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.

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.

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.

Comparison of the effects of mesquite pod and Leucaena extracts with phytoestrogens on the reproductive physiology and sexual behavior in the male rat.

Mesquite (Prosopis sp.) and Leucaena leucocephala are widespread legumes, widely used to feed several livestock species and as food source for human populations in several countries. Both mesquite and Leucaena contain several phytoestrogens which might have potential estrogenic effects. Thus, the aim of this study was to evaluate the effects of mesquite pod and Leucaena extracts on several aspects of behavior and reproductive physiology of the male rat. The effects of the extracts were compared with those of estradiol (E2) and of two isoflavones: daidzein (DAI) and genistein (GEN). The following treatments were given to groups of intact male rats: vehicle; mesquite pod extract; Leucaena extract; E2; DAI; GEN. The results indicate that mesquite pod and Leucaena extracts disrupt male sexual behavior in a similar way to DAI and GEN, but less than E2. The main disruptor of sexual behavior was E2, however after 40 and 50days of administration, both extracts and phytoestrogens disrupted sexual behavior in a similar way to E2. The extracts also increased testicular germ cell apoptosis, decreased sperm quality, testicular weight, and testosterone levels, as phytoestrogens did, although these effects were less than those caused by estradiol. The number of seminiferous tubules with TUNEL-positive germ cells increased in extracts treated groups in a similar way to phytoestrogens groups, and E2 caused the greatest effect. The number of TUNEL-positive cells per tubule increased only in Leucaena extract and E2 groups, but not in mesquite- and phytoestrogens-treated groups. Spermatocytes and round spermatids were the TUNEL-positive cells observed in all experimental groups. This effect was associated with smaller testicular weights without atrophy in experimental groups compared with control. Testicular atrophy was only observed in estradiol-treated males. Testosterone decreased in males of all experimental groups, compared with control, this androgen was undetectable in E2 treated males. These results suggest that mesquite pod and Leucaena extracts cause effects similar to those of phytoestrogens in male rat reproduction, these effects were lower than those caused by E2.

Insecticidal and Nematicidal Activities of Novel Mimosine Derivatives.

Mimosine, a non-protein amino acid, is found in several tropical and subtropical plants, which has high value for medicine and agricultural chemicals. Here, in continuation of works aimed to development of natural product-based pesticidal agents, we present the first significant findings for insecticidal and nematicidal activities of novel mimosine derivatives. Interestingly, mimosinol and deuterated mimosinol (D-mimosinol) from mimosine had strong insecticidal activity which could be a result of tyrosinase inhibition (IC50 = 31.4 and 46.1 µM, respectively). Of synthesized phosphoramidothionate derivatives from two these amino alcohols, two compounds (1a and 1b) showed high insecticidal activity (LD50 = 0.5 and 0.7 µg/insect, respectively) with 50%-60% mortality at 50 µg/mL which may be attributed to acetylcholinesterase inhibition. Compounds 1a and 1b also had strong nematicidal activity with IC50 = 31.8 and 50.2 µM, respectively. Our results suggest that the length of the alkyl chain and the functional group at the C5-position of phosphoramidothionates derived from mimosinol and d-mimosinol are essential for the insecticidal and nematicidal activities. These results reveal an unexplored scaffold as new insecticide and nematicide.

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor in dental pulp-derived cells.

INTRODUCTION: Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp-derived cells. METHODS: To test the response of dental pulp-derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, (3)[H]thymidine, and (3)[H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). RESULTS: We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp-derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. CONCLUSIONS: These results show that PHD inhibitors can stimulate VEGF production of dental pulp-derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Xanthine oxidase/tyrosinase inhibiting, antioxidant, and antifungal oxindole alkaloids from Isatis costata.

Phytochemical investigations on the ethyl acetate soluble fraction of the whole plant of Isatis costata Linn. (Brassicaseae) led to the isolation of the oxindole alkaloids costinones A (1), B (2), isatinones A (3), B (4), indirubin (5), and trisindoline (6). Compounds 1-6 displayed significant to moderate inhibition against xanthine oxidase enzyme with IC50 values ranging from 90.3+/-0.06 to 179.6+/-0.04 microM, whereas the standard inhibitor of xanthine oxidase (allopurinol) had an IC(50) value of 7.4+/-0.07 microM. Compounds 1 (IC50 7.21+/-0.05 microM), 2 (IC50 9.40+/-0.03 microM), 3 (IC50 11.51+/-0.07 microM), 4 (IC50 12.53+/-0.06 microM), 5 (IC50 14.29+/-0.09 microM), and 6 (IC50 17.34+/-0.04 microM) exhibited pronounced activities when compared with the standard tyrosinase inhibitor L-mimosine (IC50 3.70+/-0.03 microM), along with DPPH radical scavenging activity with IC50 226, 270, 300, 320, 401, and 431 microM, respectively. The crude extract and compounds 1, 2, 5, and 6 showed significant antifungal activity against Trichophyton schoen leinii, Aspergillus niger, Candida albicans, Trichophyton simii, and Macrophomina phaseolina.

Mimosine mitigates oxidative stress in selenium deficient seedlings of Vigna radiata. Part II: mitochondrial uptake of 75selenium and mimosine.

During the growth of selenium (Se)-deficient seedlings of Vigna radiata, exposure to mimosine [2-amino-3-(3-hydroxy-4-oxo-1H-pyridin-1-yl)-propanoic acid], a nonprotein plant amino acid, effectively mitigated stress at 0.1 mM, as reflected in enhancement of growth and efficiency of mitochondrial functions. Since the changes in the seedlings elicited by exposure to mimosine were similar to those effected by Se at an optimal exposure level of 0.75 ppm (Sreekala et al., Biol Trace Elem Res 70:193-207, 1999), the uptake of Se and that of mimosine itself was individually studied in the respiring mitochondria of Se-deficient seedlings (-Se-stressed group) in comparison with those exposed to mimosine during growth at 0.1 mM (Mim 0.1 group). In both groups, the mitochondrial uptake of (75)Se at 10 microM added Na(2)(75)SeO(3), increased linearly up to 2 min, attaining steady-state levels thereafter. Uptake levels were 2.3-fold higher in the Mim 0.1 group than in the -Se-stressed group. Double-reciprocal plots of mitochondrial (75)Se uptake against 2-20 microM Na(2)(75)SeO(3) in the medium were nonlinear and negative cooperative effects during the uptake were confirmed by Scatchard plots, whereas Hill coefficients were 0.8 and 0.85 for the two groups. Mitochondrial uptake of mimosine, at added levels of 25 or 50 microM, increased linearly up to 1 min and decelerated thereafter. Initial uptake levels of mimosine at 1 min were higher by 6.5-fold at 25 microM and 4-fold at 50 microM in the Mim 0.1 group than those in the -Se-stressed group. Initial uptake levels with added mimosine up to 50 or 100 microM yielded nonlinear double-reciprocal plots; and kinetic analyses at 5 to 50 microM revealed the prevalence of positive cooperativity in the -Se-stressed group and negative cooperativity in the Mim 0.1 group. Involvement of active thiol groups in the uptake of both Se and mimosine were indicated by inhibition studies. Evidence presented for mimosine mediated increase in mitochondrial Se uptake and cooperative interactions thereof underscores the metabolic significance of mimosine.

Mimosine mitigates oxidative stress in selenium deficient seedlings of Vigna radiata--Part I: Restoration of mitochondrial function.

Mimosine, a non-protein plant amino acid found in Mimosa pudica and certain species of Leucaena, was beneficial for the growth of seedlings of Vigna radiata germinated under selenium-deficient stressed condition (-Se stressed) despite the recognized toxicity of the allelochemical. Exposure of mimosine at 0.1 mM (Mim-0.1) promoted the growth of the seedlings and significantly enhanced mitochondrial functional efficiency. Growth-related parameters including root and shoot lengths and dry weight were increased by 44-58% in the Mim-0.1 group compared to that of the -Se-stressed group. Oxygen uptake by mitochondria of Mim-0.1 group, studied with different substrates, revealed enhanced State 3 respiratory rates with regulated State 4 rates, resulting in high respiratory control ratio (RCR) of 3.4 to 3.9 indicative of a high degree of oxidative coupling. Specific activities of mitochondrial electron transport enzymes, nicotinamide adenine dinucleotide (reduced form) (NADH)-cytochrome (cyt) c oxidoreductase, succinate dehydrogenase, and cyt c oxidase in the Mim-0.1 group were enhanced by 53% to threefold over those of the Se-stressed group. Marked decreases in the extent of mitochondrial lipid peroxidation ensued upon mimosine exposure, indicative of its antioxidant function. Mitochondrial 45Ca2+ uptake was notably augmented twofold in the Mim-0.1 group, compared to the Se-stressed group. Detailed kinetic analyses of Ca2+ uptake revealed positive cooperative interactions in both -Se-stressed group and Mim-0.1 groups with Hill coefficient (nH) values of 1.7 and 2, respectively. The present study establishes the beneficial effects of mimosine exposure at 0.1 mM on the growth and mitochondrial function of the seedlings grown under selenium-deficient stressed condition and a significant physiological role can be ascribed to mimosine.

Antidermatophytic and bacterial activity of mimosine.

Mimosine, a non-protein aromatic amino acid was tested at 100, 50 and 25 microg/mL on some human pathogenic bacteria and fungi. Mimosine exhibited total lethality towards Trichophyton tonsurans and Trichophyton rubrum at 100 microg/mL. Among the tested bacteria Staphylococcus aureus was inhibited to a larger extent than the other bacteria. The studies revealed mimosine to be potent against fungi rather than bacteria. This study reports the effect of mimosine on dermatophytes for the first time.

Mimosine prevents the death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite.

Immunostimulated astrocytes become highly vulnerable to glucose deprivation (Choi and Kim: J Neurosci Res 54:870-875, 1998a). The increased vulnerability is caused by the enhanced level of peroxynitrite endogenously produced in glucose-deprived immunostimulated astrocytes. In the present study, we report that the plant amino acid mimosine can attenuate the increased death by scavenging peroxynitrite. Treatment with mimosine blocked the increase of nitrotyrosine immunoreactivity, a marker of peroxynitrite, in glucose-deprived immunostimulated astrocytes. Furthermore, mimosine directly inhibited the nitration of tyrosine residues of bovine serum albumin and the oxidation of dihydrorhodamine-123 to rhodamine-123 by peroxynitrite. Mimosine has been used experimentally as a cell cycle G1/S phase transition blocker (Lalande: Exp Cell Res 186:332-339, 1990; Hoffman et al.: Cytometry 12:26-32, 1991). Flow cytometry analysis, however, showed that the cytoprotective effect of mimosine was not attributed to its inhibition of cell cycle progression. Furthermore, under our experimental conditions, mimosine did not alter the levels of cell cycle regulatory proteins, including p21(WAF1/CIP1), cyclins D1 and E, and proliferating cell nuclear antigen. In addition, cyclin-dependent kinase inhibitors olomoucine and roscovitine did not block the increased death. These results indicate that mimosine inhibits the augmented death of glucose-deprived immunostimulated astrocytes by scavenging peroxynitrite rather than suppressing the cell cycle progression.

Uptake of 45Ca by mitochondria of Trigonella foenum-graecum as influenced by selenium and mimosine--detailed kinetic analyses.

Mitochondria from Trigonella foenum-graecum seedlings grown independently in the presence of either selenium (0.75 ppm) or mimosine (0.1 mM) exhibited respiration-stimulated energy-dependent uptake of Ca2+. Uptake studies were carried out independently at a series of Ca2+ concentrations at two different levels: (1) 1-20 MM and (2) 25-1,500 microM. Levels of uptake were 50-100% higher in the mitochondria of seedlings of both the Se and mimosine groups. Detailed kinetic analyses revealed negative cooperative effects operative during uptake of Ca2+ at 25-1,500 microM given in the medium. Hill coefficients for Ca2+ uptake by the mitochondria of different groups remained unchanged (nH, 0.75). Biphasic Scatchard plots were concave upward, suggestive of two classes of binding sites. High-affinity binding sites were estimated to be 16 nmol/mg protein with dissociation constant (KCa) of 2.5 x 10(9) L/mol. In contrast, graphical analyses of the uptake of Ca2+ in the range 1-20 microM in the medium revealed cooperative effects of positive nature. The present study demonstrates mixed cooperative effects during Ca2+ uptake by mitochondria from seedlings of T. foenum-graecum.

Kinetic analyses of mitochondrial 75selenium uptake in Trigonella foenum-graecum seedlings exposed to selenium and mimosine.

Uptake of (75Se) added in vitro was followed in mitochondria isolated from Trigonella foenum-graecum seedlings grown under different Se status (0.5-1.0 ppm) and with added mimosine (0.1 mM). Uptake of 75Se followed with added Na2 75SeO3 upto 20 microM in the medium was nonlinear in all the groups. Kinetic analyses of the uptake of 75Se for 1 min were carried out for all the groups. The results indicated a cooperative effect during Se transport. Graphical analyses using the Hill plot and Scatchard plot confirmed the existence of negative cooperativity during 75Se uptake. Scatchard plots were biphasic, suggesting the probable presence of two classes of binding sites. The presence of succinate or ATP in the incubation medium inhibited 75Se uptake by 40%. Studies with mitochondrial respiratory inhibitors indicated the uptake to be energy independent. A decrease in the uptake of 75Se by 40% effected by HgCl2, N-ethyl maleimide, and iodoacetate confirmed the interaction of active thiols in the process. The present study confirms the controlled nature of 75Se uptake by plant mitochondria.

Oxidative stress during selenium deficiency in seedlings of Trigonella foenum-graecum and mitigation by mimosine. Part I. Hydroperoxide metabolism.

Oxidative stress during selenium (Se) deficiency in the seedlings of Trigonella foenum-graecum grown for 72 h was investigated and the response to supplemented levels of Se (0.5-1 ppm) and mimosine (0.05-1 mM) was evaluated. Beneficial effects of Se was maximal at 0.75 ppm. Mimosine, a toxic amino acid, was also found to be beneficial to the growth of the seedlings exposed up to 0.2 mM. When compared to the stressed seedlings, mitochondrial oxygen uptake from seedlings of Se (0.75 ppm) group and mimosine (0.2 mM) group exhibited threefold enhancement in state 3 respiration rate and a controlled state 4 rate, with respiratory control ratios of 5-8. Upon supplementation at the optimal levels, superoxide dismutase (SOD) activities were enhanced fourfold with Se and eightfold with mimosine in the mitochondria. The soluble activity in mimosine groups increased twofold, but only by 75% in Se groups. Peroxidase activity registered a significant increase by threefold in mitochondria and fourfold in soluble fraction in both Se and mimosine groups. Exposure to Se or mimosine exhibited a differential response in the mitochondrial catalase and ascorbate peroxidase (Asc-Px) activities. In the Se groups, both catalase and Asc-Px in mitochondria decreased by 50-60%, which was contrasted by 60% increase in Asc-Px activity and 40% in catalase activity in mimosine groups. Supplementation with either Se or mimosine evoked similar responses of increases with respect to soluble catalase by twofold to threefold and Asc-Px by 90%. The results of the present study reveal (1) the prevalence of oxidative stress in T. foenum-graecum during Se deficiency, (2) enhanced mitochondrial functional efficiency mediated by Se and mimosine independently, and (3) an antioxidative role for mimosine during Se deficiency. The study demonstrates for the first time that mimosine, a naturally occurring toxic amino acid, could be a beneficial growth factor in concentrations between 0.1 and 0.2 mM.

Oxidative stress during selenium deficiency in seedlings of Trigonella foenum-graecum and mitigation by mimosine Part II. Glutathione metabolism.

Adaptive alterations in glutathione (GSH) metabolism were studied during oxidative stress induced by selenium (Se) deficiency in germinating seedlings of Trigonella foenum-graecum grown for 72 h and the response to supplementation individually of Se or mimosine was explored. Growth enhancement with improved mitochondrial efficiency was elicited by supplementation of Se at 0.5-0.75 ppm or mimosine at 0.1-0.2 mM. Total thiol and protein levels of mitochondrial and soluble fractions, in general, did not vary significantly with supplementation of either Se or mimosine except that the mitochondrial protein levels in mimosine groups (0.1-0.2 mM) decreased by 20-30%. Mitochondrial glutathione peroxidase (GSH-Px) increased by twofold in activity toward H2O2, cumene hydroperoxide (CHP), and t-butyl hydroperoxide (tBHP) in Se groups, and by 50-60% increase toward H2O2 and CHP but by a twofold enhancement in enzyme activity with tBHP in mimosine groups. Soluble GSH-Px activity increased by 30-40% only in mimosine groups and remained unaltered in Se groups. Glutathione S-transferase activity (GST) in the soluble fraction of both Se and mimosine groups increased dramatically by fivefold to sixfold. Distinct differences were noted in the response of the stressed seedlings toward exposure to Se or mimosine and included a decline in glutathione reductase (GR) activity by 50-60% in both mitochondria and soluble fractions of Se groups and an increase in GR activity of the mitochondria by twofold and of the soluble enzyme activity by 30% in the mimosine groups. Mimosine exposure resulted in a dose-dependent decrease in the gamma-glutamyl transpeptidase levels, but, in contrast, a significant enhancement by 50% was noted in the Se group at 0.75 ppm. The results including the differential response of GR activity to Se or mimosine supplementation are reflective of an effective reductive environment in Se groups and increased turnover of GSH in the presence of mimosine.

Enhancement of beta-glucosidase and beta-galactosidase of Trigonella foenum-graecum by exposure to the allelochemical mimosine.

Glycohydrolases assume significance in the metabolism of biological systems and have important industrial applications in the areas of pharmaceuticals, food, and medicine. Glycosidases were screened in germinating seeds, and attempts were made to enhance their levels. Screening of glycosidases in the seedlings during a 72 h germination period revealed higher levels of beta-glucosidase and beta-galactosidase in Trigonella foenum-graecum compared to Cicer arietinum and Vigna radiata. Activity of beta-galactosidase was in general higher than that of beta-glucosidase in all the seedlings tested. During growth, exposure of the seedlings to an allelochemical, mimosine, at 0.1 mM resulted in the enhancement of enzyme levels by 50% in the seedlings of T. foenum-graecum, whereas the addition of mimosine to the assay medium in vitro did not affect the enzyme activities. Hydrolytic activity was enhanced by addition of glycerol in the medium up to 0.1 M in the case of beta-glucosidase and with 0.05 M in the case of beta-galactosidase. In general, the hydrolytic rate was higher by about 30% in the seedlings exposed to mimosine compared to that of the control. Concomitant enhancement in the rates of transgalactosidation by 51% and transglucosidation by 23% was also noted, underscoring the relevance of plant glycohydrolases for appropriate applications.

Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S period in CHO cells synchronized with the plant amino acid mimosine.

In previous studies, we used two complementary two-dimensional gel electrophoretic methods to examine replication intermediates in the 240-kb amplified dihydrofolate reductase (DHFR) domain of methotrexate-resistant CHOC 400 cells (J. P. Vaughn, P. A. Dijkwel, and J. L. Hamlin, Cell 61:1075-1087, 1990). Surprisingly, in both asynchronous and early-S-phase cultures, initiation bubbles were detected in several contiguous fragments from a previously defined 28-kb initiation locus. However, because of the low levels of bubblelike structures observed on gels, it has been suggested that these structures might represent artifacts, possibly unrelated to replication per se. In this study, we have achieved much more synchronous entry into S phase by using a novel inhibitor and have isolated replication intermediates by a new procedure that largely eliminates branch migration and shear. Under these conditions, we find that (i) the relative number of bubblelike structures detected in fragments from the initiation locus is markedly increased, (ii) bubbles are detected at multiple sites scattered throughout the region lying between the DHFR and 2BE2121 genes, and (iii) bubbles appear and disappear in this region with the kinetics expected of an early-firing origin. These data strengthen the proposal that in vivo, initiation can occur at any of a large number of sites scattered throughout a broad zone in the DHFR domain.

The effect of synthetic iron chelators on bacterial growth in human serum.

The effect of synthetic iron chelators of the 1-alkyl-3-hydroxy-2-methylpyrid-4-one class (the L1 series) and 1-hydroxypyrid-2-one (L4) on bacterial growth in human serum was compared with those of the plant iron chelators mimosine and maltol and of the microbial siderophore desferrioxamine. None of the synthetic chelators enhanced growth of 3 Gram-negative organisms (Yersinia enterocolitica, Escherichia coli and Pseudomonas aeruginosa); in some cases they were even inhibitory. L4 strongly stimulated growth of Staphylococcus epidermidis, but the L1 series had only a marginal effect. Maltol was mildly inhibitory to all 4 bacterial species, while mimosine enhanced the growth of S. epidermidis and Y. enterocolitica but had little effect on E. coli or P. aeruginosa. Desferrioxamine enhanced the growth of all except E. coli. These results suggest that the chelators of synthetic or plant origin may carry less risk of increasing susceptibility to bacterial infection in patients undergoing chelation therapy for iron overload than does desferrioxamine, the drug currently in clinical use.

The goitrogen 3-hydroxy-4(1H)-pyridone, a ruminal metabolite from Leucaena leucocephala: effects in mice and rats.

Mice fed a diet containing 1% (w/w) 3-hydroxy-4(1H)-pyridone (DHP) developed goitre even with a diet high in iodine whereas mimosine (0.5% w/w) did not produce goitre even with a low-iodine diet. Thyroid enlargement was apparent (measured morphometrically) by the 7th week and was advanced by the 11th week. Histologically the goitre was hyperplastic in type. No marked histological changes were found in other organs of mice fed DHP or any organs of mice fed mimosine, except for some atrophy of hair follicles. A single intragastric dose of DHP inhibited the uptake of 125I by the thyroid in the rat but an equivalent dose of mimosine did not. Evidence is presented that the inhibition occurs at the iodine binding step, as with methyl thiouracil, rather than at the iodide trapping step, as with thiocyanate. Chronic treatment of mice with DHP, as with 6-methyl thiouracil, increased the avidity of the thyroid in taking up 125I. The major conjugated form of DHP in mammals, DHP-3-O-glucuronide, was almost as effective a goitrogen as the unconjugated compound when given by mouth but considerably less active than the free form in the blood stream. It was concluded that DHP is a potent antithyroid compound of the thiouracil type with low general toxicity, since mammals can tolerate a level of intake sufficient to produce goitre in spite of iodine supplementation.

Studies on the inhibition of synthesis of the tyrosine-rich proteins of wool.

Three treatments known to produce weak wool were imposed on sheep, and the effects on the synthesis of high-tyrosine wool proteins were noted. The treatments were: intravenous infusion of the amino acid mimosine (a potential chemical defleecing agent), intravenous injection of the synthetic steroid Opticortenol (dexamethasone-21-trimethylacetate), and the abomasal infusion of methionine into sheep consuming a diet of wheat. All three treatments caused a partial suppression of high-tyrosine protein synthesis. The inhibition caused by mimosine could not be prevented by the simultaneous infusion of tyrosine or phenylalanine, suggesting that in this system mimosine is not acting as a tyrosine antagonist. The role of phenylalanine in controlling the synthesis of the high-tyrosine proteins in wool was also investigated. Although the infusion of an amino acid mixture minus phenylalanine reduces the level of these proteins, supplements of phenylalanine or tyrosine do not stimulate their synthesis, irrespective of the initial level in the fibre. The improtance of aromatic amino acids in the regulation of the high-tyrosine proteins is therefore uncertain. Suppression of the high-tyrosine proteins is usually accompanied by a stimulation in the synthesis of the ultra-high-sulphur proteins, although there does not seem to be a simple stoichiometric relationship between the two protein types.