Indian Journal of Biochemistry & Biophysics

CODEN : IJBBBQ  ISSN : 0301-1208

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VOLUME 44

NUMBER 3

JUNE 2007

 
CONTENTS

 

 

Minireview

 

Modularity: A New Perspective in Biology

133

        Diksha N Dani and Jayashree K Sainis*

 

 

 

Papers

 

Oral and parenteral immunization with synthetic retro-inverso peptides induce antibodies that cross-react with native peptides and parent antigens

 

140

        Peter Fischer, Alfio Comis, Margaret Tyler and Merlin Howden*

 

 

 

Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis

 

145

        Zhao Yunxue, Li Jing, Wang Jingjian, Xing Yanli and Geng Meiyu*

 

 

 

Added inositol regulates invertase secretion and glucose-repressed SUC2 gene expression in Saccharomyces sp. W4

 

152

        Zhenming Chi*, Liyan Ma, Lingmei Gao and Xiaohui Duan

 

 

 

Dietary supplementation of vitamin A, C and E prevents p-dimethylamino-azobenzene induced hepatic DNA damage in rats

 

157

  A Antony Joseph Velanganni, S Dharaneedharan, P Geraldine and C Balasundram*

 

 

 

Triiodothyronine and melatonin influence antioxidant defense mechanism in a teleost Anabas testudineus (Bloch): In vitro study

 

164

        P Sreejith, R S Beyo, L Divya, A S Vijayasree, M Manju and O V Oommen*

 

 

 

Comparative QSAR modeling of COX-2 inhibitor 1,2-diarylimidazoles using
E-state and physicochemical parameters

 

169

        Santanu Chakraborty, Chandana Sengupta and Kunal Roy*

 

 

 

Notes

 

An alpha-1 antitrypsin genetic variant from India

176

        Ajit K Shasany*, Ashutosh K Shukla, Mahendra P Darokar, Megha Saraiya, Nidarshana Chaturvedi, Lankeshwar Tewari and Suman P S Khanuja

 

 

 

Estimation of hydrogen sulphide in the human lymphocytes

179

        S Barathi, Prasanna Vadhana, N Angayarkanni* and S Ramakrishnan

 

 

 

Role of pectin from Cucumber (Cucumis sativus) in modulation of protein kinase C activity and regulation of glycogen metabolism in rats

 

183

        S Sudheesh* and N R Vijayalakshmi

 

 

 

Studies on glucose metabolizing enzymes in cytosolic and bacteroidal fractions of mungbean (Vigna radiata L.) and lentil (Lens culinaris L.) nodules

 

186

        N Munjral, A K Gupta and N Kaur*

 

 

 

Instructions to Authors

190

 

 

 

 

——————

*Author for correspondence

 


MINIREVIEW

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp. 133-139

 

Modularity: A New Perspective in Biology

Diksha N Dani and Jayashree K Sainis*

Molecular Biology Division, Bhabha Atomic Research Center, Mumbai 400085, India

 

Received 28 November 2006; revised 05 April 2007

Several decades of research in biochemistry and molecular biology have been devoted for studies on isolated enzymes and proteins. Recent high throughput technologies in genomics and proteomics have resulted in avalanche of information about several genes, proteins and enzymes in variety of living systems. Though these efforts have greatly contributed to the detailed understanding of a large number of individual genes and proteins, this explosion of information has simultaneously brought out the limitations of reductionism in understanding complex biological processes. The genes or gene products do not function in isolation in vivo. A delicate and dynamic molecular architecture is required for precision of the chemical reactions associated with “life”. In future, a paradigm shift is, therefore, envisaged, in biology leading to exploration of molecular organizations in physical and genomic context, a subtle transition from conventional molecular biology to modular biology. A module can be defined as an organization of macromolecules performing a synchronous function in a given metabolic pathway. In modular biology, the biological processes of interest are explored as complex systems of functionally interacting macromolecules. The present article describes the perceptions of the concept of modularity, in terms of associations among genes and proteins, presenting a link between reductionist approach and system biology.

Keywords: Genome, Modular biology, Molecular machines, Metabolon, Proteome, STRINGS, Systems biology

*E-mail: jksainis@barc.gov.in

 

 

 

PAPERS

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp. 140-144

 

Oral and parenteral immunization with synthetic retro-inverso peptides induce antibodies that cross-react with native peptides and parent antigens

Peter Fischer, Alfio Comis, Margaret Tyler and Merlin Howden*

aDeakin Research Limited, Hawkesbury Campus, University of Western Sydney, Richmond, NSW 2753, Australia

bDeakin Research Limited, CSIRO Division of Food Science and Technology, North Ryde, NSW 2113, Australia

cUNSW Foundation Year, University of New South Wales, Kensington, NSW 2033, Australia

Received 31 August 2006 ; revised 20 April 2007

 

The objective of this study was to determine whether certain retro-inverso peptides have the potential to act as synthetic vaccines in mice, when immunized by injection or orally. Immunization of mice parenterally with conjugates of three such retro-inverso peptides and orally with the unconjugated peptides elicited generally high titres of anti-peptide antibodies. Antibodies against the same three peptides cross-reacted by binding strongly in ELISA to the native peptides and vice versa, regardless of the mode of immunization. Antibodies against a retro-inverso diphtheria peptide also reacted strongly with diphtheria toxin. Seven of 8 mice, immunized by injection of the conjugate of a retro-inverso derivative of robustoxin [a lethal spider (Atrax robustus) venom toxin] were protected from challenge involving injection with twice the minimum lethal dose of A. robustus venom containing the toxin.

Keywords: Retro-inverso peptides, Immunogenicity, Cross-reactivity, Oral immunization, Synthetic vaccines

*Email: m.howden@unsw.edu.au

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp. 145-151

 

Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis

Zhao Yunxue, Li Jing, Wang Jingjian, Xing Yanli and Geng Meiyu*

Department of Molecular Pharmacology and Glycobiology, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, P. R. China

 

Received 26 October 2006; revised 16 May 2007

 

Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo — the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, β-1,6-GlcNAc-branching, α-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(β-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis.

Keywords:    Oligosaccharides, Mouse mammary tumor cell lines 67NR and 4TO7, Glycosylation, Cancer metastasis,Cell adhesion, Cell migration, Cell invasion, Lectins binding assay, Sialic acids

*Email: gengmy@ouc.edu.cn

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp. 152-156

 

Added inositol regulates invertase secretion and glucose-repressed SUC2 gene expression in Saccharomyces sp. W4

Zhenming Chi*, Liyan Ma, Lingmei Gao and Xiaohui Duan

UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China

 

Received 13 September 2006; revised 25 April 2007

The effect of inositol supplementation on glucose derepression, invertase secretion and SUC2 gene expression in Saccharomyces sp. W4 was studied. Invertase secretion was repressed, when the yeast cells, grown the synthetic medium without inositol (I- medium) contained more than 0.2% (w/v) initial concentration of glucose. However, in the same medium plus inositol (I+ medium, inositol conc. 100 mg/100 ml), invertase secretion was repressed only at glucose concentrations higher than 2.0% (w/v). Results showed that secreted invertase activity increased only in the I+ medium, whereas intracellular invertase activity remained constant in both media during the cell, growth. The mRNA encoding secreted invertase was higher in the glucose-derepressed cells grown in the I+ medium than in the glucose-repressed cells grown in the I- medium. Similarly, phosphatidylinositol (PI) content was significantly higher in the cells grown in the I+ medium than in the I- medium. These results indicated that PI might be involved in the glucose derepression, invertase secretion and SUC2 gene expression at the transcriptional level in the yeast.

Keywords:             Saccharomyces sp. W4, Invertase secretion, Phosphatidylinositol, SUC2 gene expression, Inositol, Glucose derepression

*E-mail: zhenming@sdu.edu.cn

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp. 157-163

 

Dietary supplementation of vitamin A, C and E prevents p-dimethylaminoazobenzene induced hepatic DNA damage in rats

A Antony Joseph Velanganni, S Dharaneedharan, P Geraldine and C Balasundram*

Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024, India

Department of Biochemistry, J. J. College of Arts and Science, Pudukkottai 622 404, India

 

Received 04 September 2006; revised 25 April 2007

The preventive effect of antioxidant vitamins A, C, E and their analogues against DNA damage induced by a hepatocarcinogen p-dimethylaminoazobenzene (DAB) was assessed by comet assay. For genotoxicity (DNA damage) study, male albino rats were divided into 11 groups, consisting of four rats each. Group I served as control. Group II to VII received 1, 10, 100, 200, 300 and 400 mg per kg body wt of DAB respectively; group VIII to XI received 500 mg/kg body wt of DAB. They were sacrificed by cervical decapitation 3, 6, 12 and 24 h after treatment; livers were excised immediately and subjected to comet assay to measure DNA damage. To study the effect of vitamins, experiments were conducted on a group of 275 rats divided into 3 sets of 25 rats each. First set served as control; second set received 0.06% DAB and third set received 0.06 % DAB, along with analogues of vitamins A, C and E. Rats fed with 0.06% DAB were provided water ad libitum for a period of 4 months, followed by a normal (basal) diet for further 2 months. Vitamins A (10,000-50,000 IU), C (75-1000 mg) and E (50-500 mg) and their analogues were given (per kg body wt) to the third set of rats by gavage route once in a week for a period of 6 months. The DAB induced DNA damage only at the highest tested dose of 500 mg/kg body wt. Administration of high doses of vitamin A acid, L-ascorbic acid and vit. E succinate individually prevented the DNA damage. However, administration of a mixture of these vitamins at low doses prevented the DAB-induced DNA damage, which may be due to their synergistic effect. The results indicate that there is a significant advantage in mixed vitamins therapy at low dose over the treatment with individual vitamins.

Keywords: Genotoxicity, p-Dimethylaminoazobenzene, Hepatic DNA damage, Comet assay, Analogues of vitamin A, C and E

*E-mail: bal213@rediffmail.com

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp. 164-168

 

Triiodothyronine and melatonin influence antioxidant defense mechanism in a teleost Anabas testudineus (Bloch): In vitro study

 

P Sreejith, R S Beyo, L Divya, A S Vijayasree, M Manju and O V Oommen*

Department of Zoology, University of Kerala, Kariavattom, Thiruvananthapuram- 695 581, Kerala, India

 

Received 04 June 2006; revised 17 April 2007

The effect of the hormones triiodothyronine (T3) and melatonin on antioxidant defense system was studied in 6-propyl thiouracil (6-PTU)-treated or photoperiod-exposed teleost Anabas testudineus. 6-PTU (2 µg/g) treatment or photoperiod exposure (24 h) increased malondialdehyde (MDA) and conjugated dienes (CD) concentrations, indicating increased lipid peroxidation (LPO) in the experimental conditions. T3 or melatonin (10-6 M) treatment for 15 min in vitro in PTU-treated fish reversed the activity of superoxide dismutase (SOD), catalase and glutathione content. T3-treated group showed no change in glutathione peroxidase (GPx) activity, whereas melatonin treatment decreased its activity. T3 inhibited glutathione reductase (GR) activity. Photoperiod exposure (physiological pinealotomy) induced a stressful situation in this teleost, as evidenced by LPO products and antioxidant enzyme activities. Melatonin and T3 treatment for 15 min in vitro also reversed the effect of photoperiod on peroxidation products and the SOD and catalase activities. GR activity decreased in photoperiod-exposed group and melatonin and T3 treatment reversed the activities. The antioxidant enzymes responded to the stress situation after 6-PTU treatment and photoperiod exposure by altering their activities. The study suggested an independent effect of T3 and melatonin on antioxidant defence mechanism in different physiological situations in fish.

KeywordsAnabas testudineus, Antioxidant enzyme, Fish, Free radicals, Lipid peroxidation, Melatonin, Photoperiod,
6-Propyl thiouracil, Triiodothyronine

*Email: oommenvo@yahoo.com

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp.169-175

 

Comparative QSAR modeling of COX-2 inhibitor 1,2-diarylimidazoles using E-state and physicochemical parameters

Santanu Chakraborty, Chandana Sengupta and Kunal Roy*

Drug Theoretics and Cheminformatics Lab, Division of Medicinal and Pharmaceutical Chemistry, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700 032, India

 

Received 16 October 2006; revised 23 May 2007

Considering importance of developing selective COX-2 inhibitors, COX-2 binding affinity data of 4-(2-aryl-1-imidazolyl)-phenyl methyl sulfones and sulfonamides (n = 83) have been modeled using electrotopological state (E-state) index as electronic parameter, hydrophobic substituent constant (π) and molar refractivity (MR) of aryl ring substituents as lipophilic and steric parameters, respectively. Additionally, suitable dummy parameters have been used for the development of multiple regression equations in a stepwise manner. The study suggests that lipophilicity of ortho, meta and para substituents of the aryl ring increases the binding affinity, while molar refractivity (MR) of ortho and meta substituents of the aryl ring decreases the binding affinity. Again, electron-withdrawing substituents at meta and para positions of the aryl ring increase the binding affinity. Additionally, a 4-fluoro substituent on the aryl ring, a trifluoromethyl substituent at R3 position and simultaneous presence of 3-chloro and 4-methyl groups on the aryl ring are conducive to the binding affinity. Also, an amino substituent is preferred over a methyl group at R2 position suggesting preference of the sulfonamide moiety over the methyl sulfone moiety for the COX-2 binding affinity. Furthermore, importance of E-state values of different atoms in the generated relations suggests the influence of electron density distribution over the 1,2-diarylimidazole nucleus for the binding affinity. For this data set, E-state parameters perform better as electronic parameters in comparison to Hammett sigma parameters. When lipophilic whole molecular descriptor (ClogP) is used, instead of hydrophobic substituent constant (π), the former performs better than the latter.

Keywords:    QSAR, COX-2 binding affinity, 4-(2-Aryl-1-imidazolyl)-phenyl methyl sulfones and sulfonamides, E-state index, Physicochemical parameters

*E-mail: kunalroy_in@yahoo.com

 

 

 

NOTES

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp.176-178

 

An alpha-1 antitrypsin genetic variant from India

Ajit K Shasany*, Ashutosh K Shukla, Mahendra P Darokar, Megha Saraiya, Nidarshana Chaturvedi, Lankeshwar Tewari+ and Suman P S Khanuja

Genetic Resources and Biotechnology Division, Central Institute of Medicinal and Aromatic Plants, Lucknow 226 015, India

+Department of Biochemistry, King George’s Medical University, Lucknow 226 003, India

Received 11 October 2006; revised 10 May 2007

The highly polymorphic human alpha-1 antitrypsin (AAT) gene codes for the most abundant circulating plasma serine protease inhibitor. Previously, genetic variants of the AAT gene were reported from different regions of the world. In the present study, the AAT gene was characterized in an Indian sample. The AAT gene was isolated and cloned from a liver biopsy sample through RT-PCR and the full-length gene was sequenced. Nucleotide sequence comparison with the human genome and the AAT sequences available in the GenBank (NCBI) demonstrated four unique variations—(i) an A to G variation at position 286 (Thr96Ala), (ii) an A to G variation at position 839 (Asp280Gly), (iii) a T to C variation at position 1182 that did not result in any change in the protein sequence (TTT to TTC both code for Phe) and (iv) an A to C variation at position 1200 (Glu400Asp) that resulted in replacement by an amino acid of similar nature. Other variations found were T to C at position 710 (Val237Ala) and T to A at position 863 (Val288Glu), which were also reported earlier. In conclusion, this study reports the entire 1257 bp nucleotide sequence of protein coding region of the human AAT gene from an Indian sample. This preliminary finding is significant, as it reports for the first time the AAT gene sequence in the Indian sample.

Keywords: Human alpha-1 antitrypsin, RT-PCR, Gene variants, Gene sequence

*Email: akshasany@yahoo.com

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44,June 2007, pp.179-182

 

Estimation of hydrogen sulphide in the human lymphocytes

 

S Barathi1, Prasanna Vadhana2, N Angayarkanni1* and
S Ramakrishnan1

1Biochemistry Research Department, Vision Research Foundation, Sankara Nethralaya, Chennai 600 006, India

2BITS, Pilani, Rajasthan 333 031, India

 

Received 20 September 2006; revised 07 March 2007

Hydrogen sulphide (H2S), a signaling gasotransmitter and a potent vasorelaxant is endogenously produced by the enzymes cystathionine-b-synthase (CBS) and cystathionine-g-lyase (CSE). CBS is a predominant source of H2S in the central nervous system, while CSE is the major H2S producing enzyme in the brain and other nervous tissues. Though the expression of these enzymes in the blood lymphocytes is known, H2S formation in the lymphocytes has not been reported so far. In the present study, H2S levels in the lymphocytes of healthy control subjects were estimated, after suitable modifications in a routine method [Stipanuk M H & Beck P W (1982) Biochem J 206, 267-277] used for detecting tissue levels of H2S. In this method, homocysteine (Hcys) due to its higher solubility was used as the substrate in place of l-cysteine and NaOH was used in place of zinc acetate to increase the entrapment of H2S in the central well. A mean H2S level of 11.64 ± 6.36 µM/min/mg protein was detected in the lymphocytes of 8 subjects (mean age, 24 ± 2; 2 male, 6 female). The modified method was found to be more sensitive for H2S estimation in human lymphocytes. As endogenous H2S is reported to be involved in the pathogenesis of various cardiovascular and pulmonary diseases, the levels of H2S in lymphocytes can be a marker of the endogenous tissue levels.

Keywords: Homocysteine, Hydrogen sulphide, Lymphocytes

*E-mail: drak@snmail.org

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp. 183-185

 

Role of pectin from Cucumber (Cucumis sativus) in modulation of protein kinase C activity and regulation of glycogen metabolism in rats

 

S Sudheesh* and N R Vijayalakshmi Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram, 695 581, Kerala, India

 

Received 29 May 2006; revised 29 March 2007

The regulatory role of protein kinase C (PKC) in glycogen metabolism in pectin fed rats was investigated. Administration of pectin (5 g/kg body wt/day) from cucumber (Cucumis sativius L.) led to inhibitory effects on PKC activity in the liver of rats. In the brain and pancreas, PKC activity was significantly higher in pectin-treated rats as compared to the control group. Level of blood glucose was significantly lowered and the level of glycogen in the liver was significantly increased in pectin-administered rats. Glycogen synthase activity was enhanced, while glycogen phosphorylase enzyme showed inhibition in pectin-treated rats. Results indicated that pectin administration might have caused an increase in the secretion of the insulin, which, in turn, had a stimulatory effect on the PKC activity in the pancreas. The decreased PKC activity in the liver and increased PKC activity in the brain and pancreas on pectin administration indicated enhanced glycogenesis and reduced glycogenolysis.

Keywords: Cucumis sativus L., Glycogen synthase, Pectin, Phosphorylase, Protein kinase C

*E mail: sudheeshatl@yahoo.co.uk

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 44, June 2007, pp. 186-189

 

Studies on glucose-metabolizing enzymes in cytosolic and bacteroidal fractions of mungbean (Vigna radiata L.) and lentil (Lens culinaris L.) nodules

N Munjral, A K Gupta and N Kaur*

Department of Biochemistry and Chemistry
Punjab Agricultural University, Ludhiana 141 004, India

 

Received 03 May 2006; revised 25 May 2007

Nitrogen is exported in the form of ureides or amides from the nodules in pulse crops. In order to understand the carbon metabolism in ureide and amide exporting nodules, activities of enzymes involved in glucose metabolism were compared in cytosolic and bacteroidal fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules during development. Activities of hexokinase, fructokinase, phosphoglucomutase, fructose-1,6-bisphosphatase, phosphohexose isomerase and UDP-glucose pyrophosphorylase were detected in cytosolic fraction of nodules of both the crops during development. Out of these enzymes, specific activity of phosphohexose isomerase was the highest in nodules of both the crops, in comparison with other enzymes. In comparison with mungbean, activities of various enzymes were less in cytosolic fraction of lentil. Activities of hexokinase, fructokinase, phosphoglucomutase were present only in cytosolic fraction of mungbean (Vigna radiata L.), however, low activity of these enzymes was also observed in lentil (Lens culinaris L.) bacteroids. Activities of phosphohexose isomerase and fructose-1,6-bisphosphatase were higher in bacteroids of lentil, as compared to mungbean during early nodule development, but this pattern was reversed with progress of crop development. Higher activities of phosphoglucomutase and fructose-1,6-phosphatase in mungbean cytosolic fraction could lead to increased flow of carbon towards pentose phosphate pathway.

Keywords: Lens culinaris, Lentil, Vigna radiata, Mungbean, Hexokinase, Fructokinase, Phosphoglucomutase, Fructose-1,6-bisphosphatase, Phosphohexose isomerase, UDP-glucose pyrophosphorylase, Bacteroids

 

*E mail: nkaur@rediffmail.com

 

 

 

 

 

 

 

 

AUTHOR INDEX

 

 


Angayarkanni N                   179

 

Balasundram C                      157

Barathi S                                 179

Beyo R S                                 164

 

Chakraborty S                        169

Chaturvedi N                          176

Chi Z                                        152

Comis A                                  140

 

Dani D N                                133

Darokar M P                          176

Dharaneedharan S               157

Divya L                                  164

Duan X                                  152

 

Fischer P                                140

 

Gao L                                      152

Geraldine P                            157

Gupta A K                             186

 

Howden M                            140

 

Jing LiJingjian W                 145                            

Kaur N                                   186

Khanuja S P S                       176

 

Ma L                                       152

Manju M                                164

Meiyu G                                  145

Munjral N                               186

 

Oommen O V                         164

 

Ramakrishnan S                    179

Roy K                                     169

 

Sainis J K                               133

Saraiya M                              176

Sengupta C                           169

Shasany A K                        176

Shukla A K                           176

Sreejith P                               164

Sudheesh S                           183

 

Tewari L                                 176

Tyler M                                  140

 

Vadhana P                             179

Velanganni A A J                 157

Vijayalakshmi N R                183

Vijayasree A S                      164

 

Yanli X                                    145

Yunxue Z                                145