Exponential growth in the number of patients suffering from diseases caused by the consumption of sugar has become a threat to mankind's health. Artificial low calorie sweeteners available in the market may have severe side effects. It takes time to figure out the long term side effects and by the time these are established, they are replaced by a new low calorie sweetener. Saccharine has been used for centuries to sweeten foods and beverages without calories or carbohydrate. It was also used on a large scale during the sugar shortage of the two world wars but was abandoned as soon as it was linked with development of bladder cancer. Naturally occurring sweet and taste modifying proteins are being seen as potential replacements for the currently available artificial low calorie sweeteners. Interaction aspects of sweet proteins and the human sweet taste receptor are being investigated.

Sweet and taste modifying proteins The prevalence of obesity and diabetes has increased dramatically in recent years in the United States, with similar patterns seen in several other countries including India [ 1 ] as well. Diabetes mellitus is a chronic disease caused by inherited or acquired deficiency in production of insulin by the pancreas or by the ineffectiveness of the insulin produced [ 2 ]. Artificial sweeteners like Saccharin, Aspartame, Cyclamate and AcesulfameK are used world-wide as low calorie sweeteners by patients affected by diseases linked to the consumption of sugar, e.g. diabetes, hyperlipemia, caries, obesity etc. but they have side effects such as psychological problems, mental disorders, bladder cancer, heart failure and brain tumors [ 3 - 7 ]. Sweet proteins have the potential to replace these artificial sweeteners, by acting as natural, good, low calorie sweeteners, as we know that proteins do not trigger a demand for insulin in these patients whereas sucrose does. In humans, the sweet taste is mainly due to the recently discovered T1R2-T1R3 receptor [ 8 - 10 ], two of the three members of the T1R class [ 8 - 10 ] of taste-specific proteins hypothesized to function in combination as a heterodimer. The human T1R2-T1R3 receptor recognizes natural and synthetic sweetness and T1R1-T1R3 recognizes umami taste [ 11 , 12 ]. So far there are seven known sweet and taste-modifying proteins, namely Brazzein [ 13 ], Thaumatin [ 14 ], Monelin [ 15 ], Curculin [ 16 ], Mabinlin [ 17 ], Miraculin [ 18 ] and Pentadin [ 19 ]. Properties and characteristics of these proteins are illustrated in Table  1 . The key residues on the protein surface responsible for biological activity have not yet been identified with certainty for any of these proteins [ 20 ]. Monellin was found to be 100000 times sweeter than sucrose on a molar basis [ 21 ], followed by Brazzein and Thaumatin which are 500 times [ 13 ] and 3000 times sweeter then sucrose [ 14 ] respectively (the latter two on a weight basis). All of these proteins have been isolated from plants that grow in tropical rainforests. Although most of them share no sequence homology or structural similarity, Thaumatin shares extensive homology with certain non-sweet proteins found in other plants [ 15 ]. The potential industrial applications of these proteins are the low calorie sweetener industry and the cola, snacks, food and chocolate industries.
Sweet and taste modifying proteins
The prevalence of obesity and diabetes has increased dramatically in recent years in the United States, with similar patterns seen in several other countries including India [ 1 ] as well. Diabetes mellitus is a chronic disease caused by inherited or acquired deficiency in production of insulin by the pancreas or by the ineffectiveness of the insulin produced [ 2 ]. Artificial sweeteners like Saccharin, Aspartame, Cyclamate and AcesulfameK are used world-wide as low calorie sweeteners by patients affected by diseases linked to the consumption of sugar, e.g. diabetes, hyperlipemia, caries, obesity etc. but they have side effects such as psychological problems, mental disorders, bladder cancer, heart failure and brain tumors [ 3 - 7 ]. Sweet proteins have the potential to replace these artificial sweeteners, by acting as natural, good, low calorie sweeteners, as we know that proteins do not trigger a demand for insulin in these patients whereas sucrose does.In humans, the sweet taste is mainly due to the recently discovered T1R2-T1R3 receptor [ 8 - 10 ], two of the three members of the T1R class [ 8 - 10 ] of taste-specific proteins hypothesized to function in combination as a heterodimer. The human T1R2-T1R3 receptor recognizes natural and synthetic sweetness and T1R1-T1R3 recognizes umami taste [ 11 , 12 ]. So far there are seven known sweet and taste-modifying proteins, namely Brazzein [ 13 ], Thaumatin [ 14 ], Monelin [ 15 ], Curculin [ 16 ], Mabinlin [ 17 ], Miraculin [ 18 ] and Pentadin [ 19 ]. Properties and characteristics of these proteins are illustrated in Table  1 . The key residues on the protein surface responsible for biological activity have not yet been identified with certainty for any of these proteins [ 20 ]. Monellin was found to be 100000 times sweeter than sucrose on a molar basis [ 21 ], followed by Brazzein and Thaumatin which are 500 times [ 13 ] and 3000 times sweeter then sucrose [ 14 ] respectively (the latter two on a weight basis). All of these proteins have been isolated from plants that grow in tropical rainforests. Although most of them share no sequence homology or structural similarity, Thaumatin shares extensive homology with certain non-sweet proteins found in other plants [ 15 ].The potential industrial applications of these proteins are the low calorie sweetener industry and the cola, snacks, food and chocolate industries.Brazzein Brazzein is the smallest, most heat-stable [ 13 ] and pH-stable member of the set of proteins known to have intrinsic sweetness. The protein, consisting of 54 amino acid residues, is reported to be between 500 and 2000 times sweeter than sucrose [ 22 ] and represents an excellent alternative to available low calorie sweeteners. It was originally isolated from the fruit of an African plant  Pentadiplandra brazzeana  Baillon [ 23 ]. Heat and pH stability of the protein make it an ideal system for investigating the chemical and structural requirements of a sweet-tasting protein. Based on the wild-type brazzein, 25 mutants were produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29–33 and 39–43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness [ 24 ]. Second, charge plays an important role in its interaction with the sweet taste receptor [ 24 ].
Brazzein
Brazzein is the smallest, most heat-stable [ 13 ] and pH-stable member of the set of proteins known to have intrinsic sweetness. The protein, consisting of 54 amino acid residues, is reported to be between 500 and 2000 times sweeter than sucrose [ 22 ] and represents an excellent alternative to available low calorie sweeteners. It was originally isolated from the fruit of an African plant  Pentadiplandra brazzeana  Baillon [ 23 ]. Heat and pH stability of the protein make it an ideal system for investigating the chemical and structural requirements of a sweet-tasting protein. Based on the wild-type brazzein, 25 mutants were produced to identify critical regions important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29–33 and 39–43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness [ 24 ]. Second, charge plays an important role in its interaction with the sweet taste receptor [ 24 ].Pentadiplandra brazzeanaThaumatin The thaumatins are a class of intensely sweet proteins isolated from the fruit of the tropical plant  Thaumatococcus danielli . The protein crystallizes in a hexagonal lattice after a temperature shift from 293 to 277 K. The structure has been solved at 1.6 Å resolution. Its fold was found to be identical to that found in three other crystal forms grown in the presence of crystallizing agents of differing chemical natures [ 25 ]. It consists of 207 amino acid residues with eight intramolecular disulfide bonds and contains no free cysteine residues. It aggregates upon heating at pH 7.0 above 70 degrees C, whereupon its sweetness disappears [ 26 , 27 ]. The protein is approximately 10000 times sweeter than sugar on a molar basis [ 28 ]. It is a protein that tastes intensely sweet only to Old World monkeys and to higher primates, including man [ 29 ], as it has been found that the protein binds to certain elements in taste pores of Rhesus monkey foliate papillae [ 30 ]. Thaumatin has been approved for use in many countries as both a flavor enhancer and a high-intensity sweetener [ 31 ].
Thaumatin
The thaumatins are a class of intensely sweet proteins isolated from the fruit of the tropical plant  Thaumatococcus danielli . The protein crystallizes in a hexagonal lattice after a temperature shift from 293 to 277 K. The structure has been solved at 1.6 Å resolution. Its fold was found to be identical to that found in three other crystal forms grown in the presence of crystallizing agents of differing chemical natures [ 25 ]. It consists of 207 amino acid residues with eight intramolecular disulfide bonds and contains no free cysteine residues. It aggregates upon heating at pH 7.0 above 70 degrees C, whereupon its sweetness disappears [ 26 , 27 ]. The protein is approximately 10000 times sweeter than sugar on a molar basis [ 28 ]. It is a protein that tastes intensely sweet only to Old World monkeys and to higher primates, including man [ 29 ], as it has been found that the protein binds to certain elements in taste pores of Rhesus monkey foliate papillae [ 30 ]. Thaumatin has been approved for use in many countries as both a flavor enhancer and a high-intensity sweetener [ 31 ].Thaumatococcus danielliMonellin Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains, an A chain of 44 amino acid residues and a B chain of 50 amino acid residues [ 32 ]. The protein can be purified from the fruit of  Dioscoreophyllum cumminsii  grown in West Africa and is approximately 100,000 times sweeter than sugar on a molar basis and several thousand times sweeter on a weight basis [ 28 ]. Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has proven to be as sweet as native two-chain monellin, and is more stable than the native monellin at high temperature and in acidic environments [ 33 ]. Native monellin is relatively sensitive to heat or acid treatment, which may cause separation of the sub-units and denaturation of the protein. Despite misgivings about the stability of the protein to heat and acid, downstream processes have been established. Its D-enantiomer has been crystallized and analyzed by X-ray crystallography at 1.8 Å resolution. Two crystal forms (I and II) were found under crystallization conditions similar, but not identical, to the crystallization conditions of natural L-monellin [ 34 ]. One NMR study of a non-sweet analog in which the Asp B7  of protein was replaced by Abu B7  (L-2-Aminobutylicacid), showed similar 3-dimensional structures of these two proteins, indicating that the lack of the beta-carboxyl group in the Abu B7  analog is responsible for the loss of sweetness [ 35 ]. Recent research on identifying binding sites on the receptor by means of structure-taste relationships, found that four monellin analogues, [AsnA16]-, [AsnA22]-, [GlnA25]-, and [AsnA26]-monellin were 7500, 750, 2500, and 5500 times as sweet as sucrose on a weight basis, respectively. Thus, among them, [AsnA22]-monellin and [GlnA25]-monellin were less sweet than the native monellin [ 36 ].
Monellin
Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains, an A chain of 44 amino acid residues and a B chain of 50 amino acid residues [ 32 ]. The protein can be purified from the fruit of  Dioscoreophyllum cumminsii  grown in West Africa and is approximately 100,000 times sweeter than sugar on a molar basis and several thousand times sweeter on a weight basis [ 28 ]. Single-chain monellin (SCM), which is an engineered 94-residue polypeptide, has proven to be as sweet as native two-chain monellin, and is more stable than the native monellin at high temperature and in acidic environments [ 33 ]. Native monellin is relatively sensitive to heat or acid treatment, which may cause separation of the sub-units and denaturation of the protein. Despite misgivings about the stability of the protein to heat and acid, downstream processes have been established. Its D-enantiomer has been crystallized and analyzed by X-ray crystallography at 1.8 Å resolution. Two crystal forms (I and II) were found under crystallization conditions similar, but not identical, to the crystallization conditions of natural L-monellin [ 34 ]. One NMR study of a non-sweet analog in which the Asp B7  of protein was replaced by Abu B7  (L-2-Aminobutylicacid), showed similar 3-dimensional structures of these two proteins, indicating that the lack of the beta-carboxyl group in the Abu B7  analog is responsible for the loss of sweetness [ 35 ]. Recent research on identifying binding sites on the receptor by means of structure-taste relationships, found that four monellin analogues, [AsnA16]-, [AsnA22]-, [GlnA25]-, and [AsnA26]-monellin were 7500, 750, 2500, and 5500 times as sweet as sucrose on a weight basis, respectively. Thus, among them, [AsnA22]-monellin and [GlnA25]-monellin were less sweet than the native monellin [ 36 ].Dioscoreophyllum cumminsiiCurculin Curculin which is extracted from  Curculigo latifolia  acts as a good low calorie sweetener. Its maximum sweetness is equal to 0.35 M of sucrose. It has taste modifying abilities since water and sour substances elicit a sweet taste after consumption of curculin [ 37 ]. There is no other protein currently available with both sweet taste and taste modifying abilities [ 38 ]. The taste modifying activity of the protein (discussed below) remains unchanged when it is incubated at 50°C for 1 hr between pH 3 and 11 [ 39 ]. The molecular weight of Curculin was determined by low angle laser light scattering and was found to be 27800 [ 38 ]. Its three-dimensional model has been built from the X-ray coordinates of GNA, a mannose-binding lectin from snowdrop ( Galanthus nivalis ) [ 38 ]. The three mannose-binding sites present in GNA were found in curculin but were not functional. Some well exposed regions on the surface of the three-dimensional model of the said protein could act as epitopes responsible for the sweet-tasting properties of the protein [ 40 ]. The protein can be crystallized by the vapor diffusion method using polyethylene glycol 400 as a precipitant. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions: a = 105 Å, b = 271 Å, c = 48.7 Å. The crystals diffract X-rays to resolution of 3.0 Å and are suitable for X-ray crystallographic studies [ 41 ].
Curculin
Curculin which is extracted from  Curculigo latifolia  acts as a good low calorie sweetener. Its maximum sweetness is equal to 0.35 M of sucrose. It has taste modifying abilities since water and sour substances elicit a sweet taste after consumption of curculin [ 37 ]. There is no other protein currently available with both sweet taste and taste modifying abilities [ 38 ]. The taste modifying activity of the protein (discussed below) remains unchanged when it is incubated at 50°C for 1 hr between pH 3 and 11 [ 39 ].Curculigo latifoliaThe molecular weight of Curculin was determined by low angle laser light scattering and was found to be 27800 [ 38 ]. Its three-dimensional model has been built from the X-ray coordinates of GNA, a mannose-binding lectin from snowdrop ( Galanthus nivalis ) [ 38 ]. The three mannose-binding sites present in GNA were found in curculin but were not functional. Some well exposed regions on the surface of the three-dimensional model of the said protein could act as epitopes responsible for the sweet-tasting properties of the protein [ 40 ]. The protein can be crystallized by the vapor diffusion method using polyethylene glycol 400 as a precipitant. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions: a = 105 Å, b = 271 Å, c = 48.7 Å. The crystals diffract X-rays to resolution of 3.0 Å and are suitable for X-ray crystallographic studies [ 41 ].Galanthus nivalisMabinlin Mabinlin is a sweet protein with the highest known thermostablility [ 42 ]. It is derived from  Capparis masaikai  and its sweetness was estimated to be around 400 times that of sucrose on weight basis. It consists of an A chain with 33 amino acid residues and a B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges [ 43 ]. Its heat stability is due to the presence of these four disulfide bridges [ 44 ]. The sweetness of Mabinlin-2 is unchanged after 48 hr incubation at boiling point [ 17 ]and of Mabinlin-3 and -4 are unchanged after 1 hr at 80°C [ 45 ].
Mabinlin
Mabinlin is a sweet protein with the highest known thermostablility [ 42 ]. It is derived from  Capparis masaikai  and its sweetness was estimated to be around 400 times that of sucrose on weight basis. It consists of an A chain with 33 amino acid residues and a B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges [ 43 ]. Its heat stability is due to the presence of these four disulfide bridges [ 44 ]. The sweetness of Mabinlin-2 is unchanged after 48 hr incubation at boiling point [ 17 ]and of Mabinlin-3 and -4 are unchanged after 1 hr at 80°C [ 45 ].Capparis masaikaiMiraculin Miraculin is a taste-modifying protein that belongs to the class of sweet proteins. It is extracted from  Richadella dulcifica  an evergreen shrub native of West Africa. The protein is a single polypeptide with 191 amino acid residues [ 46 ]. It modifies the sweet receptor in such a way that it can be stimulated by acid [ 47 ]. Thus, miraculin has the unusual property of modifying sour taste into sweet taste [ 46 ]. Taste-modifying protein modifies the sweet taste receptor on binding and this behavior of these proteins is responsible for modification in taste of sour substance [ 46 , 47 ]. All acids (which are normally sour) taste sweet after consumption of these proteins. The effects of these proteins exist for around half an hour after consumption and intake of any sour substance will therefore taste sweet during this period of time. The taste buds come to there normal state with time.
Miraculin
Miraculin is a taste-modifying protein that belongs to the class of sweet proteins. It is extracted from  Richadella dulcifica  an evergreen shrub native of West Africa. The protein is a single polypeptide with 191 amino acid residues [ 46 ]. It modifies the sweet receptor in such a way that it can be stimulated by acid [ 47 ]. Thus, miraculin has the unusual property of modifying sour taste into sweet taste [ 46 ].Richadella dulcificaTaste-modifying protein modifies the sweet taste receptor on binding and this behavior of these proteins is responsible for modification in taste of sour substance [ 46 , 47 ]. All acids (which are normally sour) taste sweet after consumption of these proteins. The effects of these proteins exist for around half an hour after consumption and intake of any sour substance will therefore taste sweet during this period of time. The taste buds come to there normal state with time.Pentadin Pentadin is a sweet protein extracted from the plant  Pentadiplandra brazzeana , a shrub found in tropical forests of a few African countries. Not much information is available about the protein despite its isolation several years ago, in 1989 [ 48 ]. The protein was reported to be around 500 times sweeter then sucrose on a weight basis. It also consists of subunits coupled by disulfide bonds [ 49 ].
Pentadin
Pentadin is a sweet protein extracted from the plant  Pentadiplandra brazzeana , a shrub found in tropical forests of a few African countries. Not much information is available about the protein despite its isolation several years ago, in 1989 [ 48 ]. The protein was reported to be around 500 times sweeter then sucrose on a weight basis. It also consists of subunits coupled by disulfide bonds [ 49 ].Pentadiplandra brazzeanaInteraction of sweet proteins with their receptor Humans detect taste with taste receptor cells. These are clustered in taste buds. Each taste bud has a pore that opens out to the surface of the tongue enabling molecules and ions taken into the mouth to reach the receptor cells inside. There are five primary taste sensations salty, sour, sweet, bitter and umami. Sweet and umami (the taste of monosodium glutamate) are the main pleasant tastes in humans. T1Rs are mammalian taste receptors that assemble two heteromeric G-protein-coupled receptor complexes T1R1+T1R3, an umami sensor, and T1R2+T1R3, a sweet receptor [ 50 ]. Sweet and taste-modifying proteins interact with the T1R2-T1R3 receptor with a different mechanism compared to small molecular weight compounds [ 51 ]. Recently, it has been shown that the T1R2-T1R3 receptor has many characteristics similar to the mGluR [ 52 ], apart from some minor differences in the active site region. The major work by Kunishima et al. [ 52 ] solving the crystal structure of the N-terminal active site region of the subtype 1 of mGluR both free and complexed with glutamate has helped a lot in understanding the mechanism of interaction between ligand and T1R2-T1R3 receptor. Their structural work on mGluR and its N-terminal domain [ 52 , 53 ] showing considerable conformational change induced by the glutamate complexation. The 'Active' and 'resting' conformations of m1-LBR, an extracellular ligand binding region of mGluR, is modulated by the dimer interface. The protomer can form 'open' or 'closed' confirmations and is made up of two domains namely LB1 and LB2. The population of active conformers depends on ligand binding, i.e. the so called 'closed-open_A'. The ligand-free receptor exists as two different structures, free form I (open-open_R), the 'resting' conformation with two open protomers and free form II (closed-open_A), nearly identical to the complexed form (Figure  1 , references 52, 54). The mechanism suggested by these structures is that the receptor is in dynamic equilibrium, and that ligand binding stabilizes the 'active' dimer. There are thus two ways, in principle, to activate the receptor: first, to complexate form I with the proper ligand (glutamate for the mGluR, aspartame or any other small molecular weight sweetener for the T1R2-T1R3 receptor) and second, by shift the equilibrium between free form I and free form II in favor of free form II. The exact mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated [ 51 ]. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor[ 52 ]. Studies have shown that the T1R3 receptor protein is encoded by the Tas1r3 gene involved in transduction of sweet taste [ 55 ]. Recently it has been found that T1R3-independent sweet- and umami-responsive receptors and/or pathways also exist in taste cells [ 56 ].
Interaction of sweet proteins with their receptor
Humans detect taste with taste receptor cells. These are clustered in taste buds. Each taste bud has a pore that opens out to the surface of the tongue enabling molecules and ions taken into the mouth to reach the receptor cells inside. There are five primary taste sensations salty, sour, sweet, bitter and umami. Sweet and umami (the taste of monosodium glutamate) are the main pleasant tastes in humans. T1Rs are mammalian taste receptors that assemble two heteromeric G-protein-coupled receptor complexes T1R1+T1R3, an umami sensor, and T1R2+T1R3, a sweet receptor [ 50 ].Sweet and taste-modifying proteins interact with the T1R2-T1R3 receptor with a different mechanism compared to small molecular weight compounds [ 51 ]. Recently, it has been shown that the T1R2-T1R3 receptor has many characteristics similar to the mGluR [ 52 ], apart from some minor differences in the active site region.The major work by Kunishima et al. [ 52 ] solving the crystal structure of the N-terminal active site region of the subtype 1 of mGluR both free and complexed with glutamate has helped a lot in understanding the mechanism of interaction between ligand and T1R2-T1R3 receptor. Their structural work on mGluR and its N-terminal domain [ 52 , 53 ] showing considerable conformational change induced by the glutamate complexation. The 'Active' and 'resting' conformations of m1-LBR, an extracellular ligand binding region of mGluR, is modulated by the dimer interface. The protomer can form 'open' or 'closed' confirmations and is made up of two domains namely LB1 and LB2. The population of active conformers depends on ligand binding, i.e. the so called 'closed-open_A'. The ligand-free receptor exists as two different structures, free form I (open-open_R), the 'resting' conformation with two open protomers and free form II (closed-open_A), nearly identical to the complexed form (Figure  1 , references 52, 54). The mechanism suggested by these structures is that the receptor is in dynamic equilibrium, and that ligand binding stabilizes the 'active' dimer. There are thus two ways, in principle, to activate the receptor: first, to complexate form I with the proper ligand (glutamate for the mGluR, aspartame or any other small molecular weight sweetener for the T1R2-T1R3 receptor) and second, by shift the equilibrium between free form I and free form II in favor of free form II.The exact mechanism of interaction of sweet proteins with the T1R2-T1R3 sweet taste receptor has not yet been elucidated [ 51 ]. Low molecular mass sweeteners and sweet proteins interact with the same receptor, the human T1R2-T1R3 receptor[ 52 ]. Studies have shown that the T1R3 receptor protein is encoded by the Tas1r3 gene involved in transduction of sweet taste [ 55 ].Recently it has been found that T1R3-independent sweet- and umami-responsive receptors and/or pathways also exist in taste cells [ 56 ].Conclusion and scope of further work As it has been found that sweet proteins are thousands of times sweeter than sucrose and are of low calorie value, these proteins can be used as natural low calorie sweeteners by people suffering from diseases linked to consumption of sugar e.g. obesity, diabetes and hyperlipemia. Candidate proteins can be checked for biological activity with the human taste receptor. Also mutations can be induced in candidate sweet proteins to analyze changes in their physical, chemical and biological properties. The work can be taken forward by solving the structures of the proteins and taste receptors with a view to increasing the efficiency of these sweeteners.
Conclusion and scope of further work
As it has been found that sweet proteins are thousands of times sweeter than sucrose and are of low calorie value, these proteins can be used as natural low calorie sweeteners by people suffering from diseases linked to consumption of sugar e.g. obesity, diabetes and hyperlipemia.Candidate proteins can be checked for biological activity with the human taste receptor. Also mutations can be induced in candidate sweet proteins to analyze changes in their physical, chemical and biological properties. The work can be taken forward by solving the structures of the proteins and taste receptors with a view to increasing the efficiency of these sweeteners.


Background In order to optimize the potential benefits of neural stem cell (NSC) transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different  in vitro  conditions. In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT 1  and MT 2  receptors are expressed in NSCs.
Background
In order to optimize the potential benefits of neural stem cell (NSC) transplantation for the treatment of neurodegenerative disorders, it is necessary to understand their biological characteristics. Although neurotrophin transduction strategies are promising, alternative approaches such as the modulation of intrinsic neurotrophin expression by NSCs, could also be beneficial. Therefore, utilizing the C17.2 neural stem cell line, we have examined the expression of selected neurotrophic factors under different  in vitro  conditions. In view of recent evidence suggesting a role for the pineal hormone melatonin in vertebrate development, it was also of interest to determine whether its G protein-coupled MT 1  and MT 2  receptors are expressed in NSCs.in vitroResults RT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT 1  receptor mRNA was detected in NSCs maintained for two days in culture, but the MT 2  receptor was not seen. An immature MT 1  receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 – 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT 1  receptor is expressed in both neural (β-tubulin III positive) and glial (GFAP positive) progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours.
Results
RT-PCR analysis revealed robust expression of glial cell-line derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in undifferentiated cells maintained for two days in culture. After one week, differentiating cells continued to exhibit high expression of BDNF and NGF, but GDNF expression was lower or absent, depending on the culture conditions utilized. Melatonin MT 1  receptor mRNA was detected in NSCs maintained for two days in culture, but the MT 2  receptor was not seen. An immature MT 1  receptor of about 30 kDa was detected by western blotting in NSCs cultured for two days, whereas a mature receptor of about 40 – 45 kDa was present in cells maintained for longer periods. Immunocytochemical studies demonstrated that the MT 1  receptor is expressed in both neural (β-tubulin III positive) and glial (GFAP positive) progenitor cells. An examination of the effects of melatonin on neurotrophin expression revealed that low physiological concentrations of this hormone caused a significant induction of GDNF mRNA expression in NSCs following treatment for 24 hours.Conclusions The phenotypic characteristics of C17.2 cells suggest that they are a heterogeneous population of NSCs including both neural and glial progenitors, as observed under the cell culture conditions used in this study. These NSCs have an intrinsic ability to express neurotrophic factors, with an apparent suppression of GDNF expression after several days in culture. The detection of melatonin receptors in neural stem/progenitor cells suggests involvement of this pleiotropic hormone in mammalian neurodevelopment. Moreover, the ability of melatonin to induce GDNF expression in C17.2 cells supports a functional role for the MT 1  receptor expressed in these NSCs. In view of the potency of GDNF in promoting the survival of dopaminergic neurons, these novel findings have implications for the utilization of melatonin in neuroprotective strategies, especially in Parkinson's disease.
Conclusions
The phenotypic characteristics of C17.2 cells suggest that they are a heterogeneous population of NSCs including both neural and glial progenitors, as observed under the cell culture conditions used in this study. These NSCs have an intrinsic ability to express neurotrophic factors, with an apparent suppression of GDNF expression after several days in culture. The detection of melatonin receptors in neural stem/progenitor cells suggests involvement of this pleiotropic hormone in mammalian neurodevelopment. Moreover, the ability of melatonin to induce GDNF expression in C17.2 cells supports a functional role for the MT 1  receptor expressed in these NSCs. In view of the potency of GDNF in promoting the survival of dopaminergic neurons, these novel findings have implications for the utilization of melatonin in neuroprotective strategies, especially in Parkinson's disease.

Background Neural stem cells are multipotent cells which are capable of self-replication and differentiation into neurons, astrocytes or oligodendrocytes in the central nervous system [ 1 ]. Because of their intrinsic plasticity and multipotency, there are great expectations that NSC transplantation will ultimately provide immense benefits in the treatment of neurodegeneration. However, it is essential to fully understand the cellular and molecular mechanisms involved in the differentiation and function of NSCs, in order to fully harness their therapeutic potential. Because of the very limited availability of NSCs in the central nervous system (CNS), neural stem cell lines are very useful for the study and characterization of NSC biology. For example, transplantation studies with the C17.2 neural stem cell line [ 2 ] have revealed that these cells express diverse neurotransmitter phenotypes, depending on the environment prevailing in the CNS area of engraftment [ 3 , 4 ]. Recently, transplanted C17.2 NSCs, genetically modified to express glial cell line-derived neurotrophic factor (GDNF), were found to engraft in the 6-hydroxydopamine-lesioned mouse striatum and to express therapeutic levels of this neurotrophin, with consequent protection of dopaminergic neurons in this model of Parkinson's disease [ 5 ]. Although this and other similar approaches are promising, limitations including the stability and regulation of transduced genes await resolution. Therefore, it was of interest to determine whether C17.2 cells have the intrinsic ability to express neurotrophins or neurotrophic factors, which would make them amenable to modulation by appropriate agents  in vitro or in vivo . In addition, we examined whether these NSCs express receptors for the pineal hormone melatonin, which can induce GDNF mRNA and protein expression [ 6 , 7 ] and which has been implicated in the development of vertebrates including humans [ 8 - 10 ]. Initially, different concentrations and types of sera were used for cell culture in order to select optimal conditions for gene expression studies. We now report that C17.2 NSCs exhibit heterogeneous phenotypes and express neurotrophic factors and melatonin MT 1  receptors.
Background
Neural stem cells are multipotent cells which are capable of self-replication and differentiation into neurons, astrocytes or oligodendrocytes in the central nervous system [ 1 ]. Because of their intrinsic plasticity and multipotency, there are great expectations that NSC transplantation will ultimately provide immense benefits in the treatment of neurodegeneration. However, it is essential to fully understand the cellular and molecular mechanisms involved in the differentiation and function of NSCs, in order to fully harness their therapeutic potential. Because of the very limited availability of NSCs in the central nervous system (CNS), neural stem cell lines are very useful for the study and characterization of NSC biology. For example, transplantation studies with the C17.2 neural stem cell line [ 2 ] have revealed that these cells express diverse neurotransmitter phenotypes, depending on the environment prevailing in the CNS area of engraftment [ 3 , 4 ]. Recently, transplanted C17.2 NSCs, genetically modified to express glial cell line-derived neurotrophic factor (GDNF), were found to engraft in the 6-hydroxydopamine-lesioned mouse striatum and to express therapeutic levels of this neurotrophin, with consequent protection of dopaminergic neurons in this model of Parkinson's disease [ 5 ]. Although this and other similar approaches are promising, limitations including the stability and regulation of transduced genes await resolution. Therefore, it was of interest to determine whether C17.2 cells have the intrinsic ability to express neurotrophins or neurotrophic factors, which would make them amenable to modulation by appropriate agents  in vitro or in vivo . In addition, we examined whether these NSCs express receptors for the pineal hormone melatonin, which can induce GDNF mRNA and protein expression [ 6 , 7 ] and which has been implicated in the development of vertebrates including humans [ 8 - 10 ]. Initially, different concentrations and types of sera were used for cell culture in order to select optimal conditions for gene expression studies. We now report that C17.2 NSCs exhibit heterogeneous phenotypes and express neurotrophic factors and melatonin MT 1  receptors.in vitro or in vivoResults Effects of culture conditions on neurotrophic factor and cell-specific marker mRNA expression in C17.2 NSCs Following two days in culture, C17.2 cells remain in an undifferentiated state, as indicated by their flat and rounded appearance (Fig.  1A ) and high expression of the stem cell/progenitor cell marker, nestin (Fig.  1C,1E,1G ). These cells also expressed the early neuronal marker, β-tubulin III, but there was little or no expression of the mRNA for the glial marker, glial fibrillary acidic protein (GFAP). After seven days in culture, differentiating C17.2 cells exhibit an elongated shape with an extension of neurite-like processes, as shown in Fig.  1B . However, as observed in undifferentiated cells after two days, there was still strong expression of nestin and β-tubulin III, with little or no detectable GFAP mRNA (Fig.  1D,1F,1H ). An examination of neurotrophin mRNA expression in undifferentiated C17.2 cells, revealed a robust expression of GDNF, BDNF and NGF, regardless of the type or concentration of serum used for culturing (Fig.  1C,1E,1G ). A similar strong expression of BDNF and NGF was observed in differentiating cells after seven days, but GDNF mRNA was relatively lower in cells maintained in 1% fetal bovine serum (FBS) or 10% FBS + 5% horse serum (Fig.  1F,1H ). The mRNA levels of the control gene, GAPDH, did not change under the conditions examined (Fig.  1I ). Detection of MT 1  receptor mRNA and protein in C17.2 NSCs Melatonin MT 1  receptor mRNA was detected by RT-PCR in NSCs maintained for two days, especially in cells cultured in 1% FBS, as shown in Fig.  2A . GAPDH mRNA expression did not change under the conditions examined (Fig.  2B ). C17.2 NSCs maintained for indicated periods in 1% FBS or 10% FBS + 5% horse serum, expressed the MT 1  receptor protein, as revealed by western analysis (Fig.  2C,2D ). Interestingly, when cells were cultured for 2–3 days, the MT 1  protein detected had a molecular weight of about 30 kDa, which is less than the predicted size of the mature receptor. However, when cells were cultured for 10–12 days, a mature MT 1  receptor of about 40–45 kDa was present, as shown in Fig.  2D . The MT 2  receptor transcript was not detected under any of the conditions used in this study. Immunocytochemical detection of the MT 1  receptor and cell-specific markers in C17.2 NSCs MT 1  receptor immunoreactivity was detected within C17.2 cells maintained in 1% FBS for two days, as shown in Figure  3A,3B . Omission of the primary antibody or its preincubation with a blocking peptide (CIDtech Research Inc., Cambridge, ON) abolished MT 1  immunoreactivity (Fig.  3C ), indicating the specificity of MT 1  detection. In keeping with RT-PCR results, nestin (Fig.  3D,3E ) and β-tubulin III (Fig.  3F ), were detected by immunocytochemical analysis. Double- labeling studies indicated that the MT 1  receptor is coexpressed with the stem /progenitor cell marker, nestin (Fig.  4A,4B,4C ), the glial marker, GFAP (Fig.  4D,4E,4F ) and the early neuronal marker, β-tubulin III (Fig.  4G,4H,4I ). Induction of GDNF mRNA expression by melatonin in C17.2 NSCs In order to assess the potential functionality of the MT 1  receptor detected in C17.2 NSCs, the effect of low physiological concentrations of melatonin on GDNF mRNA expression was examined. Cells were grown as described in Methods and treated with melatonin or vehicle (0.001% DMSO) for 24 hours. Following RT-PCR analysis, GDNF mRNA levels were converted to optical density (OD) values and normalized to GAPDH OD levels, as reported previously [ 6 ]. After conversion of GDNF/GAPDH OD ratios to percentage values, one-way ANOVA indicated a significant treatment effect (F 3,7  = 7.03, p < 0.04). A Neuman-Keuls test indicated a significant increase in relative GDNF mRNA expression in cells treated with 0.05, 0.1 and 1 nM melatonin as shown in Figure  5 .
Results
Effects of culture conditions on neurotrophic factor and cell-specific marker mRNA expression in C17.2 NSCs Following two days in culture, C17.2 cells remain in an undifferentiated state, as indicated by their flat and rounded appearance (Fig.  1A ) and high expression of the stem cell/progenitor cell marker, nestin (Fig.  1C,1E,1G ). These cells also expressed the early neuronal marker, β-tubulin III, but there was little or no expression of the mRNA for the glial marker, glial fibrillary acidic protein (GFAP). After seven days in culture, differentiating C17.2 cells exhibit an elongated shape with an extension of neurite-like processes, as shown in Fig.  1B . However, as observed in undifferentiated cells after two days, there was still strong expression of nestin and β-tubulin III, with little or no detectable GFAP mRNA (Fig.  1D,1F,1H ). An examination of neurotrophin mRNA expression in undifferentiated C17.2 cells, revealed a robust expression of GDNF, BDNF and NGF, regardless of the type or concentration of serum used for culturing (Fig.  1C,1E,1G ). A similar strong expression of BDNF and NGF was observed in differentiating cells after seven days, but GDNF mRNA was relatively lower in cells maintained in 1% fetal bovine serum (FBS) or 10% FBS + 5% horse serum (Fig.  1F,1H ). The mRNA levels of the control gene, GAPDH, did not change under the conditions examined (Fig.  1I ).
Effects of culture conditions on neurotrophic factor and cell-specific marker mRNA expression in C17.2 NSCs
Following two days in culture, C17.2 cells remain in an undifferentiated state, as indicated by their flat and rounded appearance (Fig.  1A ) and high expression of the stem cell/progenitor cell marker, nestin (Fig.  1C,1E,1G ). These cells also expressed the early neuronal marker, β-tubulin III, but there was little or no expression of the mRNA for the glial marker, glial fibrillary acidic protein (GFAP). After seven days in culture, differentiating C17.2 cells exhibit an elongated shape with an extension of neurite-like processes, as shown in Fig.  1B . However, as observed in undifferentiated cells after two days, there was still strong expression of nestin and β-tubulin III, with little or no detectable GFAP mRNA (Fig.  1D,1F,1H ). An examination of neurotrophin mRNA expression in undifferentiated C17.2 cells, revealed a robust expression of GDNF, BDNF and NGF, regardless of the type or concentration of serum used for culturing (Fig.  1C,1E,1G ). A similar strong expression of BDNF and NGF was observed in differentiating cells after seven days, but GDNF mRNA was relatively lower in cells maintained in 1% fetal bovine serum (FBS) or 10% FBS + 5% horse serum (Fig.  1F,1H ). The mRNA levels of the control gene, GAPDH, did not change under the conditions examined (Fig.  1I ).Detection of MT 1  receptor mRNA and protein in C17.2 NSCs Melatonin MT 1  receptor mRNA was detected by RT-PCR in NSCs maintained for two days, especially in cells cultured in 1% FBS, as shown in Fig.  2A . GAPDH mRNA expression did not change under the conditions examined (Fig.  2B ). C17.2 NSCs maintained for indicated periods in 1% FBS or 10% FBS + 5% horse serum, expressed the MT 1  receptor protein, as revealed by western analysis (Fig.  2C,2D ). Interestingly, when cells were cultured for 2–3 days, the MT 1  protein detected had a molecular weight of about 30 kDa, which is less than the predicted size of the mature receptor. However, when cells were cultured for 10–12 days, a mature MT 1  receptor of about 40–45 kDa was present, as shown in Fig.  2D . The MT 2  receptor transcript was not detected under any of the conditions used in this study.
Detection of MT 1  receptor mRNA and protein in C17.2 NSCs
Melatonin MT 1  receptor mRNA was detected by RT-PCR in NSCs maintained for two days, especially in cells cultured in 1% FBS, as shown in Fig.  2A . GAPDH mRNA expression did not change under the conditions examined (Fig.  2B ). C17.2 NSCs maintained for indicated periods in 1% FBS or 10% FBS + 5% horse serum, expressed the MT 1  receptor protein, as revealed by western analysis (Fig.  2C,2D ). Interestingly, when cells were cultured for 2–3 days, the MT 1  protein detected had a molecular weight of about 30 kDa, which is less than the predicted size of the mature receptor. However, when cells were cultured for 10–12 days, a mature MT 1  receptor of about 40–45 kDa was present, as shown in Fig.  2D . The MT 2  receptor transcript was not detected under any of the conditions used in this study.Immunocytochemical detection of the MT 1  receptor and cell-specific markers in C17.2 NSCs MT 1  receptor immunoreactivity was detected within C17.2 cells maintained in 1% FBS for two days, as shown in Figure  3A,3B . Omission of the primary antibody or its preincubation with a blocking peptide (CIDtech Research Inc., Cambridge, ON) abolished MT 1  immunoreactivity (Fig.  3C ), indicating the specificity of MT 1  detection. In keeping with RT-PCR results, nestin (Fig.  3D,3E ) and β-tubulin III (Fig.  3F ), were detected by immunocytochemical analysis. Double- labeling studies indicated that the MT 1  receptor is coexpressed with the stem /progenitor cell marker, nestin (Fig.  4A,4B,4C ), the glial marker, GFAP (Fig.  4D,4E,4F ) and the early neuronal marker, β-tubulin III (Fig.  4G,4H,4I ).
Immunocytochemical detection of the MT 1  receptor and cell-specific markers in C17.2 NSCs
MT 1  receptor immunoreactivity was detected within C17.2 cells maintained in 1% FBS for two days, as shown in Figure  3A,3B . Omission of the primary antibody or its preincubation with a blocking peptide (CIDtech Research Inc., Cambridge, ON) abolished MT 1  immunoreactivity (Fig.  3C ), indicating the specificity of MT 1  detection. In keeping with RT-PCR results, nestin (Fig.  3D,3E ) and β-tubulin III (Fig.  3F ), were detected by immunocytochemical analysis. Double- labeling studies indicated that the MT 1  receptor is coexpressed with the stem /progenitor cell marker, nestin (Fig.  4A,4B,4C ), the glial marker, GFAP (Fig.  4D,4E,4F ) and the early neuronal marker, β-tubulin III (Fig.  4G,4H,4I ).Induction of GDNF mRNA expression by melatonin in C17.2 NSCs In order to assess the potential functionality of the MT 1  receptor detected in C17.2 NSCs, the effect of low physiological concentrations of melatonin on GDNF mRNA expression was examined. Cells were grown as described in Methods and treated with melatonin or vehicle (0.001% DMSO) for 24 hours. Following RT-PCR analysis, GDNF mRNA levels were converted to optical density (OD) values and normalized to GAPDH OD levels, as reported previously [ 6 ]. After conversion of GDNF/GAPDH OD ratios to percentage values, one-way ANOVA indicated a significant treatment effect (F 3,7  = 7.03, p < 0.04). A Neuman-Keuls test indicated a significant increase in relative GDNF mRNA expression in cells treated with 0.05, 0.1 and 1 nM melatonin as shown in Figure  5 .
Induction of GDNF mRNA expression by melatonin in C17.2 NSCs
In order to assess the potential functionality of the MT 1  receptor detected in C17.2 NSCs, the effect of low physiological concentrations of melatonin on GDNF mRNA expression was examined. Cells were grown as described in Methods and treated with melatonin or vehicle (0.001% DMSO) for 24 hours. Following RT-PCR analysis, GDNF mRNA levels were converted to optical density (OD) values and normalized to GAPDH OD levels, as reported previously [ 6 ]. After conversion of GDNF/GAPDH OD ratios to percentage values, one-way ANOVA indicated a significant treatment effect (F 3,7  = 7.03, p < 0.04). A Neuman-Keuls test indicated a significant increase in relative GDNF mRNA expression in cells treated with 0.05, 0.1 and 1 nM melatonin as shown in Figure  5 .Discussion The expression of nestin in undifferentiated C17.2 cells is consistent with the presence of this intermediate filament protein in stem and progenitor cells in the mammalian CNS [ 11 ]. However, as noted above, nestin mRNA was also readily detected in cells exhibiting morphological changes characteristic of differentiation, after one week in culture. Similarly, mRNA for the early neuronal marker, β-tubulin III, was found under all conditions examined, whereas GFAP mRNA was detected only in some cultures. These observations suggest that the C17.2 cells examined in this study are an heterogeneous population of stem and progenitor cells in keeping with evidence that NSCs exhibit morphological and phenotypic heterogeneity [ 12 , 13 ]. The expression of diverse neurotrophins by NSCs is consistent with the role of these factors in the differentiation and development of the CNS. Presumably, the robust mRNA expression observed, particularly in cells maintained in 10% FBS + 5% HS, is driven by the serum-enriched milieu of potential inducers including neurotransmitters, hormones and growth factors, such as basic fibroblast growth factor and epidermal growth factor, which can stimulate C17.2 cell growth  in vitro  [ 14 ]. In contrast to BDNF and NGF, which exhibited strong mRNA expression under all conditions examined, GDNF expression was weaker or not detectable in differentiating cells after seven days in culture. The suppression of GDNF expression might have been due to the prolonged exposure of NSCs to regulatory factors in the serum, as its decline appears to be inversely correlated with the concentration or enrichment of serum used for cell culture. Thus, moderate, weak or no expression of GDNF was observed in cells cultured for 1 week in 1% CS, 1% FBS or 10% FBS + 5% HS, respectively (see Fig.  1D,1F,1H ). Various biological agents or pathways have been implicated in the regulation of GDNF expression. For example, fibroblast growth factor-2 and proinflammatory cytokines such as interleukin(IL)-1β, IL-6 and tumor necrosis factor-α stimulate GDNF synthesis and secretion [ 15 ]. Activation of protein kinase C by phorbol esters increases GDNF expression [ 15 , 16 ], whereas the adenylate cyclase activator, forskolin, inhibits GDNF production in cultured cells, suggesting an inhibitory role for the cyclic AMP- protein kinase A pathway [ 15 ]. The cAMP pathway and its transcriptional factor cAMP response element binding protein (CREB) have been shown to induce differentiation in neuronal progenitor cells [ 17 , 18 ]. Therefore, it is possible that activation of this pathway was involved in both the initiation of differentiation and the inhibition of GDNF expression observed in C17.2 cells after seven days. While this work was in progress, it was reported that C17.2 neural stem cells constitutively secrete BDNF, GDNF and NGF, but do not label for GFAP or neuronal markers like β-tubulin III [ 19 ]. Our findings are in agreement with these observations with regard to neurotrophin expression. However, in contrast to their findings, β-tubulin III mRNA and immunoreactivity were readily detected in our study. In addition, although GFAP mRNA was weakly expressed or not detectable in some cultures, immunoreactivity for this glial cell marker is present in C17.2 cells, as shown in Figure  4 . These differences may be due to our examination of β-tubulin III and GFAP expression in cells maintained for 2–12 days in culture, whereas their C17.2 cells were examined after 2–3 weeks [ 19 ]. Other factors, such as our use of low serum concentrations, as compared with the enriched culture medium used by Lu et al. [ 19 ], may also be involved. The detection of melatonin MT 1  receptor mRNA in C17.2 cells after 2 days but not after 7 days, presumably involves downregulation of this receptor. There is considerable evidence that many G protein-coupled receptors are downregulated by their agonists [ 20 ]. More importantly, melatonin, which is present in serum, has been found to suppress MT 1  transcription  in vitro  [ 21 ]. Interestingly, our immunocytochemical studies revealed MT 1  immunoreactivity within C17.2 cell bodies and extensions, as shown in Figure  3A,3B . Although an intracellular localization could result from internalization of receptors [ 20 ], it is also possible that the immunoreactivity detected within these neural stem/progenitor cells is due to the presence of newly synthesized MT 1  receptors. In accordance with this view, the MT 1  protein detected in short-term (2-day) cultures is about 30 kDa, which is less than the approximately 37–45 kDa molecular weight observed in various mammalian tissues [ 22 - 24 ]. Moreover, when cells were cultured for 10–12 days, a MT 1  receptor of about 40–45 kDa was detected, as shown in Fig.  2D . The mammalian MT 1  contains two glycosylation sites in its N-terminal [ 24 ] and it may exist in more than one glycosylated form, as has been reported for other G protein-coupled receptors [ 25 , 26 ]. Thus, the above cytochemical observations suggest that newly synthesized immature MT 1  receptors,which have yet to undergo posttranslational modification and translocation to the plasma membrane, were detected in cells cultured for 2 days in 1% FBS, whereas a mature glycosylated receptor was present in cells grown for longer periods. Although the MT 2  receptor transcript was not detected under any of the conditions used in this study, additional studies are required before the possibility of its expression in these cells can be ruled out. It is possible that MT 2  mRNA may undergo rapid turnover/degradation, while a functional protein may still be present. This is the first evidence that melatonin receptors are expressed in neural stem or progenitor cells and raises the obvious question of whether this hormone plays a role in neuronal development. Although studies in this field are limited, there is increasing evidence that melatonin is involved in the early development of vertebrates. For example, melatonin is produced in chick embryos as early as the 7 th  day of embryonic development [ 27 ], and a physiological concentration of this hormone has been shown to significantly enhance mouse embryogenesis  in vitro  [ 8 ]. Similarly, when sheep blastocysts were treated with melatonin for 24 hr  in vitro , there was a significant increase in the percentage of embryonic survival [ 28 ]. Other studies have shown that functional G i  protein-coupled melatonin receptors, which mediate inhibition of the adenylate-cyclase-cAMP pathway, are present in the embryonic (day 8) neural retina [ 29 ]. Melatonin receptor transcripts for all the known G i  protein-coupled receptor subtypes have been found in 24 hr-old embryos from Japanese quail [ 30 ]. Various studies have detected melatonin receptors in human fetal brain [ 31 , 32 ] and peripheral tissues [ 33 ]. Moreover, recent autoradiographic and  in situ  hybridization studies indicate that the melatonin MT 1  receptor is expressed in diverse areas of the human fetal brain [ 9 ]. Thus, the presence of MT 1  receptors in NSCs is in keeping with the foregoing, and supports the view that melatonin is involved in neurodevelopment. Colocalization evidence that the MT 1  receptor is present in both neural and glial progenitor cells is consistent with a neurodevelopmental role for melatonin, and suggests that in addition to the presence of the MT 1  in mammalian neurons [ 34 ], it may also be expressed in astrocytes, as observed in similar cells from rat [ 6 ] and chick brain [ 35 ]. The detection of nestin in some cells expressing the MT 1  receptor is consistent with its presence not only in neural progenitor cells but also in GFAP positive glial progenitors [ 36 ]. Preliminary evidence that melatonin induces GDNF mRNA expression in C17.2 NSCs, as we have observed previously in C6 glioma cells [ 6 ], supports the foregoing as this neurotrophic factor plays a critical role in both central and peripheral neurodevelopment [ 37 , 38 ]. GDNF also exerts neuroprotective effects in the CNS, including a potent role in the survival of dopaminergic neurons in the midbrain [ 39 , 40 ]. Therefore, modulation of GDNF expression may be one of the mechanisms underlying physiological neuroprotection by melatonin in the CNS [ 6 ].
Discussion
The expression of nestin in undifferentiated C17.2 cells is consistent with the presence of this intermediate filament protein in stem and progenitor cells in the mammalian CNS [ 11 ]. However, as noted above, nestin mRNA was also readily detected in cells exhibiting morphological changes characteristic of differentiation, after one week in culture. Similarly, mRNA for the early neuronal marker, β-tubulin III, was found under all conditions examined, whereas GFAP mRNA was detected only in some cultures. These observations suggest that the C17.2 cells examined in this study are an heterogeneous population of stem and progenitor cells in keeping with evidence that NSCs exhibit morphological and phenotypic heterogeneity [ 12 , 13 ].The expression of diverse neurotrophins by NSCs is consistent with the role of these factors in the differentiation and development of the CNS. Presumably, the robust mRNA expression observed, particularly in cells maintained in 10% FBS + 5% HS, is driven by the serum-enriched milieu of potential inducers including neurotransmitters, hormones and growth factors, such as basic fibroblast growth factor and epidermal growth factor, which can stimulate C17.2 cell growth  in vitro  [ 14 ]. In contrast to BDNF and NGF, which exhibited strong mRNA expression under all conditions examined, GDNF expression was weaker or not detectable in differentiating cells after seven days in culture. The suppression of GDNF expression might have been due to the prolonged exposure of NSCs to regulatory factors in the serum, as its decline appears to be inversely correlated with the concentration or enrichment of serum used for cell culture. Thus, moderate, weak or no expression of GDNF was observed in cells cultured for 1 week in 1% CS, 1% FBS or 10% FBS + 5% HS, respectively (see Fig.  1D,1F,1H ). Various biological agents or pathways have been implicated in the regulation of GDNF expression. For example, fibroblast growth factor-2 and proinflammatory cytokines such as interleukin(IL)-1β, IL-6 and tumor necrosis factor-α stimulate GDNF synthesis and secretion [ 15 ]. Activation of protein kinase C by phorbol esters increases GDNF expression [ 15 , 16 ], whereas the adenylate cyclase activator, forskolin, inhibits GDNF production in cultured cells, suggesting an inhibitory role for the cyclic AMP- protein kinase A pathway [ 15 ]. The cAMP pathway and its transcriptional factor cAMP response element binding protein (CREB) have been shown to induce differentiation in neuronal progenitor cells [ 17 , 18 ]. Therefore, it is possible that activation of this pathway was involved in both the initiation of differentiation and the inhibition of GDNF expression observed in C17.2 cells after seven days.in vitroWhile this work was in progress, it was reported that C17.2 neural stem cells constitutively secrete BDNF, GDNF and NGF, but do not label for GFAP or neuronal markers like β-tubulin III [ 19 ]. Our findings are in agreement with these observations with regard to neurotrophin expression. However, in contrast to their findings, β-tubulin III mRNA and immunoreactivity were readily detected in our study. In addition, although GFAP mRNA was weakly expressed or not detectable in some cultures, immunoreactivity for this glial cell marker is present in C17.2 cells, as shown in Figure  4 . These differences may be due to our examination of β-tubulin III and GFAP expression in cells maintained for 2–12 days in culture, whereas their C17.2 cells were examined after 2–3 weeks [ 19 ]. Other factors, such as our use of low serum concentrations, as compared with the enriched culture medium used by Lu et al. [ 19 ], may also be involved. The detection of melatonin MT 1  receptor mRNA in C17.2 cells after 2 days but not after 7 days, presumably involves downregulation of this receptor. There is considerable evidence that many G protein-coupled receptors are downregulated by their agonists [ 20 ]. More importantly, melatonin, which is present in serum, has been found to suppress MT 1  transcription  in vitro  [ 21 ]. Interestingly, our immunocytochemical studies revealed MT 1  immunoreactivity within C17.2 cell bodies and extensions, as shown in Figure  3A,3B . Although an intracellular localization could result from internalization of receptors [ 20 ], it is also possible that the immunoreactivity detected within these neural stem/progenitor cells is due to the presence of newly synthesized MT 1  receptors. In accordance with this view, the MT 1  protein detected in short-term (2-day) cultures is about 30 kDa, which is less than the approximately 37–45 kDa molecular weight observed in various mammalian tissues [ 22 - 24 ]. Moreover, when cells were cultured for 10–12 days, a MT 1  receptor of about 40–45 kDa was detected, as shown in Fig.  2D . The mammalian MT 1  contains two glycosylation sites in its N-terminal [ 24 ] and it may exist in more than one glycosylated form, as has been reported for other G protein-coupled receptors [ 25 , 26 ]. Thus, the above cytochemical observations suggest that newly synthesized immature MT 1  receptors,which have yet to undergo posttranslational modification and translocation to the plasma membrane, were detected in cells cultured for 2 days in 1% FBS, whereas a mature glycosylated receptor was present in cells grown for longer periods. Although the MT 2  receptor transcript was not detected under any of the conditions used in this study, additional studies are required before the possibility of its expression in these cells can be ruled out. It is possible that MT 2  mRNA may undergo rapid turnover/degradation, while a functional protein may still be present. This is the first evidence that melatonin receptors are expressed in neural stem or progenitor cells and raises the obvious question of whether this hormone plays a role in neuronal development. Although studies in this field are limited, there is increasing evidence that melatonin is involved in the early development of vertebrates. For example, melatonin is produced in chick embryos as early as the 7 th  day of embryonic development [ 27 ], and a physiological concentration of this hormone has been shown to significantly enhance mouse embryogenesis  in vitro  [ 8 ]. Similarly, when sheep blastocysts were treated with melatonin for 24 hr  in vitro , there was a significant increase in the percentage of embryonic survival [ 28 ]. Other studies have shown that functional G i  protein-coupled melatonin receptors, which mediate inhibition of the adenylate-cyclase-cAMP pathway, are present in the embryonic (day 8) neural retina [ 29 ]. Melatonin receptor transcripts for all the known G i  protein-coupled receptor subtypes have been found in 24 hr-old embryos from Japanese quail [ 30 ]. Various studies have detected melatonin receptors in human fetal brain [ 31 , 32 ] and peripheral tissues [ 33 ]. Moreover, recent autoradiographic and  in situ  hybridization studies indicate that the melatonin MT 1  receptor is expressed in diverse areas of the human fetal brain [ 9 ]. Thus, the presence of MT 1  receptors in NSCs is in keeping with the foregoing, and supports the view that melatonin is involved in neurodevelopment. Colocalization evidence that the MT 1  receptor is present in both neural and glial progenitor cells is consistent with a neurodevelopmental role for melatonin, and suggests that in addition to the presence of the MT 1  in mammalian neurons [ 34 ], it may also be expressed in astrocytes, as observed in similar cells from rat [ 6 ] and chick brain [ 35 ]. The detection of nestin in some cells expressing the MT 1  receptor is consistent with its presence not only in neural progenitor cells but also in GFAP positive glial progenitors [ 36 ]. Preliminary evidence that melatonin induces GDNF mRNA expression in C17.2 NSCs, as we have observed previously in C6 glioma cells [ 6 ], supports the foregoing as this neurotrophic factor plays a critical role in both central and peripheral neurodevelopment [ 37 , 38 ]. GDNF also exerts neuroprotective effects in the CNS, including a potent role in the survival of dopaminergic neurons in the midbrain [ 39 , 40 ]. Therefore, modulation of GDNF expression may be one of the mechanisms underlying physiological neuroprotection by melatonin in the CNS [ 6 ].in vitroin vitroin vitroin situConclusions In summary, the NSCs utilized in this study exhibited an intrinsic ability to express neurotrophins under various cell culture conditions. This ability was not affected by their morphological state, except in the case of GDNF mRNA expression which was lower in cells undergoing differentiation in FBS-supplemented media. Novel evidence that neural stem/progenitor cells express MT 1  receptors adds to the increasing evidence that NSCs can respond to diverse modulators [ 41 ], and suggests an early role for melatonin in CNS development. Moreover, since melatonin induces GDNF expression in NSCs, its potential  in vivo  modulation of this and/or other neurotrophic factors, via its G protein-coupled receptors in the brain or on transplanted NSCs, could have important implications for optimizing therapeutic strategies in neurodegenerative disorders such as Parkinson's disease.
Conclusions
In summary, the NSCs utilized in this study exhibited an intrinsic ability to express neurotrophins under various cell culture conditions. This ability was not affected by their morphological state, except in the case of GDNF mRNA expression which was lower in cells undergoing differentiation in FBS-supplemented media. Novel evidence that neural stem/progenitor cells express MT 1  receptors adds to the increasing evidence that NSCs can respond to diverse modulators [ 41 ], and suggests an early role for melatonin in CNS development. Moreover, since melatonin induces GDNF expression in NSCs, its potential  in vivo  modulation of this and/or other neurotrophic factors, via its G protein-coupled receptors in the brain or on transplanted NSCs, could have important implications for optimizing therapeutic strategies in neurodegenerative disorders such as Parkinson's disease.in vivoMethods Cell culture The C17.2 cell line was derived by retrovirus-mediated oncogene ( v-myc ) transduction of cells from the external germinal layer of neonatal mouse cerebellum [ 2 ]. C17.2 cells were grown on 10 cm Corning culture dishes (Fisher Scientific Ltd., Nepean, ON, Canada) in DMEM supplemented with 2 mM glutamine and calf serum, fetal bovine serum or horse serum (Invitrogen Canada Inc., Burlington, ON) in the concentrations indicated. Cells were maintained in a humidified 5% CO 2  – 95% air incubator at 37°C and routinely split at approximately 90% confluency [ 3 ]. RT-PCR Total RNA was isolated from C17.2 cells with TRIzol as described by the supplier (Invitrogen Canada Inc., Burlington, ON). After DNase treatment, cDNA was synthesized from 1–2 μg of total RNA using the Omniscript reverse transcriptase kit (Qiagen Inc., Mississauga, ON) and oligo dT primers. PCR was carried out using 1.5 μl (or 3 μl for melatonin MT 1  and MT 2  receptors) of the RT product and the HotStarTaq master mix kit (Qiagen Inc., Mississauga, ON), together with appropriate primers (Table  1 ). Following a hot start at 95°C for 15 min, samples were amplified for 36 cycles (or 38 cycles for MT 1  and MT 2 ) at 94°C for 30 s, 57°C for 30 s and 72°C for 1 min, followed by a final incubation at 72°C for 10 min. Treatment of C17.2 NSCs with melatonin For semi-quantitative examination of the effects of melatonin on GDNF mRNA expression, cells were grown in 10% FBS + 5% horse serum (HS) for 1 week. After subculture, cells were kept in 10% FBS + 5% HS for 2 days followed by another subculture to 1% FBS for 2 or 3 days. The cells were then treated with vehicle (0.001% DMSO) or melatonin (0.05, 0.1, and 1 nM) for 24 hours. Following RNA extraction, RT-PCR was performed as described above, except that an annealing T m  of 55°C was used and samples were amplified for 30 cycles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was amplified with intron-spanning primers [ 42 ], in order to control for DNA contamination. Immunocytochemistry After fixation for 15 minutes in 4% paraformaldehyde on poly L-ornithine-coated glass cover slips, cells were incubated overnight at 4°C with anti-melatonin MT 1  receptor serum (1:100; CIDtech Research Inc., Cambridge, ON). Cells were washed three times with PBS and then incubated with a fluorescein (FITC)-conjugated donkey anti- rabbit IgG (1:100 dilution; Jackson ImmunoResearch Labs. Inc.,West Grove, PA). In some experiments, the primary antibody was omitted or it was preincubated with the corresponding peptide immunogen (CIDtech Research Inc., Cambridge, ON), before use. In order to examine cell marker expression, mouse monoclonal antibodies against nestin (1: 500), β-tubulin III (1:200) or GFAP (1:400; Chemicon International, Temecula, CA) were used together with a FITC-conjugated donkey anti-mouse IgG (1:100; Jackson ImmunoResearch Labs.Inc.,West Grove, PA). For double- labeling studies of the MT 1  and cell markers, a rhodamine (TRITC)-conjugated donkey anti-mouse IgG (1: 100; Jackson ImmunoResearch Labs. Inc.,West Grove, PA) was used to detect nestin, GFAP and β-tubulin III. Digital images were recorded on a Zeiss confocal microscope. Western analysis C17.2 NSCs were grown as described in Figure  2 , and proteins were extracted in a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) supplemented with PMSF (1 mM), aprotinin (2 μg/ml), leupeptin (2 μg/ml), and sodium orthovanadate (2 mM). Extracted proteins (80 μg per lane) were separated by SDS- polyacrylamide gel electrophoresis and transblotted to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk in TBS-T buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20; pH 8.5) for 1 hour at room temperature, and then incubated overnight with a 1:100 dilution of rabbit anti-MT 1  antibody (CIDTech Research Inc., Cambridge, ON) at 4°C. After washing, membranes were incubated with a horseradish peroxidase-conjugated second antibody (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 hour. Following washing and exposure to enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Inc., Baie d'Urfé, Québec) for about 5 minutes, proteins were detected by autoradiography, as described previously [ 43 ]. Buffer reagents and protease inhibitors were obtained from Sigma- Aldrich Canada Ltd. (Oakville, ON).
Methods
Cell culture The C17.2 cell line was derived by retrovirus-mediated oncogene ( v-myc ) transduction of cells from the external germinal layer of neonatal mouse cerebellum [ 2 ]. C17.2 cells were grown on 10 cm Corning culture dishes (Fisher Scientific Ltd., Nepean, ON, Canada) in DMEM supplemented with 2 mM glutamine and calf serum, fetal bovine serum or horse serum (Invitrogen Canada Inc., Burlington, ON) in the concentrations indicated. Cells were maintained in a humidified 5% CO 2  – 95% air incubator at 37°C and routinely split at approximately 90% confluency [ 3 ].
Cell culture
The C17.2 cell line was derived by retrovirus-mediated oncogene ( v-myc ) transduction of cells from the external germinal layer of neonatal mouse cerebellum [ 2 ]. C17.2 cells were grown on 10 cm Corning culture dishes (Fisher Scientific Ltd., Nepean, ON, Canada) in DMEM supplemented with 2 mM glutamine and calf serum, fetal bovine serum or horse serum (Invitrogen Canada Inc., Burlington, ON) in the concentrations indicated. Cells were maintained in a humidified 5% CO 2  – 95% air incubator at 37°C and routinely split at approximately 90% confluency [ 3 ].v-mycRT-PCR Total RNA was isolated from C17.2 cells with TRIzol as described by the supplier (Invitrogen Canada Inc., Burlington, ON). After DNase treatment, cDNA was synthesized from 1–2 μg of total RNA using the Omniscript reverse transcriptase kit (Qiagen Inc., Mississauga, ON) and oligo dT primers. PCR was carried out using 1.5 μl (or 3 μl for melatonin MT 1  and MT 2  receptors) of the RT product and the HotStarTaq master mix kit (Qiagen Inc., Mississauga, ON), together with appropriate primers (Table  1 ). Following a hot start at 95°C for 15 min, samples were amplified for 36 cycles (or 38 cycles for MT 1  and MT 2 ) at 94°C for 30 s, 57°C for 30 s and 72°C for 1 min, followed by a final incubation at 72°C for 10 min.
RT-PCR
Total RNA was isolated from C17.2 cells with TRIzol as described by the supplier (Invitrogen Canada Inc., Burlington, ON). After DNase treatment, cDNA was synthesized from 1–2 μg of total RNA using the Omniscript reverse transcriptase kit (Qiagen Inc., Mississauga, ON) and oligo dT primers. PCR was carried out using 1.5 μl (or 3 μl for melatonin MT 1  and MT 2  receptors) of the RT product and the HotStarTaq master mix kit (Qiagen Inc., Mississauga, ON), together with appropriate primers (Table  1 ). Following a hot start at 95°C for 15 min, samples were amplified for 36 cycles (or 38 cycles for MT 1  and MT 2 ) at 94°C for 30 s, 57°C for 30 s and 72°C for 1 min, followed by a final incubation at 72°C for 10 min.Treatment of C17.2 NSCs with melatonin For semi-quantitative examination of the effects of melatonin on GDNF mRNA expression, cells were grown in 10% FBS + 5% horse serum (HS) for 1 week. After subculture, cells were kept in 10% FBS + 5% HS for 2 days followed by another subculture to 1% FBS for 2 or 3 days. The cells were then treated with vehicle (0.001% DMSO) or melatonin (0.05, 0.1, and 1 nM) for 24 hours. Following RNA extraction, RT-PCR was performed as described above, except that an annealing T m  of 55°C was used and samples were amplified for 30 cycles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was amplified with intron-spanning primers [ 42 ], in order to control for DNA contamination.
Treatment of C17.2 NSCs with melatonin
For semi-quantitative examination of the effects of melatonin on GDNF mRNA expression, cells were grown in 10% FBS + 5% horse serum (HS) for 1 week. After subculture, cells were kept in 10% FBS + 5% HS for 2 days followed by another subculture to 1% FBS for 2 or 3 days. The cells were then treated with vehicle (0.001% DMSO) or melatonin (0.05, 0.1, and 1 nM) for 24 hours. Following RNA extraction, RT-PCR was performed as described above, except that an annealing T m  of 55°C was used and samples were amplified for 30 cycles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was amplified with intron-spanning primers [ 42 ], in order to control for DNA contamination.Immunocytochemistry After fixation for 15 minutes in 4% paraformaldehyde on poly L-ornithine-coated glass cover slips, cells were incubated overnight at 4°C with anti-melatonin MT 1  receptor serum (1:100; CIDtech Research Inc., Cambridge, ON). Cells were washed three times with PBS and then incubated with a fluorescein (FITC)-conjugated donkey anti- rabbit IgG (1:100 dilution; Jackson ImmunoResearch Labs. Inc.,West Grove, PA). In some experiments, the primary antibody was omitted or it was preincubated with the corresponding peptide immunogen (CIDtech Research Inc., Cambridge, ON), before use. In order to examine cell marker expression, mouse monoclonal antibodies against nestin (1: 500), β-tubulin III (1:200) or GFAP (1:400; Chemicon International, Temecula, CA) were used together with a FITC-conjugated donkey anti-mouse IgG (1:100; Jackson ImmunoResearch Labs.Inc.,West Grove, PA). For double- labeling studies of the MT 1  and cell markers, a rhodamine (TRITC)-conjugated donkey anti-mouse IgG (1: 100; Jackson ImmunoResearch Labs. Inc.,West Grove, PA) was used to detect nestin, GFAP and β-tubulin III. Digital images were recorded on a Zeiss confocal microscope.
Immunocytochemistry
After fixation for 15 minutes in 4% paraformaldehyde on poly L-ornithine-coated glass cover slips, cells were incubated overnight at 4°C with anti-melatonin MT 1  receptor serum (1:100; CIDtech Research Inc., Cambridge, ON). Cells were washed three times with PBS and then incubated with a fluorescein (FITC)-conjugated donkey anti- rabbit IgG (1:100 dilution; Jackson ImmunoResearch Labs. Inc.,West Grove, PA). In some experiments, the primary antibody was omitted or it was preincubated with the corresponding peptide immunogen (CIDtech Research Inc., Cambridge, ON), before use. In order to examine cell marker expression, mouse monoclonal antibodies against nestin (1: 500), β-tubulin III (1:200) or GFAP (1:400; Chemicon International, Temecula, CA) were used together with a FITC-conjugated donkey anti-mouse IgG (1:100; Jackson ImmunoResearch Labs.Inc.,West Grove, PA). For double- labeling studies of the MT 1  and cell markers, a rhodamine (TRITC)-conjugated donkey anti-mouse IgG (1: 100; Jackson ImmunoResearch Labs. Inc.,West Grove, PA) was used to detect nestin, GFAP and β-tubulin III. Digital images were recorded on a Zeiss confocal microscope.Western analysis C17.2 NSCs were grown as described in Figure  2 , and proteins were extracted in a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) supplemented with PMSF (1 mM), aprotinin (2 μg/ml), leupeptin (2 μg/ml), and sodium orthovanadate (2 mM). Extracted proteins (80 μg per lane) were separated by SDS- polyacrylamide gel electrophoresis and transblotted to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk in TBS-T buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20; pH 8.5) for 1 hour at room temperature, and then incubated overnight with a 1:100 dilution of rabbit anti-MT 1  antibody (CIDTech Research Inc., Cambridge, ON) at 4°C. After washing, membranes were incubated with a horseradish peroxidase-conjugated second antibody (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 hour. Following washing and exposure to enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Inc., Baie d'Urfé, Québec) for about 5 minutes, proteins were detected by autoradiography, as described previously [ 43 ]. Buffer reagents and protease inhibitors were obtained from Sigma- Aldrich Canada Ltd. (Oakville, ON).
Western analysis
C17.2 NSCs were grown as described in Figure  2 , and proteins were extracted in a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) supplemented with PMSF (1 mM), aprotinin (2 μg/ml), leupeptin (2 μg/ml), and sodium orthovanadate (2 mM). Extracted proteins (80 μg per lane) were separated by SDS- polyacrylamide gel electrophoresis and transblotted to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk in TBS-T buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20; pH 8.5) for 1 hour at room temperature, and then incubated overnight with a 1:100 dilution of rabbit anti-MT 1  antibody (CIDTech Research Inc., Cambridge, ON) at 4°C. After washing, membranes were incubated with a horseradish peroxidase-conjugated second antibody (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 hour. Following washing and exposure to enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Inc., Baie d'Urfé, Québec) for about 5 minutes, proteins were detected by autoradiography, as described previously [ 43 ]. Buffer reagents and protease inhibitors were obtained from Sigma- Aldrich Canada Ltd. (Oakville, ON).Authors' contributions LPN conceived and planned the study, designed PCR primers, supervised all aspects of the study and wrote the manuscript. KJA cultured cells and performed initial RT-PCR experiments. LMRC treated cultured cells and carried out immunocytochemistry experiments. CVD cultured cells and carried out western blotting. RS treated cultured cells and performed RT-PCR experiments. CRM performed double- labeling of cultured cells. LCD provided the C17.2 cells, and collaborated on immunocytochemical studies. DLK assisted with initial cell culture. All authors read and approved the final manuscript.
Authors' contributions
LPN conceived and planned the study, designed PCR primers, supervised all aspects of the study and wrote the manuscript. KJA cultured cells and performed initial RT-PCR experiments. LMRC treated cultured cells and carried out immunocytochemistry experiments. CVD cultured cells and carried out western blotting. RS treated cultured cells and performed RT-PCR experiments. CRM performed double- labeling of cultured cells. LCD provided the C17.2 cells, and collaborated on immunocytochemical studies. DLK assisted with initial cell culture. All authors read and approved the final manuscript.