisplatin on human cancer cells. This study provides insights into the mechanisms underlying cisplatin-induced oto- and nephrotoxicity and compelling preclinical evidence for the potential utility of dopamine and L-mimosine in the safer administration of cisplatin.

Generation of reactive oxygen species by hydroxypyridone compound/iron complexes.

Objectives: Prooxidant properties of iron-binding hydroxypyridone compounds including deferiprone and mimosine were analyzed. Methods: Hydroxypyridone/iron-dependent production of reactive oxygen species was evidenced by the inactivation of aconitase, the most sensitive enzyme to oxidative stress in permeabilized yeast cells. Results and Discussion: Deferiprone and mimosine produced reactive oxygen species in the presence of ferrous sulfate. The inactivation required sodium azide the inhibitor of catalase, and addition of TEMPOL, a scavenger of superoxide radical, protected aconitase from the inactivation, suggesting that the superoxide radical produced from the hydroxypyridone/iron complex is responsible for the inactivation of aconitase. A principal role of superoxide radical was further supported by the finding that the hydroxypyridone/iron complex can inactivate aconitase in the presence of cyanide the inhibitor of superoxide dismutase. Deferiprone and mimosine stimulated the Fe2+ oxidation, resulting in the one-electron reduction of oxygen to form superoxide anion, which can inactivate aconitase by oxidizing the prosthetic iron-sulfur cluster. Mimosine further stimulated the ascorbate/iron-dependent formation of 8-hydroxy-2'-deoxyguanosine in DNA. Conclusion: Biological toxicity of mimosine and deferiprone reported previously can be accounted for by the prooxidant properties of hydroxypyridone compounds: coordination complex with iron generates reactive oxygen species resulting in the disturbance of mitochondrial energy metabolism and DNA damage.

Metal self-assembly mimosine peptides with enhanced antimicrobial activity: towards a new generation of multitasking chelating agents.

Mimosine is a non-protein amino acid with various properties, such as antibacterial, anti-inflammatory, anti-cancer and anti-virus among others. Due to its structural similarity with deferiprone (DFP), mimosine is a potential excellent metal chelator. In the present work, we combine experimental and theoretical (DFT) approaches in order to investigate the properties of mimosine peptides. Six different peptides were synthesized and their complex stoichiometry and stability were characterized by means of UV-Vis spectrophotometry. Then, the binding mode and self-assembly features of the peptides were evaluated using a DFT approach, taking into account different number of mimosine amino acids and varying the length of the spacer between the mimosine residues, and there was good agreement between experimental data and computational calculations. Further elucidations of the structural properties of these peptides allowed us to propose improvements in the structure of the mimosine moiety which can lead to enhanced affinity for high-valent metals. Moreover, we demonstrate that these peptides show an anti-microbial activity against Gram positive bacteria that is enhanced by the formation of a complex with iron(iii) ions. The mimosine peptides could be an alternative to antimicrobial peptides (AMPs), which are expensive and susceptible to proteolytic degradation. In summary, in the present work, we propose a new generation of multipurpose mimosine-based peptides as new metal self-assembly chelators which could be a turning point in biomedical and nanotechnological applications.

Anticancer activity of a novel methylated analogue of L-mimosine against an in vitro model of human malignant melanoma.

The anticancer activity of a series of novel synthesized, hydroxypyridone-based metal chelators (analogues of L-mimosine) was evaluated in an in vitro model of melanoma consisting of malignant melanoma (A375), non-melanoma epidermoid carcinoma (A431) and immortalized non-malignant keratinocyte (HaCaT) cells. More specifically, we have demonstrated that the L-enantiomer of a methylated analogue of L-mimosine (compound 22) can exert a potent anticancer effect in A375 cells when compared to either A431 or HaCaT cells. Moreover, we have demonstrated that this analogue has the ability to i) promote increased generation of reactive oxygen species (ROS), ii) activate both intrinsic and extrinsic apoptosis and iii) induce perturbations in cell cycle growth arrest. Our data highlights the potential of compound 22 to act as a promising therapeutic agent against an in vitro model of human malignant melanoma.

Mimosine increases the expressions of tyrosine phosphorylated protein in mouse seminal vesicles / La mimosina aumenta la expresión de la proteína tirosina fosforilada en las vesículas seminales del ratón

Acute effect of purified mimosine (MiMo) extracted from Leucaena leucocephala on testicular histopathology has been documented with seminal vesicle (SV) atrophy. Since protein phosphorylation and seminal secretions play important roles in sperm physiology, this study aimed to study the alteration of substances including tyrosine phosphorylated (TyrPho) proteins in seminal vesicle treated with MiMo. Male mice were divided into a control and experimental groups treated with purified MiMo at 3 doses of 15, 30, and 60 mg/KgBW, respectively for 35 consecutive days. The morphology and weights of SV were compared among groups. The levels of magnesium and fructosamine in SV fluid were assayed. The profiles of equally SV total proteins were compared using SDS-PAGE. The expression of seminal TyrPho proteins was detected by western blotting. Recent results showed the decreased weights of SV in MiMo treated mice compared to control. However MiMo in all doses did not affect the levels of magnesium and fructosamine in SV fluid. The SV protein expression of 130 and 55 kDas was obviously decreased in a high dose MiMo. In dose-dependent response, the expressions of 72 and 55 kDas TyrPho proteins of SV were increased. In conclusion, MiMo could affect SV morphological size and protein secretions especially TyrPho proteins.

El efecto agudo de la mimosina purificada (MiMo) extraída de Leucaena leucocephala en la histopatología testicular se ha documentado con atrofia de vesícula seminal (VS). Debido a que la fosforilación de proteínas y las secreciones seminales tienen un papel importante en la fisiología de los espermatozoides, este estudio tuvo como objetivo estudiar la alteración de sustancias como la proteína tirosina fosforilada (TyrPho) en vesículas seminales tratadas con MiMo. Los ratones se dividieron en un grupo control y un grupo experimental y se trataron con MiMo purificado en 3 dosis de 15, 30 y 60 mg / KgBW, respectivamente, durante 35 días seguidos. La morfología y los pesos de VS se compararon entre los grupos. Fueron analizados los niveles de magnesio y fructosamina en el fluido VS. Los perfiles de las proteínas totales de VS se compararon utilizando SDS-PAGE. La expresión de la proteína TyrPho en las vesículas seminales se detectó mediante transferencia de Western blot. Los resultados recientes muestran la disminución del peso de las VS en ratones tratados con MiMo, en comparación con el grupo control. Sin embargo, en ninguna de las dosis se vieron afectados por mimosina purificada los niveles de magnesio y fructosamina en el líquido de las VS. La expresión de la proteína en VS de 130 y 55 kDas disminuyó notablemente en una dosis alta de MiMo. En la respuesta dependiente de la dosis, aumentaron las expresiones de 72 y 55 kDas de las proteínas TyrPho en las VS. En conclusión, la mimosina purificada podría afectar el tamaño morfológico de las VS y la expresión de proteínas, especialmente las proteínas TyrPho.

A Bio-inspired Hypoxia Sensor using HIF1a-Oxygen-Dependent Degradation Domain.

Functional imaging has become an important tool in oncology because it not only provides information about the size and localization of the tumour, but also about the pathophysiological features of the tumoural cells. One of the characteristic features of some tumour types is that their fast growth leads to deficient intratumoral vascularization, which results in low oxygen availability. To overcome this lack of oxygen, tumoural cells activate the neoangiogenic program by upregulating the transcription factor HIF-1α. Herein we report a non-invasive in vitro detection method of hypoxia using designed fluorescent peptide probes based on the oxygen-dependent degradation domain of HIF-1α. The fluorescent probe retains the oxygen-sensing capability of HIF-1α, so that it is stabilized under hypoxia and readily degraded by the proteasome under normoxia, thus providing direct information of the cellular oxygen availability.

Angiopoietin-like 4 production upon treatment with hypoxia and L-mimosine in periodontal fibroblasts.

BACKGROUND AND OBJECTIVE: A key factor in the modulation of angiogenesis as well as in bone resorption is angiopoietin-like 4. However, the role of angiopoietin-like 4 in periodontal tissue is unknown. Here, we hypothesized that hypoxia and the hypoxia mimetic agent L-mimosine can induce the production of angiopoietin-like 4 in periodontal fibroblasts. METHODS: Human periodontal ligament fibroblasts (PDLF) were cultured in monolayer and spheroid cultures. The cultures were incubated in the presence of hypoxia or L-mimosine. Angiopoietin-like 4 mRNA and protein levels were measured by qPCR and ELISA, respectively. Also, the impact of Lipopolysaccharides of E. coli and P. gingivalis, interleukin (IL)-1ß and tumor necrosis factor (TNF)α was evaluated. Furthermore, we tested dependency on hypoxia-inducible factor (HIF)-1 activity by Western blotting for HIF-1 and inhibitor studies with echinomycin. Potential autocrine effects were assessed by exposure of PDLF to recombinant angiopoietin-like 4 in full length, C-terminal and N-terminal fragments. The impact on viability, DNA synthesis, alkaline phosphatase, and matrix mineralization was evaluated. RESULTS: Both hypoxia and L-mimosine elevated angiopoietin-like 4 mRNA and protein levels in monolayer cultures of PDLF. HIF-1 was elevated after both hypoxia and L-mimosine treatment. LPS, IL-1ß, and TNFα did not modulate angiopoietin-like 4 levels significantly. Addition of echinomycin in the cultures inhibited the production of angiopoietin-like 4. In spheroid cultures of PDLF, the increase did not reach the level of significance at mRNA and protein levels. Angiopoietin-like 4 in full length, C-terminal, and N-terminal fragments did not modulate viability, DNA synthesis, alkaline phosphatase, and matrix mineralization. CONCLUSION: Overall, we found that hypoxia and the hypoxia mimetic agent L-mimosine can stimulate angiopoietin-like 4 production in monolayer cultures of PDLF. This increase depends on HIF-1 activity. Future studies will reveal how the modulation of angiopoietin-like 4 in the periodontium contributes to periodontal disease and regeneration.

The impact of clay-based hypoxia mimetic hydrogel on human fibroblasts of the periodontal soft tissue.

Thixotropic clays have favorable properties for tissue regeneration. Hypoxia mimetic agents showed promising results in pre-clinical models for hard and soft tissue regeneration. It is unclear if clays can be used as carrier for hypoxia mimetic agent in a periodontal regenerative setting. Here, we tested the response of human fibroblasts of the periodontal soft tissue to synthetic clay hydrogels and assessed hypoxia mimetic agent release. Cells were cultured on synthetic clay hydrogels (5.00%-0.15%). We assessed viability and differentiation capacity with resazurin-based toxicity assays, MTT staining, Live-Dead staining, and alkaline phosphatase staining. To reveal the response of fibroblasts to hypoxia mimetic agent-loaded clay hydrogels, cells were exposed to clay supplemented with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2. Supernatants from hypoxia mimetic agent-loaded clay hydrogels were harvested and replaced with medium at hour 1, 3, 6, 24, 48, and 72. To reveal the hypoxia mimetic capacity of supernatants, vascular endothelial growth factor production in the fibroblasts was assessed in the culture medium. Our data show that clay did not induce relevant toxic effects in the fibroblasts which remained capable to differentiate into alkaline phosphatase-positive cells at the relevant concentrations. Fibroblasts cultured on clay hydrogel loaded with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl2 remained vital, however, no significant increase in vascular endothelial growth factor levels was found in the culture medium. Only dimethyloxalylglycine-loaded clay supernatants taken in the first hours stimulated vascular endothelial growth factor production in fibroblasts. In conclusion no pronounced toxic effects of synthetic clay were observed. Supplementation with dimethyloxalylglycine leads to hypoxia mimetic activity. This pilot study provides first insights into the impact of synthetic clay on periodontal tissue.

Cell cycle inhibitors improve seed storability after priming treatments.

Seed priming is a treatment that controls seed water content to partially activate germination processes such as metabolism but prevents full germination of the seeds. The treatment is well known to enhance seed performance, including germination, but sometimes reduces seed storability or longevity as a side effect. Toward developing a novel priming technique that can maintain seed longevity for a longer time period, chemicals that suppress the seed deterioration under a controlled condition were screened from 80 known biologically active compounds contained in the RIKEN NPDepo authentic library using Arabidopsis thaliana seeds. Seeds primed with mimosine, a cell cycle inhibitor, retained higher survival rate after a controlled deterioration treatment compared to seeds primed without the chemical. In addition, other cell cycle inhibitors such as aphidicolin, hydroxyurea and oryzalin had similar effects on the seed storability after priming. Our results suggest that progression of the cell cycle during priming is an important checkpoint that determines the storability of seeds after the treatment.

Oestrogenic activity of mimosine on MCF-7 breast cancer cell line through the ERα-mediated pathway.

Hormone replacement therapy has been a conventional treatment for postmenopausal symptoms in women. However, it has potential risks of breast and endometrial cancers. The aim of this study was to evaluate the oestrogenicity of a plant-based compound, mimosine, in MCF-7 cells by in silico model. Cell viability and proliferation, ERα-SRC1 coactivator activity and expression of specific ERα-dependent marker TFF1 and PGR genes were evaluated. Binding modes of 17ß-oestradiol and mimosine at the ERα ligand binding domain were compared using docking and molecular dynamics simulation experiments followed by binding interaction free energy calculation with molecular mechanics/Poisson-Boltzmann surface area. Mimosine showed increased cellular viability (64,450 cells/ml) at 0.1 µM with significant cell proliferation (120.5%) compared to 17ß-oestradiol (135.2%). ER antagonist tamoxifen significantly reduced proliferative activity mediated by mimosine (49.9%). Mimosine at 1 µM showed the highest ERα binding activity through increased SRC1 recruitment at 186.9%. It expressed TFF1 (11.1-fold at 0.1 µM) and PGR (13.9-fold at 0.01 µM) genes. ERα-mimosine binding energy was -49.9 kJ/mol, and it interacted with Thr347, Gly521 and His524 of ERα-LBD. The results suggested that mimosine has oestrogenic activity.

Core circadian clock gene expression in human dental pulp-derived cells in response to L-mimosine, hypoxia and echinomycin.

Core circadian clock genes set the pace for a wide range of physiological functions, including regeneration. The role of these genes and their regulation in the dental pulp, in particular under hypoxic conditions, is unknown. Here we investigated if core clock genes are expressed in human dental pulp-derived cells (DPC) and if their expression is modulated by the hypoxia mimetic agent, L-mimosine (L-MIM), hypoxia or echinomycin. Dental pulp-derived cells in monolayers and spheroids were treated with L-MIM, hypoxia or echinomycin. mRNA levels of the core circadian clock genes were analysed using quantitative PCR (qPCR) and their protein levels were analysed by western blot. All core clock genes and proteins were produced in DPC monolayer and spheroid cultures. The expression of cryptochrome circadian regulators and period circadian regulators was reduced by L-MIM, hypoxia and echinomycin at mRNA, but not at protein levels. Time course experiments indicated that modulations were based on alterations in overall mRNA levels of core circadian clock genes. Our results suggest a potential role of the core circadian clock in the response of dental pulp to hypoxia. Future studies need to consider that regulation of the core circadian clock at mRNA levels might not be paralleled by modulation of protein levels.

Acute effects of mimosine purified from Leucaena leucocephala on male reproductive system of adult mice / Efectos agudos de la mimosina purificada de Leucaena leucocephala en el sistema reproductor masculino de ratones adultos

This study attempted to examine the acute effect of purified minosine extracted from Leucaena leucocephala on male reproductive system. Adults male mice were divided into 4 groups (n =8); control and 3 experimental groups treated with purified mimosine at different doses of 15, 30, and 60 mg/KgBW, respectively for 7 consecutive days. The morphological features and weights of body and reproductive organs including testis, epididymis plus vas deferens, and seminal vesicle were compared among groups. In addition, epididymal sperm concentration and the changes of histopathology of testicular tissues in all groups were observed. The results showed that mimosine in all doses did not affect mice body weights. However, all doses of mimosine could significantly reduce the absolute and relative weights of testis and seminal vesicle but not of epididymis plus vas deferens. Significantly, mimosine at doses of 30, and 60 mg/KgBW could decrease sperm concentration. Moreover, the seminiferous atrophy and degeneration were obviously found in mimosine treated mice as compared to the control. In conclusion, consumption of Leucaena leucocephala edible parts containing mimosine could damage male reproductive organs which may cause acute male subfertility or infertility.

Este estudio intentó examinar el efecto agudo de la mimosina purificada extraída de Leucaena leucocephala en el sistema reproductivo masculino. Se dividieron ratones machos adultos en 4 grupos (n = 8): un grupo control y tres grupos experimentales tratados con mimosina purificada a diferentes dosis de 15, 30 y 60 mg / Kg por peso, respectivamente, durante 7 días consecutivos. Se compararon entre los grupos, las características morfológicas y el peso corporal, los órganos reproductivos, incluyendo los testículos, el epidídimo más conducto deferente y vesícula seminal. Además, se observó la concentración de espermatozoides epididimarios y los cambios de la histopatología de los tejidos testiculares en todos los grupos. Los resultados mostraron que la mimosina no afectó los pesos corporales de los ratones. Sin embargo, todas las dosis de mimosina podrían reducir significativamente los pesos absolutos y relativos de los testículos y las glándulas seminales, pero no así del epidídimo y los conductos deferentes. La mimosina en dosis de 30 y 60 mg / Kg por peso podría disminuir significativamente la concentración de esperma. Además, se observó la atrofia y degeneración seminífera en ratones tratados con mimosina en comparación con el grupo control. En conclusión, el consumo de partes comestibles de Leucaena leucocephala que contienen mimosina podría dañar los órganos reproductivos masculinos, lo que puede causar subfertilidad masculina aguda o infertilidad.

Do hypoxia and L-mimosine modulate sclerostin and dickkopf-1 production in human dental pulp-derived cells? Insights from monolayer, spheroid and tooth slice cultures.

BACKGROUND: To understand the responses of the dental pulp to hypoxia is of high relevance for regenerative endodontics and dental traumatology. Here, we aimed to reveal the effects of hypoxia and the hypoxia mimetic agent L-mimosine (L-MIM) on the production of sclerostin (SOST) and dickkopf-1 (DKK-1) in human dental pulp-derived cells (DPC). METHODS: DPC in monolayer, spheroid and tooth slice cultures were treated with L-MIM or hypoxia. Resazurin-based toxicity and MTT assays were performed to determine cell viability. mRNA and protein levels of SOST and DKK-1 were measured with quantitative reverse transcription PCR and ELISA, respectively. To validate the hypoxia-like response, SDF-1, VEGF and IL-8 were assessed. In addition Western blots for HIF-1α, HIF-2α and HIF-3α were done. RESULTS: Cells were vital upon treatment procedures and showed increased levels of HIF-1α, and HIF-2α. In monolayer cultures, mRNA levels of SOST and DKK-1 were downregulated by L-MIM and hypoxia, respectively. A significant downregulation of SOST by hypoxia was found at the protein level compared to untreated cells while the effect on DKK-1 and the impact of L-MIM on SOST and DKK-1 did not reach the level of significance at the protein level. In spheroid cultures, mRNA levels of SOST and DKK-1 were downregulated by L-MIM. A significant downregulation of DKK-1 upon hypoxia treatment was found at the protein level while the impact of hypoxia on SOST and the effect of L-MIM on SOST and DKK-1 did not reach the level of significance. SOST and DKK-1 were also produced in tooth slices, but no pronounced modulation by L-MIM or hypoxia was found. Evaluation of SDF-1, VEGF and IL-8 showed a hypoxia-like response in the culture models. CONCLUSIONS: There is no pronounced influence of hypoxia and L-MIM on DPC viability, SOST and DKK-1 protein production. However, the specific response depends on the culture model and the level of evaluation (mRNA or protein). These results deepen our understanding about the role of hypoxia and the potential impacts of hypoxia-based strategies on dental pulp.

L­mimosine induces caspase­9­mediated apoptosis in human osteosarcoma cells.

L-mimosine is a rare plant amino acid extracted from Mimosa or Leucaena spp., and it has been reported to exhibit antitumor activity in a number of types of cancer. However, the underlying mechanisms remain to be clarified. In the present study, the effect of L­mimosine was investigated in human osteosarcoma cells. A Cell Counting Kit­8 assay and flow cytometry were used for toxicity detection. Hoechst staining and transmission electron microscopy (TEM), in addition to western blot analysis, were used for the examination of the associated mechanisms. The results of the present study indicated that L­mimosine significantly inhibited cell proliferation by inducing cellular apoptosis in osteosarcoma cells. The Hoechst staining results and TEM revealed that nuclear damage increased with the concentration increase in L­mimosine, as did the formation of apoptotic bodies. Additionally, the results of the western blot analysis confirmed that the treatment of cells with L­mimosine was accompanied by increasing expression of cleaved caspase­9. L­mimosine­induced apoptosis was inhibited by the caspase­9 inhibitor Z­LEHD­FMK. In addition, the extracellular signal­regulated kinase (ERK) signaling pathway was suppressed following treatment with L­mimosine. In conclusion, the results of the present study suggested that L­mimosine induced apoptosis via the mitochondrial apoptotic pathway. The ERK signaling pathway was indicated to be an additional mechanism underlying apoptosis induction. The results provided evidence for the use of L­mimosine as a promising candidate for osteosarcoma therapy.