• Asian-Australasian Journal of Animal Sciences
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• v.18 no.3
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• pp.332-339
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• 2005

The effect of transinoculation of goat rumen liquor into sheep rumen on mimosine toxicity was studied. One adult Kutchi male goat having higher mimosine degradation capacity than sheep was gradually adapted to Leucaena leucocephala (Leucaena) leaves by feeding increasing level of eucaena leaves supplementation for 1 month. Six Bharat Merino rams (12-18 months of age) were divided into two equal groups with (group I) or without (group II) infusion of 200 ml of goat rumen liquor per animal. The mimosine degradation in groups I and II were 3.04 and 2.31; 3.90 and 3.73 mg per day per 10 ml rumen liquor respectively after 1 and 2 weeks of leucaena feeding leaves. Total rumen bacterial population in RGCA medium and in a selective medium containing iron showed an increasing trend in both groups, while the bacterial population growing in the presence of cellulose showed a decreasing trend. Animal performance data did not show any adverse effect. Results revealed that transinoculation of rumen liquor from leucaena leaves adapted goat to sheep rumen did not help to improve mimosine degradation in the sheep. The sheep transinoculated with goat rumen liquor displayed no in vivo improvements in nutrient utilization vis-a-vis mimosine metabolism.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.36-36
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• 2017

Out of twenty common protein amino acids, there are many kinds of non protein amino acids (NPAAs) that exist as secondary metabolites and exert ecological functions in plants. Mimosine (Mim), one of those NPAAs derived from L. leucocephala acts as an iron chelator and reversely block mammalian cell cycle at G1/S phases. Cysteine (Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur-containing secondary products. Cys biosynthesis includes consecutive two steps using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast, and mitochondria. In the first step, the acetylation of the ${\beta}$-hydroxyl of L-serine by acetyl-CoA in the existence of SAT and finally, OASTL triggers ${\alpha}$, ${\beta}$-elimination of acetate from OAS and bind $H_2S$ to catalyze the synthesis of Cys. Mimosine synthase, one of the isozymes of the OASTLs, is able to synthesize Mim with 3-hydroxy-4-pyridone (3H4P) instead of $H_2S$ for Cys in the last step. Thus, the aim of this study was to clone and characterize the cytosolic (Cy) OASTL gene from L. leucocephala, express the recombinant OASTL in Escherichia coli, purify it, do enzyme kinetic analysis, perform docking dynamics simulation analysis between the receptor and the ligands and compare its performance between Cys and Mim synthesis. Cy-OASTL was obtained through both directional degenerate primers corresponding to conserved amino acid region among plant Cys synthase family and the purified protein was 34.3KDa. After cleaving the GST-tag, Cy-OASTL was observed to form mimosine with 3H4P and OAS. The optimum Cys and Mim reaction pH and temperature were 7.5 and $40^{\circ}C$, and 8.0 and $35^{\circ}C$ respectively. Michaelis constant (Km) values of OAS from Cys were higher than the OAS from Mim. Inter fragment interaction energy (IFIE) of substrate OAS-Cy-OASTL complex model showed that Lys, Thr81, Thr77 and Gln150 demonstrated higher attraction force for Cys but 3H4P-mimosine synthase-OAS intermediate complex showed that Gly230, Tyr227, Ala231, Gly228 and Gly232 might provide higher attraction energy for the Mim. It may be concluded that Cy-OASTL demonstrates a dual role in biosynthesis both Cys and Mim and extending the knowledge on the biochemical regulatory mechanism of mimosine and cysteine.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.224-224
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• 2017

In plants, cysteine(Cys) is decisive for protein and glutathione that acts as an indispensable sulfur grantor for methionine and many other sulfur containing secondary products. Cys formation is involved in the consecutive two reactions using two enzymes-serine acetyl transferase (SAT) and O-acetylserine (thiol)lyase (OASTL) and appeared in plant cytosol, chloroplast and mitochondria. OASTL is able to produce mimosine with 3-hydroxy-4-pyridone (3H4P) in lieu of $H_2S$ for Cys. In this report, we describe the first time cloning, purification and characterization of cytosolic(cy)OASTL from M. pudica and its expression in Escherichia coli and try to find out the cross link between this OASTL and the mimosine formation and to elucidate the metabolic role of cy-OASTL in M. pudica. The purified recombinant protein was 34.7 KDa. The optimum reaction pH and temperature was 6.5 and $50^{\circ}C$, respectively. The Michaelis constant (Km) and the Vmax value of the enzyme was $252{\pm}25{\mu}M$ and $57{\pm}3{\mu}M\;cysteine\;min^{-1}\;{\mu}g\;protein^{-1}$ for sulfide and $159{\pm}21{\mu}M$ and $58{\pm}2.4{\mu}M\;cysteine\;min^{-1}\;{\mu}g\;protein^{-1}$ for OAS subsequently. After cleaving the His-tag, we tried to observe cy-OASTL to form mimosine with appropriate substrate but it was not successful. It may be concluded that cy-OASTL of the present study is only Cys specific, not mimosine.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.3
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• pp.213-217
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• 1995

Leucaena leucocephala (lam. de Wit) is a multipurpose leguminous tree/shrub, promoted extensively for reforestation and rural development programmes, as well as in ruminant production. After a year of establishment in the plots, the overall leaves and twigs yield was on average 0.92 kg/plant, containing an average 23.34, 10.61, 5.32 and 10.13 percent CP, CF, and ash, respectively. The proximate and mineral compositions are affected by factors such as stage of maturity, type of cultivar, seasons, cutting intervals, different plant parts. Though leucaena contains toxic mimosine and tannins, these are not always deleterious for ruminants and rumen microbes can often degrade toxic factors into a nontoxic utilizable product Mimosine content is generally higher in the seed than other plant parts. Leucaena supplementation in ruminant ration showed higher digestibility co-efficient of proximate component, which may results in higher productivity. Considering its productivity, composition and nutritive value. L. Leucocephala could be more widely used as a protein source for ruminants in tropical and sub-tropical countries.

• Journal of Animal Science and Technology
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• v.57 no.11
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• pp.34.1-34.5
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• 2015

Himalayan dock (Rumex nepalensis) was evaluated for forage value and antinutrients under three, five and seven weeks cutting intervals in the temperate environment. Dry matter (DM) content was measured for each cutting interval. Forage quality parameters such as Crude Protein (CP), Acid Detergent fiber (ADF), Neutral Detergent Fiber (NDF), Calcium (Ca) and Phosphorus (P) were analyzed. Plants with seven weeks cutting interval gave higher DM yield. CP and P content were significantly higher for three weeks cutting intervals. Average CP contents were 31.38 %, 30.73 % and 27.32 % and average P content 0.58 %, 0.52 % and 0.51 % for three, five and seven weeks cutting intervals, respectively. Ca content did not differ significantly between cutting intervals. The average Ca content were 0.91 %, 0.90 % and 90 %, for three, five and seven weeks cutting intervals, respectively. Tannin and mimosine contents were not significantly different between cutting intervals. Average tannin contents were 1.32 %, 1.27 % and 1.26 % and mimosine 0.38 %, 0.30 % and 0.28 % for three, five and seven weeks cutting intervals, respectively. The study concluded that R. nepalensis could be a potential source of protein for livestock. The study also suggests seven weeks harvesting interval to provide plants with high dry matter yield, high forage quality and very low levels of anti-nutrients.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.4
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• pp.337-343
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• 1998

In a feeding trial, Jamunapari male kids (18) of about 4 months age were equally divided into two groups of nine animals each. Goats in the experimental group were fed sun-dried pelleted Leucaena leucocephala leaves and those in the control group were offered a conventional diet without Leucaena leaves as per Kearl (1982) recommendations for a period of 6 months. Daily dry matter intake DMI/100 kg BW was $3.13{\pm}0.04kg$ in the Leucaena group and $3.30{\pm}0.05kg$ in the control. There were significant (p < 0.01) differences in the apparent digestibilities of DM, OM, CP, EE, CF and NFE being lower in the Leucaena group. Contents of digestible crude protein (DCP) and total digestible nutrients (TDN) were 11.40 and 52.20%, respectively, in the Leucaena group and 14.04 and 66.10%, respectively in the control. The nitrogen in the Leucaena group was not well utilized as compared to the control, though kids were in positive nitrogen balance in both the groups. The average daily weight gain of kids on pelleted Leucaena was $29.95{\pm}2.60g$ as against $42.09{\pm}3.24g$ observed in the control. The mean DMI/kg LW gain was significantly (p < 0.01) higher in the Leucaena group ($14.70{\pm}0.78kg$) as compared to the control ($11.55{\pm}0.46kg$). The Hb, BUN, SGOT and SGPT concentrations were statistically similar in both the groups. Histopathological examination of thyroid gland of goats sacrificed at the end of experiment did not reveal any signs of colloidal goitre associated with mimosine toxicity. No significant pathological alterations were observed in vital organs irrespective of dietary treatment. Sundried, pelleted Leucaena foliage appears to be a promising potential feed for growing goats without any significant deleterious effect.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.8
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• pp.1136-1144
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• 2016

Leucaena silage was supplemented with different levels of molasses and urea to study its nutritive value and in vitro rumen fermentation efficiency. The ensiling study was randomly assigned according to a $3{\times}3$ factorial arrangement in which the first factor was molasses (M) supplement at 0%, 1%, and 2% of crop dry matter (DM) and the second was urea (U) supplement as 0%, 0.5%, and 1% of the crop DM, respectively. After 28 days of ensiling, the silage samples were collected and analyzed for chemical composition. All the nine Leucaena silages were kept for study of rumen fermentation efficiency using in vitro gas production techniques. The present result shows that supplementation of U or M did not affect DM, organic matter, neutral detergent fiber, and acid detergent fiber content in the silage. However, increasing level of U supplementation increased crude protein content while M level did not show any effect. Moreover, the combination of U and M supplement decreased the content of mimosine concentration especially with M2U1 (molasses 2% and urea 1%) silage. The result of the in vitro study shows that gas production kinetics, cumulation gas at 96 h and in vitro true digestibility increased with the increasing level of U and M supplementation especially in the combination treatments. Supplementation of M and U resulted in increasing propionic acid and total volatile fatty acid whereas, acetic acid, butyric acid concentrations and methane production were not changed. In addition, increasing U level supplementation increased $NH_3$-N concentration. Result from real-time polymerase chain reaction revealed a significant effect on total bacteria, whereas F. succinogenes and R. flavefaciens population while R. albus was not affected by the M and U supplementation. Based on this study, it could be concluded that M and urea U supplementation could improve the nutritive value of Leucaena silage and enhance in vitro rumen fermentation efficiency. This study also suggested that the combination use of M and U supplementation level was at 2% and 1%, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.10
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• pp.1433-1438
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• 2002

The study was carried out to evaluate leucaena seeds as a protein replacement of mustard seed cake (MSC) in the concentrate mixture of growing lambs. Fifteen owing male lambs (Local${\times}$Corridale) with an average body weight of 16.3 kg were allocated into three dietary treatments (T1, T2, and T3) with five animals in each group. Animals were offered dry mixed grass, berseem hay and concentrate mixture to meet their nutrient requirements. In concentrate mixture of T1, (Control) MSC was used as protein source, while in T2 and T3 groups, 25 and 50% of MSC was replaced by leucaena leucocephala seeds. On completion of three months (90 days) of feeding, a digestion cum-metabolism trial was conducted to determine DMI, nutrient utilization, and nitrogen balance. Changes in body weight were recorded at 15 day internals and eating patterns were recorded for 3 consecutive days at the end of the feeding trial. MSC had higher CP contents than leucaena seeds (27.0%). Mimosine contents in leucaena seeds were 1.1 compared to 0.2 and 0.4% in concentrate mixture of T2 and T3 group, respectively. Dry matter intake varied non-significantly ($79.3{\pm}1.2$ to $83.4{\pm}1.3g/kg$ $w^{0.75}$) across the dietary treatments. Digestibility of DM and cell wall polysaccharides (NDF, ADF. Cellulose and hemicellulose) were comparable, however CP digestibility was relatively lower in leucaena luecocephala seeds based groups (T2 $45.5{\pm}1.7$ and T3 $46.7{\pm}3.5$) compared to MSC supplemented group (T1 $47.7{\pm}0.9%$). The growth rate of lambs was non-significantly higher in T1 ($79.2{\pm}5.4$) compared to T2 ($73.8{\pm}8.8$) and T3 ($73.9{\pm}7.0$), respectively. The animals were in positive nitrogen balance and N-balance varied from 1.8 to 2.9 g/d across treatment groups. The eating rate (% of total offered) of concentrate up-to 15 min was relatively higher in T1 (82.4) than T2 (74.2) and T3 (77.8%). However no effect of leucaena seeds was recorded on total DMI of animals. The results of the study revealed that the inclusion of up to 50% leucaena seeds, as protein source in concentrate mixture of lambs had no adverse effect on DMI, nutrient utilization, eating patterns, nitrogen balance and growth performance of lambs.

• Animal cells and systems
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• v.6 no.2
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• pp.171-180
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• 2002

Nitric oxide has high affinity for iron, and thus it can cause intracellular iron loss. We tested the idea that intracellular iron can be the primary target of NO toxicity by comparing the signaling mechanisms involved in cell death caused by iron depletion and that caused by NO. Treatment of HL-60 cells with a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), decreased the intracellular iron level rapidly as that observed with the iron chelator deferoxamine (DFO). Iron chelators such as DFO and mimosine could induce death of human leukemic HL-60 cells by a mechanism requiring activation of p38 kinase, c-Jun N-terminal kinase, caspase-3 and caspase-8. DFO and SNAP also caused release of cytochrome c from mitochondria. Inhibition of p38 kinase by a selective inhibitor, SB203580, abolished the NO and DFO-induced cell death, release of cytochrome c, and activation of caspase-3 and caspase-8, thus indicating that p38 kinase lies upstream in the cell death processes. In a parallel situation, the cells that are sensitive to NO showed similar sensitivity to DFO. Moreover, simultaneous addition of ferric citrate, an iron-containing compound, inhibited the SNAP and DFO-induced activation of caspases and also blocked the NO-mediated cell cycle arrest at $G_1$ phase. Collectively, our data implicate that the NO-induced cell death of tumor cells including HL-60 cells is mediated by depletion of iron and further suggest that activation of p38 kinase lies upstream of cytochrome c release and caspase activation involved in this apoptotic process.