Present and future of horseshoe crab in biomedical research

ANIL CHATTERJI & FAIZAH SHAHAROM

INSTITUTE OF TROPICAL AQUACULTURE UNIVERSITY MALAYSIA TERENGGANU
[[email protected]]

LAL Milestones
1880 W. H. Lowell first studies coagulation of L. polyphemus blood at Johns Hopkins University.

1956

Bang discovers that horseshoe crab blood forms clots when bacteria (Vibrio sp.) are present.

1964

Levin and Bang discover that the blood clotting agent in horseshoe crabs is an amoebocyte.

1968

Scientists discover that an endotoxin causes blood-clot reaction in horseshoe crabs. The method for preparing LAL from horseshoe crab blood is developed.

1971

The standard LAL assay is developed.

1977

The FDA replaces the standard rabbit test for endotoxins with the LAL test.

1983

The FDA accepts LAL as a standard test for bacterial endotoxin contamination.

1987

The FDA establishes guidelines for LAL testing of pharmaceuticals and medical devices.

Collection of blood (haemolymph)

Conventional New technique

Companies producing Amoebocyte Lysate Associates of Cape Cod: http://www.acciusa.com/ Cambrex: http://www.cambrex.com/default.asp Charles River Endosafe: http://www.criver.com/products/endotoxin/index.html

[Indian Patent NO. 192,300 (2004)]

USES OF AMOEBOCYTE LYSATE

LAL provides annual revenue of $60 m It generates a social welfare benefit over $150 m, yearly
Horseshoe crab

Extraction of blood

Separation of amoebocytes

Amoebocyte Lysate (LAL)

PHARMACEUTICAL PRODUCTS
[Injectable, intravenous drugs and for screening prosthetic devices like heart valves or hip replacements (FDA)]

FOOD PRODUCTS

DIAGNOSIS / TREATMENTS

Clinical diagnosis
Eye disease

Homeopathic treatments (Limulus)

Body & mental exhaustion Drowsiness after sea bathing

Spinal meningitis

Gastroentric symptoms

Urinary infections (Neiserria gonorrhea)

Rheumatism

Pain at right side of body

Regeneration of gill lamellae of the Indian horseshoe crab

[Chatterji et al. (2004), Current Science, 87 (11), 1511-1512]

Optical Stack of Gill flaps of T. gigas

In vivo

In vitro

[In vitro Cellular & Developmental Biology Animal, 38, 255-257 (2002].

[US Patent No 6,790,659 (2004)]

Recent advances in various researches carried out on PVF

PVF

A developing embryo between 39 and 42 day after fertilization

List of main researches on biomedical applications and PVF

Jorgenson, J. H. and Smith, R. F. (1973) Preparation, sensitivity, and specificity of Limulus lysate for endotoxin assay. Appl. Microbiol., 26:43-48. Cohen, E. (1979) Biomedical Applications of the Horseshoe Crab (Limulidae). Alan. R. Liss. Inc., New york, 668 pp. Cooper, J. F. (1979) Acceptance of the limulus test as alternative pyrogen test for radio pharmaceuticals and interathecal drugs. Progress Clin. Biol. Res. 29:345. Rudloe, A. (1979). Limulus polyphemus. A review of the ecologically significant literature. In : Biomedical application of the Horseshoe crab (Limulidae). E. Cohen ecol. Alan R. Liss. Inc., New York, 27-35. Ross, V. C. and Bruck, C. W. (1982). Endotoxin testing of medical devices with LAL:FDA requirements. In:Endotoxin and their detection with the Limulus Amebocyte Lysate Test, Watson, S. W., Levin, J. and Novitsky, T. J. (Eds). Alan R. Liss. Inc., NewYork, 39-48. Srimal, S. and Bachhwat, B. K. (1985) Blood Brother-a living fossil helps to save human life. Sci. Age, 51-55. Srimal, S., Miyata, T. Kawabata, S. and Iwanaga, S. (1985). The complete amino acid sequence of coagulogen isolated from south-east Asian horseshoe crab, Carcinoscorpius rotundicauda. J. Biochem. (Tokyo), 98: 305-318. Toh, Y., Mizutani, A., Tokunaga, F., Muta, T., and Iwanaga, S. (1991). Morphology of the granular hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances. Cell Tissue Res., 266, 137147. Friber, J. A., Weathers, P.J., Gibson, D.G. III (1992). Culture of amoebocytes in a nutrient mist bioreactor. In vitro Cell. Dev. Biol., 28A(3): 215-217. Muta, T., and Iwanaga, S. (1996) The role of hemolymph coagulation in innate immunity Curr. Opin. Immunol. 8, 41 47. Sugita, H. and Sekiguchi, K. (1979) Protein components in the peri-vitelline fiuid of the embryo of the horseshoe crab. Dev. Biol., 73, 183-192. Nagai,T., Kawabata, S. I., Shishikura, F., Sugita, H. (1999). Purification and characterization of an amino acid sequence of an embryonic lactin in peri-vitelline fluid of the embryo of the horseshoe crab. J. Biol. Chem., 274:37673-37678. Parab, P. B., Chatterji. A. (2003) Isolation of peri-vitelline fluid of the fertilized eggs of horseshoe crab and use thereof, US patent #20030017212. Shishikura, F., Sekiguchi, K. (1984a). Studies on perivitelline fluid of horseshoe crab embryo. Purification and properties of agglutininfrom the perivitelline fluid of Tachypleus gigas, embryo. J. Biol. Chem., 96, 621-628. Shishikura, F., and Sekiguchi, K. (1984b). Studies on peri-vitelline fluid of horseshoe crab embryo. II. Purification of agglutinin-binding substance from the peri-vitelline fluid of Tachypleus gigas embryo. J. Biol. Chem. 96 (3):629-636.

Use of PVF in enhancing early gonad maturation in commercially important fish

Control

Experimental

Control

Experimental

Testes

Ovary

HCG?

After 7th day of treatment with 100 l PVF

PVF with proline zinc complex and diabetes

National Institute of Oceanography, Goa, India National Center for Cell Science, Pune, India

Cardiac promoting effect of PVF

Control PVF (1:10) treated

Heart [US Patent Nos 20030017212 (2003) & 7,087,429 (2006)]

FPLC Profile of PVF

Fractionation of PVF by FPLC

PEAK 7 PEAK 6
SHOULDER

7th fraction

PEAK 4
SHOULDER TUBE RESTART SPIKE

PEAK 2 PEAK 3 PEAK 1

PEAK 8
SPECIFICATIONS: BUFFER PBS pH 7.4 SAMPLE PVF (BATCH 25.02.2004) SAMPLE LOAD 500 g COLUMN SUPEROSE 12, 2 COLUMNS CONNECTED IN SERIES AUFS 0.1 CHART SPEED 0.1 FRACTION SIZE 0.5ml

Effect of PVF on enhancement of cardiac myocytes cells

Cardiac myocytes are the first cells to differentiate during developmental process in vertebrate embryo Wide variety of molecules take part in this process

PVF

Heart

{Cellular & Molecular Life Science, 65 (October 2008)}

[US Patent No:20040180324 (2004)]

Heart enlargement due to PVF Control PVF (1:10) treated

Gel profile of Fraction 7

M
97.4 KDa 66 KDa

43 KDa

27 KDa

Legends 20.1 KDa 14.3 KDa 7 M Peak 7 Protein Standard

[Fraction VII was excised and submitted to W. M. Keck Biomedical Mass Spectrometry Laboratory of Bio-molecular Research Facility (University of Virginia Health System, Charlottesville, USA]

The sequencing analysis revealed a 221-amino acid protein

Homology search using BLAST showed 100% similarity l to Limulus lectin L6, a lipopolysaccharide-binding antibacterial protein from of T. tridentatus (Saito et al., 1995) VQWHQIPGKLMHITATPHFLWGVNSNQQIYLCRQPCYDGQWTQISG SLKQVDADDHEVWGVNRNDDIYKRPVDGSGSWV RVSGKLKHVSASGYGYIWGVNSNDQIYKCPKPCNGAWTQVNGRLK QIDGGQSMVYGVNSANAIYRRPVDGSGSWQQISGSLKHITGSGLSE VFGVNSNDQIYRCTKPCSGQWSLIDG LKQCDATGNTIVGVNSVDNIYRSG

This molecule may prove to be an important tool in the study of cardiomyocyte differentiation

[US Patent Nos 20060210642 (2006) & 20060141055 (2006)]

PVF role in cardiomyocytes differentiation of bone marrow stem cells in vitro

Assessment of effective fraction of PVF by the expression of myoglobin (FACS analysis)
30 25 20 15 10 5 0 1 2 3 4 5 6 7 8 Different fractions of PVF
Mean fluorescence intensity

Assessment of effective dose of PVF by the expression of myoglobin (FACS analysis)

30 25 20 15 10 5 0 Control (Without PVF) PVF 0.1mg/ml PVF 0.01mg/ml PVF 0.05mg/ml Different concentrations of PVF

Mean fluorescence intesnity

Expression of myoglbin by differentiating CD34+ cells (FACS analysis)

30
Mean flourescence intensity

25 20 15 10 5 0 Control (Without PVF) PVF VEGF bFGF

NIO, Paris University, UMT

Horseshoe crab (PVF) for culture media & Growth factors

Demands of different growth media in stem cell research

(500,000 litres required per year)

NO
1
2 3 4 5 6

Growth Media
VEGF (Vascular Endothelial Growth Factor) b-FGF (Basic Fibroblast Growth Factor) LIF (Leukemia Inhibitory Factor) GM-CSF (Granulocyte Macrophage Colony Stimulating Factor) FCS (Foetal Calf Serum) FBS (Foetal Bovine Serum)

World Market
US$550 million to $5.3 billion in 2009
US$ 22 billion by 2012 US $500 million US$ 2.2 billion in 2009 US $ 32 billion US $ 29 billion

Horseshoe crab (PVF)-Embryonic stem cell

Influence of PVF in maintenance of undifferentiated state in ES cells

PVF LIF
PVF exhibits more compact morphology National Institute of Oceanography, Goa (India) National Center for Cell Science, Pune (India)

Role of peri-vitelline fluid in maintaining the original phenotype of CD34+ stems cells and colony formation in vitro
Human bone marrow from healthy volunteers (donors for bone marrow transplantation) was collected from the Haematology Division of the Hospital Hotel Dieu, Paris (France). The mononuclear cells from the cells were then immediately separated by Ficoll centrifugation technique Bone marrow was diluted in 40 ml of L- Glutamin enriched with RPMI 1640 medium.

CD 34+ cells differentiated from bone marrow stem cells into endothelial cells in presence of growth factor (VEGF)
Mononuclear cell ring was collected and separation of CD 34+ cells was carried out using immune-magnetic beads as per the protocol of Easy Sep Stem Cell Technologies Inc U.S.A NIO,Goa, Paris University, UMT

CD 34+ cells differentiate from bone marrow stem cells into endothelial cells in presence VEGF CD 34+ cells remain viable for 3-4 days CD 34+ cells do not multiply in vitro

Attachment of CD 34+ cells to the surface of the 22 days of culture

No of attached cells (10-6 cells /ml) 160 140 120 100 80 60 40 VEGF bFGF VEGF+bFGF VEGF+bFGF+PVF PVF

Control

20
0

Different media

Uses: Treatment of ischemic and vascular diseases

[US Patent No 0222NF2005, 6/20/2005] PVF treated

Role of peri-vitelline fluid in promoting vasculogenesis

NIO, Goa, Paris University, UMT Vasculogenesis is a process of differentiation of stem cell endothelial progenitor cells (EPC) into mature endothelial cells It contributes to new vessel formation and resembles the embryological process Cellular progenitor population for the generation of EC by a compound may serve as cellular source for therapeutic vasculogenesis

It could represent a novel approach to treat patients with ischemic disease that are not curable with conventional treatments

[US Patent No 0222NF2005, 6/20/2005]

Angiogenesis

Wound healing with PVF

Control Treated with PVF

( 24 hrs)

Migration of human bone marrow endothelial cells

Migration of human bone marrow endothelial cells (0.1 mg/ml of PVF) (Wound healing)
Control

(0 hrs)

(3 hrs)

(6 hrs)
PVF treated cells

(9 hrs)

(24 hrs)

NIO,Goa, Paris University, UMT

Artificial breeding by electrical stimuli (9 V 600 mA DC current)

Incubation of fertilized eggs in re-circulating type incubator

Part-2

Part-5 Part-6

Part-3 Part-4 Part-7

Part-1

Collection of PVF and releasing of larvae

Horseshoe crab in Malaysia

Distribution
TACHYPLEUS TRIDENTATUS (POCOCK)
WESTERN & SOUTHERN JAPAN, TAIWAN, PHILIPPINES & NORTH BORNEO, MALAYSIA

LIMULUS POLYPHEMUS (MLLER)

ATL ANTIC COAST OF NORTH AMERICA FROM MAINE TO YUCATAM

TACHYPLEUS GIGAS (MLER)

BAY OF BENGAL (NORTH -EAST COAST), THAILAND, MALAYSIA, PHILI PPINES, BORNEO & TORRES STRAITS

NEW SPECIES
[Johor Bahru (Malaysia)]

CARCINOSCORPIUS ROTUNDICAUDA (LATREILLE)

BAY OF BENGAL, THAILAND, MALAYSIA, PHILIPPINES, BORNEO & TORRES STRAITS

?
[Chatterji, 1994]

Tachypleus tridentatus Weight: 9.5 kg (a record) [J. Bombay Natural History Society, 2008]

Spawning ground of T. gigas at Balok (Malaysia) in 2009

Spawning ground at Balok (Malaysia) in 2010

Conservational measures suggested to Malaysian Government through WWF [MoU signed on 22 May 2011]
Prevention of over fishing for restoring wild population

Protection of sand dune vegetation on the beaches

Ban on removal of sand gravel from the breeding beaches for construction purpose.

Removal the polluting substances from the beaches and prevention of industrial discharge for protecting breeding beaches
Identifying and protecting the critical habitats Coordinating and promoting collaborative researches and monitoring law enforcement bodies. Generating public awareness programmes.

Location Plan
South China Sea

AASB-UMT Horseshoe Crabs Sanctuary

36

A first Asian horseshoe crab sanctuary at Kota Bharu in Malaysia

A restaurant of Johor popular only for horseshoe crabs food

38

INCLUSION OF HORSESHOE CRAB IN SCHEDULE-IV OF THE WILD LIFE PROTECTION ACT -1972 OF INDIA

PUBLICATIONS 1.Growth and morphometric characteristics of the Horseshoe crab, Carcinoscorpius rotundicauda (Latreille) from Canning (West Bengal), India. Pak. J. Sci. Ind. Res., 31 (5): 352-353. Anil Chatterji, R. Viyayakumar and A. H. Parulekar (1988). 2. Bharat Ke Utriya Purbi Taton Per Raj Kankra Ke Samishti Ka Viteran, Samudra Vigyan Ki Uplabdhiyan (1991). (in Hindi). Anil Chatterji (1991). 3. Spawning Migration of the Horseshoe crab, Tachypleus gigas (Muller), in relation to lunar cycle, Asian Fisheries Science, 5: 123-128. Anil Chatterji, R. Vijayakumar, and A. H. Parulekar (1992). 4.Fecundity of the Indian Horseshoe crab, Carcinoscorpius rotundicauda (Latreille) Tropical Ecology, 33 (1): 97-102. Anil Chatterji, and A. H. Parulekar (1992). 5.Feeding behaviour and food selection in the Horseshoe crab, Tachypleus gigas (Muller). Hydrobiology, 246: 41-48. Anil Chatterji, J. K. Mishra and A. H. Parulekar (1992). [IF=0.470] 6. Seasonal variation in the volume of the Haemolymph and body weight of the Horseshoe crab, Tachypleus gigas (Muller). Pak. J. Sci. Ind. Res., 35: 495498. Anil Chatterji, R. Vijayakumar and A. H. Parulekar (1992). 7.A freak twin Trilobite larva of the Indian horseshoe crab, Tachypleus gigas (Muller). J. Bombay. Nat. Hist. Soc., 99: 116-117. J. K. Mishra, A. Chatterji and A. H. Parulekar (1992). [IF=0.470] 8. The Indian horseshoe crab A living fossil. Indian J. Ocean. Stud., 1 (1): 42-48. Anil Chatterji and S. A. H. Abidi (1994). 9.Length- weight relationship of the Indian horseshoe crab, Tachypleus gigas (Muller). Indian J. Fish., 41 (2): 58-60. Anil Chatterji, J. K. Mishra, R. Vijaykumar and A. H. Parulekar (1994) 10. Occurrence of Diaphanosoma excisum (Sars) on a sandy beach Balramgari (Orissa), India. Hydrobiologia, 310: 157-161. Anil Chatterji, Z. A. Ansari, J. K. Mishra, P. Rattan and A. H. Parulekar (1995). [IF=0.470] 11. The Horseshoe crab, its economical importance and controlled breeding. Ocean and Industry-Realities and Expectations, P. S. Srivastava. Anil Chatterji (1995). 12. Fecundity of the Indian Horseshoe crab. Tachypleus gigas (Muller) from Balramgari (Orissa). Pak. J. Mar. Sci., 4 (2): 127-131. Anil Chatterji (1995). 13. Energy source in developing eggs of the Indian horseshoe crab, Tachypleus gigas (Muller). Journal of Aquaculture in the Tropics, 11: 271-276 Anil Chatterji, Queenie Aguair and Christine Saldanha (1996). 14. Nesting behaviour of the Indian horseshoe crab, Tachypleus gigas (Muller) (Xiphosura). In: Indias Exclusive Economic Zone, S. Z. Qasim and G. S. Roonwal (Eds), New Delhi, 142-148. Anil Chatterji, A. H. Parulekar and S. Z. Qasim (1996). 15. Raj Kankra Ka Ausidhi Vigyan Main Upyog. Vigyan Surabh, Rashtriya Vaigyanic Hindi Sangoshthi Ka Lekh Sangrah, 28-29 August, 1997, 183-185. Anil Chatterji and Umesh Singh (1997). 16.New record of the sympatric distribution of the two Asian species of the horseshoe crab, Current Science, 77, (6) 746-747. Anil Chatterji (1999). [IF=0.688] 17. Bhartiya Rajkankra Ka Manav Jeevan Main Mahatvya aim Sangrakshan, Rashtriya Vigyan Snagoshthi, Vaigyanic Vishyon Ka Hindi Main Sampreshan, CEERI, Pilani, 17. Anil Chatterji and Umesh Singh (1999). 18. Morphometric characteristics in the horseshoe crab, Tachypleus gigas (Arthropoda:Merostomata). Indian Journal of Marine Sciences, 29: 333-335. R. Vijaykumar, S. Das, A. Chatterji, A. H. Parulekar (2000). [IF=0.124] 19. Long term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Muller). In vitro Cellular & Developmental BiologyAnimal, 38, 255-257. Bhupali Joshi, Anil Chatterji and Ramesh Bhonde (2002). [IF=0.576] 20. Vichtriya Raj Kankra aur uska upyog (in Hindi). Bharat Main Vigyan Ki Uplabdhiyan-Ek Jhalak, Rashtria Antartic ayam Samudriya Anusandhan Kendra, 120-123.Anil Chatterji & Umesh Singh (2002) 21. Effect of salinity on the growth of laboratory reared trilobite larvae of the Indian horseshoe crab, Tachypleus gigas (Muller). Current Science, 37 (2), 248250. Anil Chatterji, Savita Kotnala and Reny Mathew (2004). [IF=0.688] 22,. Regeneration of gill lamellae of the Indian horseshoe crab, Tachypleus gigas (Muller). Current Science, 87 (11), 1511-1512. Anil Chatterji, Huma Alam, Bhupali K. Joshi and R. R. Bhonde (2004) [IF=0.688]

23. Enhancement of vertebrate cardiogenesis by a lectin from perivitelline fluid of horseshoe crab embryo. Cellular and Molecular Life Sciences, 65: 3312 3324. Surendra Ghaskadbi, Vidya Patwardhan, Monideepa Banerjee, Swati Agarwal, Nibedita Lenka, Mahesh Kumar Verma, Anil Chatterji and P.B. Parab (2008). [IF= 5.511] 24. Abundance of three species of the horseshoe crab along the coast of Malaysia. J. Bombay Nat. Hist. Soc. 105(3): 209-211. A. Chatterji, Z. Kassim, H. Shahuddin, F. Shaharom (2008) [IF=0.470]. 25. Unusual spawning behaviour of the Indian horseshoe crab (Tachypleus gigas, Mller) after the tsunami Pertanica Journal of Science and Technology, 17 (2): 263-268. A. Chatterji and F. Shaharom (2009). 26. A new record on variability in different body measurements of the horseshoe crab, Carcinoscorpius rotundicauda (Latreille) collected from Setiu and Gelang Patah habitats in Peninsular Malaysia. J. Bombay Nat. Hist. Soc. (Accepted). [IF=0.470] Srijaya, T.C., Pradeep, P.J., Mithun, S., Anuar Hassan, Faizah Shaharom, Anil Chatterji 27. Peri-vitelline fluid of the fertilized eggs of the Indian Horseshoe Crab (Tachypleus Gigas, Mller) helps in maintaining the original phenotype of CD 34+ stem cells and enhances colony formation in vitro. In: Horseshoe crab in Asia, Published by: Lambert Academic Publishing, Germany (Accepted). Massoud Mirshahi, Pezhman Mirshahi, Jeannette Soria, Huma Alam, Amu Therwath and Anil Chatterji 28. Growth performance of young larvae of mangrove horseshoe crab, Carcinoscorpius rotundicauda (Latreille) under different environmental conditions. In: Horseshoe crab in Asia, Published by: Lambert Academic Publishing, Germany (Accepted). Srijaya, T. C., Pradeep, P.J., Mithun Sukumaran, Faizah Shaharom and Anil Chatterji. 29. Allometric relationship in the population of horseshoe crab (Tachypleus tridentatus; Leach) collected from Papar Sabah in Malaysia. In: Horseshoe crab in Asia, Published by: Lambert Academic Publishing, Germany (Accepted). Anil Chatterji, Srijaya, T.C., Mithun Sukumaran, Pradeep, P.J. and Faizah Shaharom 30. Variation in distribution of horseshoe crabs nest along Pahang coastal area. 9th International Symposium on sustainability science and management, UMTAS 2010, Towarsd a healthier and sustainable future, 540-544. Nur Erni Atika Hadnan, Kamaliah Kasmarudin, Mohd Luthfu Omar, Anil Chatterji, Kamaruzzaman, B. Y., Mohd. Effendy, A. W., Norantonina Abdullah and Zaleha Kasim 31. A New record on the morphometric variations in the populations of horseshoe crab (Carcinoscorpius rotundicauda) obtained from two different ecological habitats of Peninsular Malaysia. Our Nature, 8: 204-211. Srijaya Thekkeparambil Chandrabose, Pradeep Padmaja Jayaprasad, Mithun Sukumaran, Anuar Hassan, Faizah Shaharom and Anil Chatterji 33. Marine living resources in the practice of traditional medicine. J. Coast. Environ., 1(1), 49-60 . A. Chatterji, Zaleha Kassim, Anuar bin Hassan, Amu Therwath and Faizah Shaharom 34. Effects of different environmental parameters on the respiratory metabolism of the larvae of Malaysian horseshoe crab, Tachypleus gigas (Mller). Pertanica Journal of Science and Technology, 19(1): 1-9.Suniza A. M. S., Zaleha bt Kassim and Anil Chatterji (2011). Popular articles 1. The amazing Horseshoe crab, Science Today, September, 1987:62-64. Anil Chatterji and R. Vijayakumar (1987) 2. Asharyajanak Raj Kankra aur Uska Anokha Bebhar, Vaigyanic, April_June, 1992, 37 pp. Anil Chatterji (1992) 3. Bharat Ke Untripurbi Taton per Raj Kankra ke Samishti ka Vitran, Samudra Vigyan Ki Uplabhdhiyan, Mahasagr Vikas Prakashan (1991): 59-61. Anil Chatterji (1992) 4. Bharatiya Raj Kankra aur Uske Neele Rakta ka Rahasya. Kendriya Sachivalya Hindi Parishad. Anil Chatterji (1995) 5. Vichtra Raj Kenkadon ka Aushidiyae vigyan main yogdan (Swagat). Huma Alam and Anil Chatterji 6. Horseshoe crab a valuable marine creature for human use, Prospects Malaysia, 50-56 (November 2008). Anil Chatterji and Noraznawati Ismail. 7. The horseshoe crab and their use in human cardiac disorders, Synthesis, USM, 23, 12 pp (December 2008). Anil Chatterji 8. Marine living resources in the age old practice of traditional medicine-an overview. Prospects Malaysia, 011, 50- 57 (July 2009). A. Chatterji, Zaleha Kassim, Anuar bin Hassan and Faizah Shaharom