Effect of Methionine Deficiency on the Thymus and the Subsets and Proliferation of Peripheral Blood T-Cell,and Serum IL-2 Contents in Broilers

The purpose of this study was to investigate the effects of methionine deficiency on cellular immune function by determining morphological and ultrastructural changes of thymus, thymic cell cycle and apoptosis, peripheral blood T-cell subsets, T- cell proliferation function and the serum interleukin-2 （IL-2） contents. 120 1-d-old broilers were randomly divided into two groups （6 replicates in each group and l0 broilers in each replicate） and fed on a control diet or methionine deficient diet for 42 d. Lesions were observed in experiment. Histopathologically, lymphopenia and congestion were observed in the medulla of thymic lobule. Ultrastructurally, there were more apoptosis lymphocytes, and the mitochondria of lymphocytes were swelled in thymus of methionine deficiency. The G0/G~ phase of the cell cycle of the thymus was much higher （P〈0.01）, and the S, G2＋M phases and proliferating index （PI） were lower （P〈0.01） in methionine deficiency than in control group. And the percentage of apoptotic cells in the thymus was significantly increased in methionine deficiency （P〈0.01）. The percentage of CD4＋ and CD8~ T-cells was decreased in methionine deficiency compared with control group. Meanwhile, the proliferation function of peripheral blood T-cell was decreased in methionine deficiency. Also, the serum IL-2 contents were decreased in methionine deficiency. It was concluded that methionine deficiency could cause pathological and ultrastructural changes of thymus, reduce the T-cell population, serum IL-2 contents and the proliferation function of T- cells, and induce increased percentage of apoptotic cells. The cellular immune function was finally impaired in broilers.

Influence of L-methionine-deprived total parenteral nutrition with 5-fluorouracil on gastric cancer and host metabolism

AIM: To investigate the influence of L-methionine-deprived total parenteral nutrition with 5-FU on gastric cancer and host metabolism. METHODS: N-methyl-N'-nitro-nitrosoguanidine (MNNG) induced gastric cancer rats were randomly divided into four groups: Met-containing TPN group (n=11), Met-deprived TPN group (n =12), Met-containing TPN+5-FU group (n=11) and Met-deprived TPN+5-FU group (n=12). Five rats in each group were sacrificed after 7 days of treatment and the samples were taken for examination. The remaining rats in each group were then fed separately with normal diet after the treatment until death, the life span was noted. RESULTS: The tumors were enlarged in Met-containing group and shrank in Met-deprived group markedly after the treatment. The DNA index (DI) of tumor cells and the body weight (BW) of rats had no significant change in the two groups, however, the ratio of tumor cells'S phase was increased. The ratio of G2M phase went up in Met-containing group, but down in Met-deprived group. In the other two groups that 5-FU was added, the BW of rats, and the diameter of tumors, the DI of tumor cells, the S and G2M phase ratio of tumor cells were all decreased, particularly in Met-deprived plus 5-FU group. Pathological examination revealed that the necrotic foci of the tumor tissue increased after Met-deprived TPN treatment, and the nucleoli of tumor cells enlarged. In MetTPN+5-FU group, severe nuclear damage was also found by karyopyknosis and karyorrhexis, meanwhile there was slight degeneration in some liver and kidney cells. The serum free Met and Cysteine decreased markedly (P【0.001), while other amino acids, such as serum free serine and glutamine increased significantly (P【0.005). All the rats died of multiple organ failure caused by cancer metastasis. The average survival time was 18.6 days in Met-containing TPN group, 31 days in Met-deprived TPN group, 27.5 days in Met-containing TPN+5-FU group, and 43 days in Met-deprived TPN+5-FU group (P【0.05). CONCLUSION: Met-deprived TPN causes methionine starvation of tumor cells, and can enhance the anti-tumor effect of 5-FU and prolong the life span of gastric cancer bearing rats.

Effects of methionine supplementation in a reduced protein diet on growth performance, oxidative status, intestinal health, oocyst shedding, and methionine and folate metabolism in broilers under Eimeria challenge

Background This study investigated effects of different methionine(Met)supplementation levels in a reduced protein diet on growth performance,intestinal health,and different physiological parameters in broilers under Eimeria challenge.A total of 600 fourteen-day-old Cobb500 male broilers were challenged with E.maxima,E.tenella,and E.acervulina,and randomly allocated in a 2×5 factorial arrangement.Birds received normal protein diets(20%crude protein,NCP)or reduced protein diets(17%crude protein,LCP),containing 2.8,4.4,6.0,7.6,and 9.2 g/kg of Met.Results On 6 and 9 days post inoculation(DPI),increasing Met level linearly improved the growth performance(P<0.05).Total oocyst shedding linearly increased as Met level increased(P<0.05).Duodenal villus height(VH):crypt depth(CD)in the LCP groups were higher on 6 DPI(P<0.01)while lower on 9 DPI(P<0.05)compared to the NCP groups.Jejunal CD and duodenal VH:CD changed quadratically as Met level increased(P<0.05).On 6 DPI,liver glutathione(GSH)and glutathione disulfide(GSSG)linearly increased as Met level increased(P<0.05).On 9 DPI,GSSG quadratically increased,whereas GSH:GSSG quadratically decreased as Met levels increased(P<0.05).The expression of amino acid transporters linearly decreased as Met level increased(P<0.05).The expression of zonula occludens 2 and claudin-1 linearly increased on 6 DPI whereas decreased on 9 DPI as Met level increased(P<0.05).The expressions of cytokines were lower in the LCP groups than the NCP groups(P<0.05).Interaction effects were found for the expression of IL-10 and TNFαon 6 DPI(P<0.05),where it only changed quadratically in the NCP group as Met level increased.The expression of Met and folate metabolism genes were lower in the LCP groups than the NCP groups on 9 DPI(P<0.05).The expression of these genes linearly or quadratically decreased as Met level increased(P<0.05).Conclusion These results revealed the regulatory roles of Met in different physiological parameters including oxidative status,intestinal health,and nutrient metabolism in birds fed reduced protein diet and challenged with Eimeria.

Determination of Carbon and Nitrogen Isotope Fractions in Asparagine, Aspartic Acid, Threonine and Methionine

The nomenclature for compounds that are modified with isotopes is growing every day. Compounds can be modified with isotopes either individually, in a functional group or groups, or completely with all atomic centers of the element. This diversity of isotope-modified compounds increases the range of researches that can be studied using them. Compounds modified with isotopes of carbon-13 or nitrogen-15 can be converted into carbon monoxide, carbon dioxide and molecular nitrogen. Currently, only the average value of carbon-13 or nitrogen-15 isotopes can be determined. However, by directly determining the atomic share of these isotopes in organic compounds modified with isotopes, information about the isotopic centers of the element can be obtained. The atomic fraction of an element is defined as a single carbon or nitrogen isotope-modified center or centers, or all centers that are isotope-modified with that element at the same time. Carbon-13 or nitrogen-15 isotopes’ atomic fraction can be determined molecularly or with fragment ions of different elemental content, or both. This makes the method self-verifying, increasing the accuracy and reliability of the results obtained. Amino acids, such as asparagine, aspartic acid, methionine, and threonine, are essential for the human body. This proposed method of isotopic analysis will increase the possibilities for scientific research using these compounds.

Improvement of glucose metabolism in middle-aged mice on a high-fat diet by whole-grain highland barley is related to low methionine levels

Methionine restriction(MR)is an effective dietary strategy to regulate energy metabolism and alleviate oxidative stress and inflammation in the body,especially in the middle-aged and elderly population.However,the high methionine content of meat products makes this dietary strategy impossible to combine with protein supplementation and MR.Highland barley(HB),a low-methionine cereal,not only provides the body with protein but also has improved glucose metabolism and antioxidant and anti-inflammatory properties.Therefore,this study evaluated the feasibility of HB as a source of methionine-restricted dietary protein and the potential mechanisms.Middle-aged C57BL/6J mice were fed a control diet(CON),a high-fat diet(HFD),a whole-grain HB high-fat diet(HBHF),or a HBHF+methionine diet(HBHFmet)for 25 weeks.The results showed that the HBHF could keep the body weight,fasting glucose,insulin,homeostasis model assessment of insulin resistance(HOMA-IR),blood lipids,inflammation,and oxidative stress of HFD mice at normal levels.Compared with the HFD groups,HBHF inhibited pancreatic cell apoptosis and improved insulin secretion while improving hepatic and skeletal muscle glucose metabolism.However,these efficacies were attenuated in HBHFmet group mice.These findings suggest that HBHF has an MR strategy.

Feeding Practices and Use of Lysine and Methionine in Pigrationing on Intensified Pig Breeding in the West Center and Hauts Bassins Regions of Burkina Faso

The aim of the study was to assess feeding practices and the use of lysine and methionine in pig rationing on intensified and semi-intensive pig breeding in the Koudougou and Bobo-Dioulasso areas. To this end, a cross-sectional survey was carried out on 87 breeding in these towns. A Discriminant Factorial Analysis (DFA) confirming a k-means classification of the data collected was used to retain 71 breeding divided into three breeding classes: Class A (32.4% of breeding), Class B (14.08%) and Class C (53.52%). The results show that the majority of pig breeders were men between the ages of 36 and 59. Average herd sizes were 35 ± 28;79 ± 42 and 89 ± 21 pigs for Classes A, B and C respectively. The main breeds of pig found on the breeding were crossbred, Large white, local, Landrace and Duroc. Class A (26.1%), B (30%) and C (15.8%) breeders were familiar with both lysine and methionine. Class A breeders distributed feed staggered (65.2%) and in rations (34.8%). Lysine (13%) and methionine (8.7%) were purchased at 5250 FCFA/kg. Those in class B distributed feed staggered (50%) and in the form of rations (50%), in which they incorporated lysine (30%) and methionine (30%) purchased at a cost of 2500 FCFA/kg and 3000 FCFA/kg respectively. Rationing and staggered feeding were practiced by 23.7% and 76.3% of Class C breeders respectively. Only lysine purchased at 3400 FCFA/kg was incorporated into rations by 10.5% of breeders. The high cost of lysine and methionine was incriminated by Class A (100%), B (33.3%) and C (50%) breeders. In conclusion, intensive pig breeding, the practice of rationing and the incorporation of the amino acids lysine and methionine are of ascending importance from classes C, A to B. The high cost of feedstuffs, particularly lysine and methionine, compromises their use in rations, which could have a negative impact on expected breeding performance. The screening and use of feeds rich in and/or enriched with these amino acids, through the development or adaptation of technologies, could improve the efficiency of rations and the productivity of intensive pig breeding in Burkina Faso.

谷氨酸棒杆菌中L-高丝氨酸转运蛋白的挖掘及功能分析

L-高丝氨酸是一种非必需手性氨基酸,在农业、化工、生物医药等领域具有重要的用途。近年来,利用微生物发酵法生产L-高丝氨酸越来越受到人们的关注。氨基酸转运蛋白不仅可以降低反馈抑制和细胞毒性,还能提高氨基酸产量,已成为代谢工程改造的重要靶点。目前,谷氨酸棒杆菌中L-高丝氨酸转运蛋白仍然未知。该研究通过生物信息学分析,从谷氨酸棒杆菌中筛选出10个L-高丝氨酸转运蛋白候选基因,经过体外和体内L-高丝氨酸耐受性生长实验,结果显示在大肠杆菌三重高丝氨酸转运缺陷突变体中,异源过表达brnFE、azlCD和lysE基因,能显著恢复大肠杆菌突变体的生长缺陷,而在谷氨酸棒杆菌体内,只有lysE敲除菌较对照生物量下降16%,推测在体内LysE蛋白具有更重要的高丝氨酸外排能力。进一步对LysE的外排活性进行分析,发现lysE敲除菌的外排能力降低了18%。最后,在高丝氨酸底盘菌中,评估了敲除或过表达lysE基因对L-高丝氨酸产量的影响,结果发现lysE基因缺失导致L-高丝氨酸的产量降低了65%,而过表达lysE使L-高丝氨酸产量提高了22%。综上所述,谷氨酸棒杆菌LysE蛋白具有转运L-高丝氨酸的能力,相关结果为开发高效的L-高丝氨酸细胞工厂提供了新靶点。

S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism

AIM： To determine the role of c-Jun N-terminal kinase （JNK） activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosylmethionine （AdoMet）. METHODS： Primary hepatocyte cultures were pretreated with 100 IJmol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays. JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by EIIman＇s method and reactive oxygen species （ROS） generation by cell cytometry. RESULTS： The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decrease in ethanol-induced apoptosis （P〈 0.05）. AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity, and prevented cytochrome c release and pro-caspase 3 cleavage.CONCLUSION： The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease （ALD） treatment.

Relative Bioavailability of DL and L-Methionine in Broilers

Studies on the relative bioavailability (RBV) of DL-Methionine (DL-Met) to L-Methionine (L-Met) have produced variable results. An experiment was conducted to determine the RBV of DL to L-Met. A total of 2268 1-day old male chicken were housed in 54 floor pens (42 bird/pen). There were 9 treatments (6 repetitions) including the basal diet (BD). The BD was deficient in Met content with 0.27, 0.26 and 0.25 in the starter, grower and finisher periods respectively. Four levels of experimental diets for each DL-Met and L-Met were created by supplementing 0.05%, 0.10%, 0.15% and 0.20% of DL- or L-Met to the BD. The feeding program consisted of starter (0-14 d, 21% CP and 2900 kcal ME/kg), grower (15 - 28 d, 20% CP and 3000 kcal ME/kg) and finisher period (29 - 37 d, 18.5% CP and 3050 kcal ME/kg). Chickens and feed were weighed at the end of each age period. Regression coefficients of a common plateau asymptotic regression were used to calculate RBV. Birds responded to gradual increase in Met levels, BW, FCR and ADG were significantly (P 0.05) higher in treatment groups as compared to control. Through the study period (37 d), the RBVs of DL-Met for BW and FCR were 89 and 77 respectively.

Hepatoprotective effects of S-adenosyl-L-methionine against alcohol-and cytochrome P450 2E1-induced liver injury

S-adenosyl-L-methionine (SAM) acts as a methyl donor for methylation reactions and participates in the synthesis of glutathione. SAM is also a key metabolite that regulates hepatocyte growth, differentiation and death. Hepatic SAM levels are decreased in animal models of alcohol liver injury and in patients with alcohol liver disease or viral cirrhosis. This review describes the protection by SAM against alcohol and cytochrome P450 2E1-dependent cytotoxicity both in vitro and in vivo and evaluates mechanisms for this protection.

Effect of low-protein diet with supplementing different levels of DL-methionine on production performance of minks in growing-furring period

A study was conducted to evaluate production performance of minks in growing-furring period with supplementing DL-Methinnine （Met） in low protein diet. Seventy healthy male minks were randomly divided into five groups of 14 minks each. The minks were fed in five kinds of experiment diets （HP, LP, LP＋M1, LP＋M2 and LP＋M3）. The dietary protein levels, expressed as percentage of dry matter （DM）, were 32% （high protein, HP） and 24% （low protein, LP）. LP was supple- mented with Met 0.4% （M1）, 0.8% （M2） and 1.2% （M3） DM. From mid of September to December 10, apparent digestibility of CP （crude pro- tein）, N intake and urinary N excretion were decreased with declining dietary protein levels （p 〈 0.05） and N retained was the highest in treat- ment LP＋M2. No significant difference was found in total serum protein （TP） and serum urea nitrogen （SUN） among all treatment groups （p 〉 0.05）. Skin length of treatment HP and LP^M2 was higher than that of other groups （p 〈 0.05）. Body length, skin weight, length of guard hair and under hair were not affected by different dietary protein levels （p〉0.05）. The best performance could be observed in treatment LP＋M2. In diet, 24% （DM） protein level with 1.54% Met supplementing was enough for minks during growing-farring period. Dietary protein lowered from 32% to 24% with supplementing Met in diets would result in a37.9% decrease in urinary N excretion. Furthermore, addition of Met in diets for minks would be beneficial in terms of reducing feed expenses and lessening nitrogen emissions to the environment.

λ-Red重组技术结合复合诱变提高大肠杆菌L-异亮氨酸合成能力

本研究旨在通过λ-Red重组技术结合复合诱变方法提高大肠杆菌L-异亮氨酸合成能力。以E. coli NXA为出发菌株,首先采用λ-Red同源重组敲除编码支链氨基酸转运蛋白基因brnQ,获得突变菌株E. coli NXA1。然后将E. coli NXA1经常温常压等离子体(ARTP)、紫外(UV)与亚硝基胍(NTG)多轮复合诱变,以α-氨基丁酸(α-AB)为结构类似物进行筛选,筛选得到突变菌株E. coli NXA2。摇瓶发酵结果表明,在37℃、200 r/min条件下发酵40 h后,E. coli NXA1的L-异亮氨酸滴度为2.76 g/L,较E. coli NXA提高了33.98%;E. coli NXA2的L-异亮氨酸滴度为3.22 g/L,较E. coli NXA1提高了16.67%,较E. coli NXA提高了56.31%。对菌株E. coli NXA2经连续传代20代后,表现出较好的遗传稳定性。λ-Red重组技术结合复合诱变对大肠杆菌提高L-异亮氨酸合成能力有明显效果,为选育L-异亮氨酸高产菌株奠定理论基础。

S-Adenosyl-L-methionine towards hepatitis C virus expression: Need to consider S-Adenosyl-L-methionine's chemistry, physiology and pharmacokinetics

S-Adenosyl-L-methionine(SAM) is a cofactor serving as a methyl donor in numerous enzymatic reactions. It has been reported that SAM has the potential to modify antioxidant-enzymes, glutathione-biosynthesis and methionine adenosyltransferases-1/2 in hepatitis C virus-expressing cells at millimolar concentrations. The efficacy of SAM at micromolar concentrations and the underlying mechanisms remain to be demonstrated.

关于L-代数中理想的一些注记

许多逻辑代数可以看成L-代数.讨论一些特殊的L-代数,给出其L-理想与格滤子之间的关系.刻画一类使得L-理想集与格滤子集相等的格L-代数,同时给出L-代数与蕴含格之间的一个等价刻画.进一步给出格序效应代数的理想与L-理想以及它的同余与L-同余的等价刻画.

S-methyl-L-cysteine Protects against Antimycin A-induced Mitochondrial Dysfunction in Neural Cells via Mimicking Endogenous Methionine-centered Redox Cycle

Mitochondrial superoxide overproduction is believed to be responsible for the neurotoxicity associated with neurodegeneration.Mitochondria-targeted antioxidants,such as MitoQ,have emerged as potentially effective antioxidant therapies.Methionine sulfoxide reductase A(MsrA)is a key mitochondrial-localized endogenous antioxidative enzyme and it can scavenge oxidizing species by catalyzing the methionine(Met)-centered redox cycle(MCRC).In this study,we observed that the natural L-Met acted as a good scavenger for antimycin A-induced mitochondrial superoxide overproduction in PC12 cells.This antioxidation was largely dependent on the Met oxidase activity of MsrA.S-methyl-L-cysteine(SMLC),a natural analogue of Met that is abundantly found in garlic and cabbage,could activate the Met oxidase activity of MsrA to scavenge free radicals.Furthermore,SMLC protected against antimycin A-induced mitochondrial membrane depolarization and alleviated 1-methyl-4-phenylpyridinium(MPP+)-induced neurotoxicity.Thus,our data highlighted the possibility for SMLC supplement in the detoxication of mitochondrial damage by activating the Met oxidase activity of MsrA.

L-谷氨酸修饰金属有机框架材料MIL-101对CO_(2)的吸附

利用L-谷氨酸(L-Glu)作为第二配体一锅合成了MIL-101-xGlu,采用XRD、N_(2)吸附-脱附、SEM及TG等方法进行了表征,考察了不同用量的L-Glu对MIL-101晶体生长的促进作用。实验结果表明,当L-Glu修饰比例为6%(x)时,晶体呈正八面体分布规则,颗粒均匀,呈(113)晶面择优取向生长,接近MIL-101的理论模拟值;微孔体积高达1.65 cm^(3)/g,比表面积高达3679.51 m^(2)/g,具有良好的N_(2)吸附-脱附性能和热稳定性。气体吸附测试表明,298 K及5 MPa下,MIL-101-6%Glu对CO_(2),CH_(4)的吸附量分别达22.94,7.10 mmol/g,对N_(2)的吸附量低至3.94 mmol/g,优于单一MIL-101。

DFT Study of Geometrical and Vibrational Features of Sulfur Containing Amino Acids in Hydrated Media： L-Cysteine and L-Methionine

Geometry optimization at the B3LYP/6-31＋＋G＊ level of theory has been undertaken on clusters containing L-Met （L-methionine） or L-Cys （L-cysteine） surrounded by eight water molecules. The comparison of the structural parameters of L-Met and L-Cys with X-ray experimental values is in good agreement within 4.8%. This result shows that the privileged positions of water molecules and the possible hydrogen bonding network formed around the backbone of both AAs （amino acids） are adequate. Subsequent calculations of the harmonic vibrational modes followed by a post-processing treatment enable us to assign the vibrational modes of L-Met and L-Cys surrounded explicitly by eight water molecules. The frequencies of the assigned modes are in good agreement with available IR （infra red） and Raman values within 5%.

温敏型L-薄荷醇微胶囊制备及性能

该研究以单硬脂酸甘油酯(glyceryl monostearate,GMS)与辛烯基琥珀酸淀粉钠(sodium starch octenyl succinate,SSO)为壁材,将芯材L-薄荷醇包埋,使用喷雾冷冻干燥法制备温敏型L-薄荷醇微胶囊。通过单因素试验、响应面试验优化温敏型L-薄荷醇微胶囊制备工艺,结合实际工艺得到最佳工艺条件为固形物含量21%、GMS与SSO质量比0.16、芯材与壁材质量比0.19、均质压力30 MPa、进料速率15 mL/min、干燥温度42℃,温敏型L-薄荷醇微胶囊包埋率可达91.89%。采用扫描电子显微镜、激光粒度仪、热重分析仪对微胶囊的形貌特征和性能进行测定,结果表明微胶囊微观结构完整、呈圆球形、无黏连、颗粒分散性好,粒径平均为1.9μm,在153℃时L-薄荷醇质量分数快速减少,温敏型L-薄荷醇微胶囊在75℃时具有良好的释放性能。

Effect of Low Temperature Stress on Sweet Potato S-Adenosyl Methionine Synthetase Gene Expression

[Objective] The mRNA expression level changes of S-adenosylmethionine synthetase （SAMS） under low temperature stress was studied. [Method] Total RNA were extracted from leaves, stem and earthnut of sweet potato 0,12,24,48 and 72 h after low temperature treatement, mRNA expression level was analyzed by reverse expression and Real-time PCR technique. [Result] The expression quality of the gene extracted from leaves, stem and earthnut increased and the expression quality reached the peak point 24,72 and 72 h after low temperature treatment respectively. The expression change of earthnut was the biggest. [Conclusion] Low temperature was good for increasing mRNA expression of relevart genes.

SELECTION OF METHIONINE-RESISTANT VARIANT OF MEDICAGO SATIVA

A variant cell line of Medicago saliva L. resistant to methionine (100 mmol/L) was selected .from calli treated with 1.5 mmol/L sodium azide. Plants were regenerated from this selected cell line. After growing for 6 months on the medium devoid of selection pressure, the variant cell line still showed high level of resistance, which was 7.2-fold higher than that of the wild type. This indicated the stability of the resistance expression. The variant cell line also expressed a 3.3-fold higher cross-resistance to ethionine, than that of the wild type. The contents of methionine, lysine, threonine and isoleucine in the variant calli were respectively 2.0, 1.43, 1.71 and 1.57 times higher than those in the wild type. However, in the variant regenerated plantlets they respectively were 2.0, 1.33, 1.22 and 1.22 times higher than those in the wild type regenerants. Four new protein bands, viz. 65 kD, 23 kD, 20 kD and 18 kD, appeared in the SDS-PAGE pattern of soluble proteins in the variant calli. In the peroxidase isoenzyme electrophoresis pattern, two new bands were also observed in the variant calli.