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American Journal of Medical and Biological Research

ISSN (Print): 2328-4080

ISSN (Online): 2328-4099

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Role of Some Metal Ions on Steady–state Kinetics of Engineered Wild–type and Manganese (II) Binding Site Mutants of Recombinant Phlebia radiata Manganese Peroxidase 3 (rPr-MnP3)

1Department of Biological Sciences, Akwa Ibom State University, P.M.B. 1167, Uyo, Akwa Ibom State, Nigeria

2Department of Chemistry, Akwa Ibom State University, P.M.B. 1167, Uyo, Akwa Ibom State, Nigeria

3Research and Development Unit, Akwa Ibom State University, P.M.B. 1167, Uyo, Akwa Ibom State, Nigeria

4Department of Biochemistry, University of Uyo, P. M. B. 1017, Uyo, Akwa Ibom State, Nigeria

American Journal of Medical and Biological Research. 2016, 4(3), 42-52
doi: 10.12691/ajmbr-4-3-2
Copyright © 2016 Science and Education Publishing

Cite this paper:
Usenobong F. Ufot, Aniefiok E. Ite, Idorenyin H. Usoh, Monday I. Akpanabiatu. Role of Some Metal Ions on Steady–state Kinetics of Engineered Wild–type and Manganese (II) Binding Site Mutants of Recombinant Phlebia radiata Manganese Peroxidase 3 (rPr-MnP3). American Journal of Medical and Biological Research. 2016; 4(3):42-52. doi: 10.12691/ajmbr-4-3-2.

Correspondence to: Usenobong  F. Ufot, Department of Biological Sciences, Akwa Ibom State University, P.M.B. 1167, Uyo, Akwa Ibom State, Nigeria. Email:


This study investigated the steady-state kinetics of engineered wild-type and manganese (II) binding site mutants of recombinant Phlebia radiata manganese peroxidase 3(rPr-MnP3). The effect (activation or inhibition) of some metal ions (Co2+, Zn2+ Cu2+ and Na+) on the activity of rPr-MnP3 enzymes was also studied. The results obtained showed that the rPr-MnP3 mutants in which the metal binding functionality has been largely lost have been created. Na+ (mono-valent ion) and Co2+showed similar characteristics by exhibiting stimulatory effects on the activity of wild-type rPr-MnP3. However, Cu2+ and Zn2+ had mixed inhibitory effects on wild-type and mutants (E40H, E44H, E40H/E44H). It was observed that Cu2+ was by far the strongest inhibitor of engineered rPr-MnP3 enzymes while Co2+ exhibited a non-competitive inhibitory effect on the double mutant (E40H/E44H) and D186H activities. In addition, Zn2+ and Cu2+also had non-competitive inhibitory effect on D186H mutant enzyme activity. The results obtained further showed that the competitive inhibitory effect of Cu2+observed in other rPr-MnP3 enzymes is largely removed in D186H mutant enzyme. Generally, histidine substitution retained a strong selectivity for Cu2+ as competitive inhibitor. Zn2+ being generally non-competitive suggest involvement of sites other than the Mn (II) binding site. This study showed that rPr-MnP3 enzymes function with alternate ligands in the Mn2+ binding site and does not have absolute obligate requirement for all carboxylate ligand set.



[1]  Hatakka, A. I., and A. K. Uusi-Rauva, “Degradation of 14C-labelled poplar wood lignin by selected white-rot fungi,” European journal of applied microbiology and biotechnology, 17 (4). 235-242, 1983.
[2]  Lundell, T., A. Leonowicz, J. Rogalski, and A. Hatakka, “Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid,” Applied and Environmental Microbiology, 56 (9). 2623-2629, 1990.
[3]  Vares, T., M. Kalsi, and A. Hatakka, “Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw,” Applied and Environmental Microbiology, 61 (10). 3515-3520, 1995.
[4]  Hatakka, A., T. Lundell, M. Hofrichter, and P. Maijala, “Manganese Peroxidase and Its Role in the Degradation of Wood Lignin,” Applications of Enzymes to Lignocellulosics, ACS Symposium Series 855, S. D. Mansfield and J. N. Saddler, eds., pp. 230-243: American Chemical Society, 2003.
[5]  Niemenmaa, O., A. Uusi-Rauva, and A. Hatakka, “Wood stimulates the demethoxylation of [O14CH3]-labeled lignin model compounds by the white-rot fungi Phanerochaete chrysosporium and Phlebia radiata,” Archives of Microbiology, 185 (4). 307-315, 2006.
Show More References
[6]  Hatakka, A., and K. E. Hammel, “Fungal Biodegradation of Lignocelluloses,” Industrial Applications, M. Hofrichter, ed., pp. 319-340, Berlin, Heidelberg: Springer Berlin Heidelberg, 2011.
[7]  Marco-Urrea, E., and C. A. Reddy, “Degradation of Chloro-organic Pollutants by White Rot Fungi,” Microbial Degradation of Xenobiotics, N. S. Singh, ed., pp. 31-66, Berlin, Heidelberg: Springer Berlin Heidelberg, 2012.
[8]  Lundell, T., “Ligninolytic System of the White-rot Fungus Phlebia radiata: Lignin Model Compound Studies,” Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, 1993.
[9]  Karhunen, E., A. Kantelinen, and M.-L. Niku-Paavola, “Mn-dependent peroxidase from the lignin-degrading white rot fungus Phlebia radiata,” Archives of Biochemistry and Biophysics, 279 (1). 25-31, 1990.
[10]  Moilanen, A. M., T. Lundell, T. Vares, and A. Hatakka, “Manganese and malonate are individual regulators for the production of lignin and manganese peroxidase isozymes and in the degradation of lignin by Phlebia radiata,” Applied Microbiology and Biotechnology, 45 (6). 792-799, 1996.
[11]  Hildén, K. S., M. R. Mäkelä, T. K. Hakala, A. Hatakka, and T. Lundell, “Expression on wood, molecular cloning and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata,” Current Genetics, 49 (2). 97-105, 2005.
[12]  Lundell, T. K., M. R. Mäkelä, and K. Hildén, “Lignin-modifying enzymes in filamentous basidiomycetes – ecological, functional and phylogenetic review,” Journal of Basic Microbiology, 50 (1). 5-20, 2010.
[13]  Hofrichter, M., R. Ullrich, M. J. Pecyna, C. Liers, and T. Lundell, “New and classic families of secreted fungal heme peroxidases,” Applied Microbiology and Biotechnology, 87 (3). 871-897, 2010.
[14]  Glenn, J. K., and M. H. Gold, “Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrysosporium,” Archives of Biochemistry and Biophysics, 242 (2). 329-341, 1985.
[15]  Paszczyński, A., V.-B. Huynh, and R. Crawford, “Enzymatic activities of an extracellular, manganese-dependent peroxidase from Phanerochaete chrysosporium,” FEMS Microbiology Letters, 29 (1-2). 37-41, 1985.
[16]  Hofrichter, M., “Review: lignin conversion by manganese peroxidase (MnP),” Enzyme and Microbial Technology, 30 (4). 454-466, 2002.
[17]  Wariishi, H., K. Valli, and M. H. Gold, “Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators,” Journal of Biological Chemistry, 267 (33). 23688-23695, 1992.
[18]  Kirk, T. K., and R. L. Farrell, “Enzymatic “Combustion”: The Microbial Degradation of Lignin,” Annual Review of Microbiology, 41 (1). 465-501, 1987.
[19]  Kersten, P., and D. Cullen, “Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium,” Fungal Genetics and Biology, 44 (2). 77-87, 2007.
[20]  Buswell, J. A., E. Odier, and T. K. Kirk, “Lignin Biodegradation,” Critical Reviews in Biotechnology, 6 (1). 1-60, 1987.
[21]  Martinez, D., L. F. Larrondo, N. Putnam, M. D. S. Gelpke, K. Huang, J. Chapman, K. G. Helfenbein, P. Ramaiya, J. C. Detter, F. Larimer, P. M. Coutinho, B. Henrissat, R. Berka, D. Cullen, and D. Rokhsar, “Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78,” Nat Biotech, 22 (6). 695-700, 2004.
[22]  Hildén, K., A. T. Martinez, A. Hatakka, and T. Lundell, “The two manganese peroxidases Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent,” Fungal Genetics and Biology, 42 (5). 403-419, 2005.
[23]  Martı́nez, A. T., “Molecular biology and structure-function of lignin-degrading heme peroxidases,” Enzyme and Microbial Technology, 30 (4). 425-444, 2002.
[24]  Sundaramoorthy, M., K. Kishi, M. H. Gold, and T. L. Poulos, “Crystal Structures of Substrate Binding Site Mutants of Manganese Peroxidase,” Journal of Biological Chemistry, 272 (28). 17574-17580, 1997.
[25]  Kusters-van Someren, M., K. Kishi, T. Lundell, and M. H. Gold, “The Manganese Binding Site of Manganese Peroxidase: Characterization of an Asp179Asn Site-Directed Mutant Protein,” Biochemistry, 34 (33). 10620-10627, 1995.
[26]  Kishi, K., M. Kusters-van Someren, M. B. Mayfield, J. Sun, T. M. Loehr, and M. H. Gold, “Characterization of Manganese (II) Binding Site Mutants of Manganese Peroxidase,” Biochemistry, 35 (27). 8986-8994, 1996.
[27]  Boucher, L. J., K. Koeber, M. Kotowski, and D. Tille, “Coordination Compounds of Manganese,” Coordination Compounds 7, H. Demmer, M. Kotowski, E. Schleitzer-Rust and D. Tille, eds., pp. 1-2, Berlin, Heidelberg: Springer Berlin Heidelberg, 1989.
[28]  Sundaramoorthy, M., K. Kishi, M. H. Gold, and T. L. Poulos, “The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-A resolution,” Journal of Biological Chemistry, 269 (52). 32759-32767, 1994.
[29]  Sundaramoorthy, M., H. L. Youngs, M. H. Gold, and T. L. Poulos, “High-Resolution Crystal Structure of Manganese Peroxidase:  Substrate and Inhibitor Complexes,” Biochemistry, 44 (17). 6463-6470, 2005.
[30]  Gold, M. H., H. L. Youngs, and M. D. Sollewijn Gelpke, “Manganese Peroxidase,” Metal Ions in Biological Systems: Volume 37: Manganese and Its Role in Biological Processes, A. Sigel and H. Sigel, eds., pp. 559-586, New York, USA: CRC Press, 2000.
[31]  Kuan, I. C., K. A. Johnson, and M. Tien, “Kinetic analysis of manganese peroxidase. The reaction with manganese complexes,” Journal of Biological Chemistry, 268 (27). 20064-20070, 1993.
[32]  Kishi, K., H. Wariishi, L. Marquez, H. B. Dunford, and M. H. Gold, “Mechanism of Manganese Peroxidase Compound II Reduction. Effect of Organic Acid Chelators and pH,” Biochemistry, 33 (29). 8694-8701, 1994.
[33]  Paszczyński, A., V.-B. Huynh, and R. Crawford, “Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium,” Archives of Biochemistry and Biophysics, 244 (2). 750-765, 1986.
[34]  Wariishi, H., H. B. Dunford, I. D. MacDonald, and M. H. Gold, “Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism,” Journal of Biological Chemistry, 264 (6). 3335-3340, 1989.
[35]  Gregory, D. S., A. C. R. Martin, J. C. Cheetham, and A. R. Rees, “The prediction and characterization of metal binding sites in proteins,” Protein Engineering, 6 (1). 29-35, 1993.
[36]  Kennedy, M. L., and B. R. Gibney, “Metalloprotein and redox protein design,” Current Opinion in Structural Biology, 11 (4). 485-490, 2001.
[37]  Maneiro, M., W. F. Ruettinger, E. Bourles, G. L. McLendon, and G. C. Dismukes, “Kinetics of proton-coupled electron-transfer reactions to the manganese-oxo “cubane” complexes containing the Mn4O and Mn4O core types,” Proceedings of the National Academy of Sciences, 100 (7). 3707-3712, 2003.
[38]  Garcia, J. S., C. S. d. Magalhães, and M. A. Z. Arruda, “Trends in metal-binding and metalloprotein analysis,” Talanta, 69 (1). 1-15, 2006.
[39]  Pecoraro, V., and W. Hsieh, “The use of model complexes to elucidate the structure and function of manganese redox enzymes,” Metals in Biological Systems, A. S. a. H. Sigel, ed., pp. 429-504, New York, USA: CRC Press, 2000.
[40]  Puglisi, A., G. Tabbı̀, and G. Vecchio, “Bioconjugates of cyclodextrins of manganese salen-type ligand with superoxide dismutase activity,” Journal of Inorganic Biochemistry, 98 (6). 969-976, 2004.
[41]  Childs, R. E., and W. G. Bardsley, “The steady-state kinetics of peroxidase with 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen,” Biochemical Journal, 145 (1). 93-103, 1975.
[42]  Ruiz-Dueñas, F. J., M. Morales, M. Pérez-Boada, T. Choinowski, M. J. Martínez, K. Piontek, and Á. T. Martínez, “Manganese Oxidation Site in Pleurotus eryngii Versatile Peroxidase:  A Site-Directed Mutagenesis, Kinetic, and Crystallographic Study,” Biochemistry, 46 (1). 66-77, 2007.
[43]  Cleland, W. W., “1 Steady State Kinetics,” The Enzymes, D. B. Paul, ed., pp. 1-65: Academic Press, 1970.
[44]  Sandler, M., and H. J. Smith, “Introduction to the use of enzyme inhibitors as drugs,” Design of Enzyme Inhibitors as Drugs, , M. Sandler and H. J. Smith, eds., pp. 1-18, Oxford, UK: Oxford University Press, 1989.
[45]  Blake, R. D., Informational biopolymers of genes and gene expression, Sausalito, CA: University Science, 2004.
[46]  Millero, F. J., Chemical Oceanography, Fourth Edition, Boca Raton: CRC Press, 2013.
[47]  Sarkar, S., A. T. Martı́nez, and M. a. J. Martı́nez, “Biochemical and molecular characterization of a manganese peroxidase isoenzyme from Pleurotus ostreatus,” Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1339 (1). 23-30, 1997.
[48]  Liu, C., and H. Xu, “The metal site as a template for the metalloprotein structure formation,” Journal of Inorganic Biochemistry, 88 (1). 77-86, 2002.
[49]  Emeléus, H. J., and J. S. Anderson, “Modern Aspects of Inorganic Chemistry,” 1956.
[50]  Arnold, F. H., and J.-H. Zhang, “Metal-mediated protein stabilization,” Trends in Biotechnology, 12 (5). 189-192, 1994.
[51]  Ufot, U. F., and M. I. Akpanabiatu, “An engineered Phlebia radiata manganese peroxidase: expression, refolding, purification and preliminary characterization,” American Journal of Molecular Biology, 2 (4). 359-370, 2012.
[52]  Ite, A. E., I. I. Udousoro, and U. J. Ibok, “Distribution of Some Atmospheric Heavy Metals in Lichen and Moss Samples Collected from Eket and Ibeno Local Government Areas of Akwa Ibom State, Nigeria,” American Journal of Environmental Protection, 2 (1). 22-31, 2014.
[53]  Ite, A. E., N. F. Hanney, and K. T. Semple, “The Effect of Hydroxycinnamic Acids on the Microbial Mineralisation of Phenanthrene in Soil,” International Journal of Environmental Bioremediation & Biodegradation, 3 (2). 40-47, 2015.
[54]  Ufot, U. F., “Expression and Characterisation of a Novel Manganese Peroxidise from Phlebia radiata,” Department of Biochemistry, University of Sussex, University of Sussex, Brighton, United Kingdom, 2010.
[55]  Johnson, F., G. H. Loew, and P. Du, “Prediction of Mn(II) binding site of manganese peroxidase from homotology modeling,” Plant peroxidases: Biochemistry and Physiology: III International Symposium 1993: proceedings, xiii, 497 p., K. G. Weirder, S. K. Rasmussen, C. Penel and H. Greppin, eds., pp. 31 – 34, Geneva, Switzerland: University of Copenhagen and University of Geneva, 1993.
[56]  Harris, R. Z., H. Wariishi, M. H. Gold, and P. R. Ortiz de Montellano, “The catalytic site of manganese peroxidase. Regiospecific addition of sodium azide and alkylhydrazines to the heme group,” Journal of Biological Chemistry, 266 (14). 8751-8758, 1991.
[57]  Whitwam, R. E., K. R. Brown, M. Musick, M. J. Natan, and M. Tien, “Mutagenesis of the Mn2+-Binding Site of Manganese Peroxidase Affects Oxidation of Mn2+ by both Compound I and Compound II,” Biochemistry, 36 (32). 9766-9773, 1997.
[58]  Da Silva, J. F., and R. J. P. Williams, The Biological Chemistry of the Elements: the Inorganic Chemistry of Life: Oxford University Press, 2001.
[59]  Sunda, W. G., and S. A. Huntsman, “Effect of competitive interactions between manganese and copper on cellular manganese and growth in estuarine and oceanic species of the diatom Thalassiosira,” Limnology and Oceanography, 28 (5). 924-934, 1983.
[60]  Sunda, W. G., and S. A. Huntsman, “Relationships among growth rate, cellular manganese and manganese transport kinetics in estuarine and oceanic species of the diatom Thalassiosira,” Journal of Phycology, 22 (3). 259-270, 1986.
[61]  Sunda, W. G., “Trace Metal Interactions with Marine Phytoplankton,” Biological Oceanography, 6 (5-6). 411-442, 1989.
[62]  Coolbear, T., J. M. Whittaker, and R. M. Daniel, “The effect of metal ions on the activity and thermostability of the extracellular proteinase from a thermophilic Bacillus, strain EA.1,” Biochemical Journal, 287 (Pt 2). 367-374, 1992.
[63]  Nieboer, E., and D. H. S. Richardson, “The replacement of the nondescript term ‘heavy metals’ by a biologically and chemically significant classification of metal ions,” Environmental Pollution Series B, Chemical and Physical, 1 (1). 3-26, 1980.
[64]  Haas, K. L., and K. J. Franz, “Application of Metal Coordination Chemistry to Explore and Manipulate Cell Biology,” Chemical Reviews, 109 (10). 4921-4960, 2009.
Show Less References


Histomorphometrical Characterization of Skin of Native Cattle (Bos indicus) in Bangladesh

1Department of Quality Assurance, Incepta Vaccine Limited, Dhaka, Bangladesh

2Department of Anatomy and Histology, Chittagong Veterinary and Animal Sciences University, Chittagong, Bangladesh

3Department of Microbiology and Parasitology, Sher-e-Bangla Agricultural University, Dhaka

4Animal Health research Division, Bangladesh Livestock Research Institute, Savar, Dhaka-1341, Bangladesh

American Journal of Medical and Biological Research. 2016, 4(3), 53-65
doi: 10.12691/ajmbr-4-3-3
Copyright © 2016 Science and Education Publishing

Cite this paper:
Md. Ershad Hossain, Mohi Uddin, S. Kumar Shil, M. H. B. kabir, M. Showkat Mahmud, Kh. Nurul Islam. Histomorphometrical Characterization of Skin of Native Cattle (Bos indicus) in Bangladesh. American Journal of Medical and Biological Research. 2016; 4(3):53-65. doi: 10.12691/ajmbr-4-3-3.

Correspondence to: M.  H. B. kabir, Department of Microbiology and Parasitology, Sher-e-Bangla Agricultural University, Dhaka. Email:


Fresh skin samples of the native cattle (Bos indicus) of 6 male and 6 female were collected from ten regions of the body immediately after slaughtering aimed to know the regional and sex variation of skin components. The skin samples were fixed, processed and stained according to the standard histological procedure. The samples of vertical sections were stained by both Hematoxylin & Eosin stain and Van Geison & Verhoff’s stain. Transparent sheet and ocular micrometer was used for histomorphometric study. Histomorphometrical study of the skin revealed that the mean thicknesses of epidermis, papillary layer, reticular layer and total skin were 45.37 ± 0.59 µm, 0.55 ± 0.01 mm, 3.03 ± 0.09 mm and 3.57 ± 0.09 mm respectively. These thicknesses were significantly (P<0.05) varied among the body regions. The highest thicknesses for epidermis and papillary layer were found in back and ventral abdomen respectively and the lowest thicknesses in thigh and neck & shoulder respectively whereas the highest thicknesses for reticular layer and total skin were found in head and the lowest thicknesses in shoulder. The mean thicknesses of the epidermis, reticular layer and total skin were significantly (P<0.05)) higher in male than the female. The mean papillary layer thickness was insignificantly (P<0.05) higher in female than the male. The mean density of collagen fibre bundle, elastic fibre bundle, sebaceous gland, sweat gland and hair follicle per mm² were 302.93 ± 9.07, 4.96 ± 0.17, 4.22 ± 0.13, 4.57 ± 0.14 and 37.25 ± 1.77 respectively. The density of collagen and elastic fibre bundle were significantly (P<0.05) varied among the body regions. The highest density of collagen bundle was found in ventral abdomen and lowest density in thigh and the highest elastic bundle density in thigh and lowest in back & ventral abdomen. The density of collagen fibre, hair follicle and sebaceous gland were significantly higher (P<0.05) in male than the female. The density of sebaceous gland was significantly (P<0.05) highest in head and lowest in lateral abdomen. The density of sweat gland was insignificantly (P>0.05) highest in neck & shoulder and lowest in loin. The density of hair follicle was significantly (P<0.05) highest in back and lowest in ventral abdomen. In conclusion, male skin may be better than the female skin for the quality leather production. Collagen bundle is more compact in ventral abdomen that regions may be better for quality leather production, but the percentage of collagen did not revealed in this study.



[1]  Mobini B, Faradonbeh KS (2012) A morphometric study on differentregions of the skin in lori-Bakhtiari sheep at different ages. International journal of plant, animal and environmental science. 2(4): 180-185.
[2]  Salehi M, Lavvaf A, Farahvash T (2013a) Skin Quality and Physical Properties of Leather Based on Sex, Age and Body Parts of Goats Reared on Sub‐Humid Hill Country. Iranian Journal of Applied Animal Science. 3(4): 853-857.
[3]  Adel R, Elboushi Y (1994) Poultry Feed from Waste. Hide and Tanning by Products. 154-156.
[4]  Widelitz RB, Tin-Xin J, Noveena A, Sheree A, Tin-Berreth EY, Hang-Sung J, Cheng-Ming C (1997) Molecular Histology in Skin Appendage Morphogenesis. Microscopy Research and Technique. 38: 452-465.
[5]  Genkovski D, Gerchev G (2007) Study of the skin histological structure in ewes from staroplaninska and thorough bred Tsigai. Biotechnology in Animal Husbandry. 23(5-6): 191–197.
Show More References
[6]  Dellmann HD, Brown EM (1987) Text book of Veterinary Histology; 3rd editionn. Lea and Febiger. Philadelphia. 382-415.
[7]  Mannan AM (1973) Micro-anatomical and histochemical investigation of the normal and pathological skin of the hump of cattle. M. Sc (Vet. Science) thesis. Bangladesh Agricultural University, Mymensingh.
[8]  Copenhaver WM, Kelly DE, Wood RL (1978) Bailye’s Textbook of Histology. 17th Edition. Asian Edition. 423-445.
[9]  Ozfiliz N, Balikcier M, Erdost H, Zik B (2002) Histological and morphometric features of the skin of native and hybrid (f2) sheep. Turkish Journal of Veterinary and Animal Sciences. 26: 429-438.
[10]  Muralidharan MR, Ramesh V (2005). Histological and biochemical studies of the skin of cattle and buffalo. Indian Journal of Animal Research. 39: 41-44.
[11]  Razvi R, Suri S, Sarma K, Sharma R (2014) Histomorphological and histochemical studies on the different layers of skin of Bakerwali goat. Journal of Applied Animal Research. 1-6.
[12]  Sharma DN, Bharadwaj RL (1993) Regional variations in the thickness of the skin of adult yak. The Indian Veterinary Journal. 70: 437-438.
[13]  GoJdsbery S, Calhoun ML (1959). The comparative histology of the skin of Hereford and Aberdeen Angus cattle. American Journal of veterinary Research. 20: 61-68.
[14]  Kurtdede N, Asti RN (1999) The investigation on the skin structure of German Black Head, HampshireDown, Lincoln Longwool, White Karaman, Awassi and Konya Merino. Veterinary Journal of Ankara University. 46: 219-230.
[15]  Hafez ESE, Badreldin AL, Shafei MM (1955b) Skin structure of Egyptian buffaloes and cattle with particular reference to sweat glands. The Journal of Agricultural Science. 46(1): 19-30.
[16]  Hole MB, Bhosle NS Kapadnis PJ (2008) Histological Study of Skin Epidermis in Red Kandhari Cows. Indian Journal Animal Research. 42(1): 69-70.
[17]  Sultan GA (2012) Comparative histological and topographical study for the skin of head of male and female native black goat. AL-Qadisiya Journal of Veterinary Medicine Science. 11: 21-33.
[18]  Kobayashi A, Takehana K, Eerdunchaolu, Iwasa K, Abe M, Yamaguchi M (1999) Morphometric analysis of collagen; a comparative study in cow and pig skins. Anatomia, histologia, embryologia. 28: 235-238.
[19]  Mobini B (2013a) A quantitative evaluation of different regions of skin in adult Iranian native sheep. Veterinarni Medicina. 58(5): 260-263.
[20]  Bilgen G, Oktay G, Tokgoz Z, Guner G, Yalcin S (1999) Collagen content and electrophoretic analysis of type I collagen in breast skin of heterozygous naked neck and normally feathered commercial broilers. Turkish Journal of Veterinary and Animal Sciences. 23(5): 483-488.
[21]  Mitra SK (1963) Indian Hides and Skins-Histological Characteristics. Part - I.
[22]  Warren GH, James PJ, Neville AM (1983) A morphometric analysis of the changes with age in the skinsurface wax and the sebaceous gland area of Merino sheep. Australian Veterinary Journal. 60: 238-240.
[23]  Mobini B (2013g) Studies on the density of various dermal structures in adult rams and ewes. Bulgarian Journal of Veterinary Medicine. 16(1): 1-6.
[24]  PanYS (1963) Quantitative and morphological variation of sweat glands, skin thickness, and skin shrinkage over various body regions of Sahiwal Zebu and Jersey cattle. Australian Journal of Agricultural Research. 14(3): 424-437.
[25]  Hafez ESE, Badreldin AL, Shafei MM (1955a) The Hair Coat in Bovinae. Empire Journal of Experimental Agriculture. 23: 34-39.
[26]  Shafie MM (1954) Skin histology of Egyptian buffaloes and cattle. M. Sc. Thesis, Faculty of Agriculture. Cairo University. U.A.R.
[27]  Renani, HRA, Salehi M, Ebadi Z, Moradi S, Baghershah HR, Renani MYA, Ameli SH (2011b) Determination of hair follicle characteristics, density and activity of Iranian cashmere goat breeds. Small Ruminant Research. 95: 128-132.
[28]  Renani HRA, Salehi M, Ebadi Z, Moradi S (2010) Identification of hair follicle characteristics and activity of one and two humped camels. Small Ruminant Research. 90: 64-70.
[29]  Abbasi M, Gharzi A, Karimi H, Khosravinia H (2008) Effects of sex on histological characteristics of various areas of skin in an Iranian native breed of sheep. Journal of Animal and Veterinary Advances. 7: 1503-1505.
[30]  Andrews RN, Beattie AE, Dodds KG, Wuliji T, Montgomery GW (1998) Wool follicle traits of 1/2 Merino ½ Romney F1, and backcross 3/4 Merino 1/4 Romney gene mapping flocks. In: Proceedings of the New Zealand Society of Animal Production, New Zealand. 262-265.
[31]  Butler LG, Orazio RD, Ahlen K (1993) Some objective skin and fleece traits relating to pelt quality of Swedishpelt sheep. Small Ruminant Research. 12: 69-78.
[32]  Turner HG (1962) Effect of clipping the coat on performance of calves in the field.Australian Journal of Agricultural Research. 13: 180-192.
[33]  Renani HRA, Moradi S, Baghershah HR, Ebadi Z, Salehi M, Momen SMS, Renani MYA (2011a) Determination of wool follicle characteristics of Iranian sheep breeds. Asian-Australian Journal of Animal Sciences. 24: 1173-1177.
[34]  Kocamis H, Aslan S (2004) Histological and histometrical study on the structure of Tuj breed sheep skin. Kafkas Universitesi Veteriner Fakultesi Dergisi. 10(1): 91-98.
Show Less References


A Study of Possible Association between Cannabinoid Receptor Gene II and Drug Dependence

1Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Alexandria University, Egypt

2Currently working at College of Biotechnology, University of Modern Sciences, Dubai

American Journal of Medical and Biological Research. 2016, 4(4), 66-72
doi: 10.12691/ajmbr-4-4-1
Copyright © 2016 Science and Education Publishing

Cite this paper:
Fouad, R. Gomaa, M. Farag. A Study of Possible Association between Cannabinoid Receptor Gene II and Drug Dependence. American Journal of Medical and Biological Research. 2016; 4(4):66-72. doi: 10.12691/ajmbr-4-4-1.

Correspondence to: R.  Gomaa, Department of Forensic Medicine and Clinical Toxicology, Faculty of Medicine, Alexandria University, Egypt. Email:


Drug dependence is considered a major contributor to both medical morbidity and mortality all over the world. It also represents an important health problem that has a great impact on the person's life both socially and economically. It was suggested that there is a substantial genetic contribution to drug dependence vulnerability. Cannabinoid receptors are known to be activated by natural as well as synthetic cannabinoids. Several evidences suggested that improved information about Cannabinoid receptor genes and their human variants might add to the understanding of vulnerabilities to drug dependence. The current study aimed at investigating the possible association between the cannabinoid receptor gene and drug dependence. The study was conducted on 150 drug dependent individuals. The diagnosis of drug dependence was based on the current Diagnostic and Statistical Manual of Mental disorders (DSM-IV) and urine screening tests. These individuals were using either Cannabis or Tramadol solely or in combination. All drug dependent individuals were males and all were current smokers. The duration of drug abuse ranged from 1 to 9 years. All participants were screened for a nucleotide polymorphism in cannabinoid receptor 2 gene (CB2) by PCR amplification and HapII Restriction Fragment Length Polymorphism analysis. The study has proved a significant association between occurrence of polymorphism in the Cannabinoid Receptor 2 gene and drug dependence, where 83.3% of drug dependents showed the polymorphism compared to 15% of the control group. A significant association was also detected between the presence of this polymorphism and family history of drug dependence and.The results of the present study confirmed the possible role of Cannabinoid Receptor 2 gene in drug dependence vulnerability.



[1]  Weiss RD. Drug abuse and dependence. In: Goldman L, Ausiello D. Cecil Medicine. 24Th ed. Philadelphia: Saunders Elsevier, 2012; 146-58.
[2]  Hyman SE. Biology of Addiction. In: Goldman L, Ausiello D. Cecil Medicine. 24 Th ed. Philadelphia: Saunders Elsevier, 2012; 140-2.
[3]  Carter A, Capps B, Wayne H. What is addiction? In: Addiction neurobiology: Ethical and social implications. 9Th ed Luxembourg: Office for Official Publications of the European Communities, 2009; 21-8.
[4]  Okasha A, Khalil A, Fahmy M. Psychological understanding of Egyptian heroin users. Egypt J Psychiatry1999; 13: 37-49.
[5]  Merikangas KR, Stolar M, Stevens DE, Goulet J, Preisig MA, Fenton B, Zhang H, O'Malley SS, Rounsaville BJ. Familial transmission of substance use disorders. Arch Gen Psychiatry 1998; 55: 973-9.
Show More References
[6]  Ball D, Collier D. Substance misuse. In: Mc Guffin P, Owen MJ, Gottesman II, eds. Psychiatric genetics and genomics. Oxford: Oxford University Press, 2002:267-302.
[7]  Ball D, Pembrey M, Stevens D. Genomics. In: Nutt D, Robbins T, Stimson G, Ince M, Jackson A, eds. Drugs and the future: Brain science, addiction and society. London: Academic Press, 2007: 89-132.
[8]  Goldman D, Oroszi G, Ducci F. The genetics of addictions: Uncovering the genes. Nature Reviews Genetics 2005; 6:521-32.
[9]  Nestler EJ. Genes and addiction. Nature Genetics 2000; 26: 277-81.
[10]  Uhl GR, Li MD, Gelertner J, Berrettini W, Pollock J. Molecular genetics of addiction vulnerability and treatment responses. Neuropsychopharmacology 2004; 29:26.
[11]  Kendler KS, Neale MC, Heath AC, Kessler RC, Eaves LJ.A twin-family study of alcoholism in women. Am J Psychiatry 1994; 151 (5):707-15.
[12]  Tyndale RF. Genetics of alcohol and tobacco use in humans. Annals of Medicine 2003; 35(2): 94-121.
[13]  Hall W, Morley KL, Lucke JC. The prediction of disease risk in genomic medicine: Scientific prospects and implications for public policy and ethics. EMBO Reports 2004; 5:22-6.
[14]  Khoury MJ, McCabe LL, McCabe ER. Genomic medicine - population screening in the age of genomic medicine. New England Journal of Medicine 2003; 348:50-8.
[15]  Khoury MJ, Yang QH, Gwinn M, Little JL, Flanders WD. An epidemiologic assessment of genomic profiling for measuring susceptibility to common diseases and targeting interventions. Genetics in Medicine 2004; 6:38-47.
[16]  Lerman C, Berrettini W. Elucidating the role of genetic factors in smoking behavior and nicotine dependence. American Journal of Medical Genetics 2003; 118:48-54.
[17]  Comings DE, Blum K. Reward deficiency syndrome: genetic aspects of behavioral disorders. Progress in Brain Research 2000; 126:325-41.
[18]  Quattrocki E, Baird A, Yurgelun-Todd D. Biological aspects of the link between smoking and depression. Harvard Review of Psychiatry 2000; 8:99-110.
[19]  Kauer J, Malenka RC. Synaptic plasticity and addiction. Nature Reviews Neuroscience 2007; 8: 844-58.
[20]  Rhee SH, Hewitt JK, Young SE, Corley RP, Crowley TJ, Stallings MC. Genetic and environmental influences on substance initiation, use, and problem use in adolescents. Arch Gen Psychiatry 2003; 60:1256-64.
[21]  Duggirala R, Almasy L, Blangero J. Smoking behavior is under the influence of a major quantitative trait locus on human chromosome 5q. Genetic Epidemiology1999; 17(1):139-44.
[22]  Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993; 365: 61-5.
[23]  Sipe JC, Arbour N, Gerber A, Beutler E. Reduced endocannabinoid immune modulation by a common cannabinoid 2 (CB2) receptor gene polymorphism: possible risk for autoimmune disorders. J Leukoc Biol 2005; 78: 231-8.
[24]  Karsak M, Cohen-Solal M, Freudenberg J, Ostertag A, Morieux C, Kornak U. The cannabinoid receptor type 2 (CNR2) gene is associated with human osteoporosis. Hum Mol Genet 2005; 4: 4.
[25]  Anonymous. Diagnostic and Statistical Manual of Mental Disorders, Text Revision. Washington, DC: American Psychiatric Association, 2000.
[26]  Pouliopoulos A, Spagou K, Raikos N, Tsoukali H. Immunoassay technologies for drugs of abuse testing - General principles - Recognized advantages and disadvantages. Aristotle University Medical Journal 2007; 34(2):19-24.
[27]  Illustra pure Taq Ready-To-Go PCR Beads. Product booklet. Code:27-9559-01.
[28]  Ishiguro H, Iwasaki S,Teasenfitz L, Higuchi S, Horiuchi Y, Saito T, Arinami T, Onaivi ES. Involvement of cannabinoid CB2 receptor in alcohol preference in mice and alcoholism in humans. The Pharmacogenomics Journal 2007; 7: 380-5.
[29]  Howlett AC. The cannabinoid receptors. Prostaglandins Other Lipid Mediat 2002; 6869:619-31.
[30]  Mackie K. Cannabinoid receptors: where they are and what they do. J. Neuroendocrinol 2008; 20: 4-10.
[31]  Graham ES, Ashton JC, Glass M. Cannabinoid receptors: a brief history and what's hot. Front Biosci 2009; 14: 944-57.
[32]  Ishiguro H, Iwasaki S,Teasenfitz L, Higuchi S, Horiuchi Y, Saito T, Arinami T, Onaivi ES. Involvement of cannabinoid CB2 receptor in alcohol preference in mice and alcoholism in humans. The Pharmacogenomics Journal 2007; 7: 380-5.
[33]  Brown SM, Wager-Miller J, Mackie K. Cloning and molecular characterization of the rat CB2 cannabinoid receptor. Biochem Biophys Acta 2002; 1576: 255-64.
[34]  Lauber J, Marsac C, Kadenbach B, Seibel P. Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases. Nucl. Acids Res 1991; 19(7):1393-7.
[35]  Rodham K, Hawton K, Evans E,Weatherall R. Ethnic and gender differences in drinking, smoking and drug taking among adolescents in England: a self-report school-based survey of 15 and16 year old. J Adolesc 2005; 28: 63-73.
[36]  Bloor R. The influence of age and gender on drug use in the United Kingdom-a review. Am J Add 2006; 15: 201- 07.
[37]  Fergusson DM, Boden, JM, Horwood LJ. The developmental antecedents of illicit drug use: Evidence from a 25-year longitudinal study. Drug and Alcohol Dependence 2008; 96: 165-77.
[38]  National Institute on Drug Abuse. Gender differences in drug abuse, risks and treatment. NIDA 2000; 15: 4.
[39]  Ghanem AA, Abdel Rahman RH, Mandour RA, Attia AM. Detection of some substances of abuse during daily practice in emergency hospital, Mansoura University. Uiversity Mansoura J. Forensic Med. Clin. Toxicol 2010; 18 (1):65-79.
[40]  Emara AM. Toxicological, biochemical, psychological study on patients with drug abuse. M.D. Thesis of Clinical Toxicology, Faculty of Medicine, Tanta University. 1998.
[41]  Brady KT, Randall CL. Gender differences in substances use disorders. Psych Clin North Am 1999; 22(2):241-52.
[42]  Elekes Z, Kovacs L. Old and new drug consumption habits in Hungary, Romania and Moldova. Eur Addict Res 2002; 8:166-9.
[43]  Cirakoglu OC, Isin G. Perception of drug addiction among Turkish university students: Causes, cures and attitudes. Addictive behaviors 2005; 30:1-8.
[44]  Kaul P, Coupey SM. Clinical evaluation of substance abuse .Ped Rev 2002; 23(3): 85-93.
[45]  SoueifMI, HanourahM, DarweeshZ, El-SayedA, YunisF, Taha H. The use of psychoactive substances by female Egyptian university students, compared with their male colleagues on selected items. Drug Alcohol Depend.1987; (19): 233-47.
[46]  Wahdan N. Social and economic effects of the phenomenon of spread of narcotics in Egypt. National Institute for Planning, Social and Cultural Planning Center and Police Research Center. 1986; 1425.
[47]  Anonymous. Use, abuse and addiction, preliminary report. National Research on Addiction. Ministry of Health. Egypt: 1996.
[48]  El-Sawy H, Abdel Hay M and Badawy A. Gender Differences in Risks and Pattern of Drug Abuse in Egypt. Egypt J Neurol psychiat Neurosug 2010; 47(3):413-18.
[49]  Bonomo Y, Proimos, J. Substance misuse: alcohol, tobacco, inhalants and other drugs. BMJ 2005; 330:777-80.
[50]  Lewis KS, Han NH. Tramadol: A new centrally acting analgesic. Am J Health Syst Pharm 1997; 54:643-652.
[51]  Hawkins JD, Catalano RF, Miller JY. Risk and protective factors for alcohol and other drug problems in adolescence and early adulthood: Implications for substance abuse prevention. Psychological Bulletin 1992; 112: 64-105.
Show Less References