Characterization of bovine collagens using capillary electrophoresis — an alternative to slab gel electrophoresis

A capillary electrophoresis method was developed and characterized for analyzing the spectrum of collagen subspecies in collagen preparations. The Bio-Rad CE-SDS protein kit was used for the dynamic sieving separation of collagen subspecies in this CE method (DSCE). The optimized method utilized a 36 cm (or 24 cm)×50 μm uncoated capillary, electrophoretic injection at 10 kV for 10 s, a run voltage of 15 kV, a capillary temperature of 20°C, and UV detection at 220 nm. A preliminary validation of the method was performed. The assay had good repeatability (RSDs for peaks were 1–5%), and responses were linear for assay solutions with collagen concentrations from 0.125 to 1.25 mg/ml. The DSCE electropherogram of bovine skin collagen provided a profile of subspecies similar in number and relative abundance to that generated by scanning of Coomassie-stained SDS-PAGE gels.     

Use of the uricase-inhibited rat as an animal model in toxicology.

An accessible, reproducible, and inexpensive animal model for toxicologic evaluation of hyperuricemic conditions has been required for some time. A number of authors have tried to develop such a model by administering high doses of uric acid to various animal species (dog, rabbit, rat) but the potent liver uricase in these species prevented development of sustained hyperuricemia. Johnson et al. [4], Stavric et al. [5], and a number of other investigators [72, 75] successfully used potassium oxonate [63] to block the effect of hepatic uricase and to produce hyperuricemia in rats [4, 5, 68, 69, 72, 74, 76, 80], rabbits [66], mongrel dogs [67], mice [65], and pigs [64]. The oxonate-treated rat can serve as a useful animal model not only in investigation of the uric acid nephropathy, but also in a number of other toxicologic evaluations connected with uric acid. This model has been used to evaluate drugs that affect uric acid excretion, to determine which dietary factors affect serum urates, or to evaluate possible therapeutic agents in certain disorders associated with uric acid. The same model could also be used by behavioral scientists, for whom research on uric acid has become increasingly popular in recent years [33, 137]. The ideal uricase inhibitor for induction of hyperuricemia would be one which is irreversible, noncompetitive, and relatively nontoxic, so that its activity would be independent of high levels of uric acid, and effective inhibition could be attained at low dosage levels. Oxonic acid is not an ideal uricase inhibitor, because it is competitive and is eliminated from the body relatively rapidly. Although relatively nontoxic, oxonic acid and its salts are foreign substances that could interfere with some other metabolic systems. The possibility exists that an ideal, or at least a better inhibitor, could be developed by appropriate substitutions on the molecule of oxonic acid or by introducing different types of compounds such as derivatives of diazohypoxanthines, barbiturates, or similar substances. Until such improvements on the uricase-inhibited rat models are available, potassium oxonate, which is easily obtainable, can be used as an effective inhibitor of uricase in vivo.     

Characterization of the rat isolated retractor penis muscle as a model for the study of nitrergic transmission

INTRODUCTION: The anococcygeus and retractor penis muscles are part of the erectile machinery in male rodents. The rat anococcygeus muscle is a widely used smooth muscle preparation for the study of the effects of test substances on adrenergic, nitrergic, and cholinergic transmission. There is, however, little information available on the process of autonomic transmission in the rat retractor penis muscle, although its autonomic innervation has generally been assumed to be similar to that of the anococcygeus muscle because of the contiguous nature of the two muscles. The present study investigated the involvement of nitrergic transmission in mediating relaxant responses of the rat retractor penis muscle to electrical field stimulation. METHODS: The retractor penis muscle was isolated from Sprague-Dawley rats and mounted in Krebs solution. Phentolamine (5 microM) was added to the bath to block the adrenergic responses of the muscle, which was then precontracted with carbachol (10 microM). RESULTS: Electrical field stimulation (20-30 V, 1 ms pulse width, at 0.5-20 Hz for 10 s) of the carbachol precontracted muscle elicited frequency-dependent relaxant responses (0.9-68%). Tetrodotoxin (1 microM), N(G)-nitro-L-arginine (L-NOARG) (50 microM), N(G)-nitro-L-arginine methylester (L-NAME) (100 microM), and haemoglobin (100 microM) inhibited these relaxant responses by 99.3%, 93.9%, 86.9%, and 77.5%, respectively. L-Arginine (250 microM) (but not its D-isomer) reversed the blockade produced by L-NOARG (72.7%) and L-NAME (81.5%). DISCUSSION: Our results provide clear evidence that the inhibitory (relaxant) responses of the rat retractor penis muscle to electrical field stimulation are mediated by nitric oxide involving the L-arginine-nitric oxide synthase-nitric oxide pathway. The rat retractor penis muscle is a versatile preparation that can replace the cumbersome preparations from the pig, ox, and horse, hitherto used as pharmacological models for the study of the retractor penis muscle.     

Characterization of a potential animal model of an idiosyncratic drug reaction: nevirapine-induced skin rash in the rat

Idiosyncratic drug reactions are difficult to study in humans due to their unpredictability. Unfortunately, this characteristic also hinders the development of animal models needed for mechanistic studies. Nevirapine, used to treat human immunodeficiency virus (HIV) infections, results in a severe idiosyncratic skin rash in some patients. We found that nevirapine can also cause a significant rash in some strains of rats. At a dose of 150 mg/kg/day, the incidence in female Sprague-Dawley rats was 6/28 (21%), in female Brown Norway rats 32/32 (100%), and in female Lewis rats 0/6 (0%) while no male Sprague-Dawley or Brown Norway rats developed a rash. Female SJL mice 0/7 also did not develop nevirapine-induced skin lesions. The first sign of a reaction in Brown Norway rats was red ears at days 7-10 followed by a rash with scabbing mainly on the back; this was a shorter time to onset than in Sprague-Dawley rats. Light microscopy of the skin revealed a primarily mononuclear inflammatory infiltrate and lesions typical of self-trauma. Immunohistochemistry results suggest that the infiltrate was composed of CD4 and CD8 T cells as well as macrophages. A lower dose of either 40 or 75 mg/kg/day did not lead to a rash and, in fact, 2 weeks of the lower doses induced tolerance to the 150 mg/kg/day dose in female Brown Norway rats. A dose of 100 mg/kg/day resulted in rash in 2/4 (50%) of female Brown Norway rats. Rechallenge of Brown Norway rats that had been allowed to recuperate after a nevirapine-induced rash led to red ears in less than 24 h followed by hair loss and occasional skin lesions. Although the skin rash was less evident on rechallenge, microscopically, the cellular infiltrate was more prominent, especially surrounding the hair follicles. Moreover, there were lesions of interface dermatitis with apoptosis and satellitosis, indicative of a cell-mediated immune attack on the epidermis. While systemic signs of illness did not accompany the rash on primary exposure, on rechallenge, the animals appeared generally unwell and this forced sacrifice after 2 weeks or less of treatment. Importantly, splenocytes isolated from rechallenged animals were able to transfer susceptibility to nevirapine-induced skin rash to na?ve female Brown Norway recipients, which was illustrated by a faster time to onset of rash in the recipients. The characteristics of this adverse reaction are similar to that seen in humans; that is, it is idiosyncratic in that it only occurs in some strains of animals, is delayed in onset, is more common in females, is dose-dependent, and appears to be immune-mediated. Therefore, it may represent a good animal model for the study of idiosyncratic drug reactions.     

Review article: targeting TNFα as a key cytokine in the inflammatory processes of Crohn's disease — the mechanisms of action of infliximab

Crohn's disease is a chronic, debilitating gastrointestinal disorder in which a variety of cellular processes and pro-inflammatory mediators influence the pathogenesis of the disease. Although the potential roles and functions of the pro-inflammatory mediators continue to be debated, several mediators, specifically tumour necrosis factor-alpha, have been clearly identified as having a pivotal role in the inflammation of the bowel mucosa of these patients. Therapies specifically focusing on the inflammatory process underlying Crohn's disease have the potential for providing disease modification and prolonged remission. Infliximab, an antitumour necrosis factor-alpha monoclonal antibody, has been demonstrated to neutralize tumour necrosis factor-alpha and restore and reset the immunological dysbalance of the inflamed mucosa. Preliminary studies with infliximab suggested that treatment resulted in a rapid and almost complete inhibition of multiple inflammatory pathways. In clinical studies of infliximab, patients with Crohn's disease achieved rapid reduction in clinical signs and symptoms, substantiated by both endoscopic and microscopic evaluation.     

Characterization of the biological and biochemical activities of F 11782 and the bisdioxopiperazines, ICRF-187 and ICRF-193, two types of topoisomerase II catalytic inhibitors with distinctive mechanisms of action

F 11782 is a newly identified catalytic inhibitor of topoisomerases I and II, without any detectable interaction with DNA. This study aimed to establish whether its catalytic inhibition of topoisomerase II was mediated by mechanisms similar to those identified for the bisdioxopiperazines. In vitro combinations of F 11782 with etoposide resulted in greater than additive cytotoxicity in L1210 cells, contrasting with marked antagonism for combinations of etoposide with either ICRF-187 or ICRF-193. All three compounds caused a G2/M blockade of P388 cells after an 18-h incubation, but by 40 h polyploidization was evident only with the bisdioxopiperazines. Gel retardation data revealed that only F 11782, and not the bisdioxopiperazines, was capable of completely inhibiting the DNA-binding activity of topoisomerase II, confirming its novel mechanism of action. Furthermore, unlike ICRF-187 and ICRF-193, the cytotoxicity of F 11782 appeared mediated, at least partially, by DNA damage induction in cultured GCT27 human teratoma cells, as judged by a fluorescence-enhancement assay and monitoring p53 activation. Finally, the major in vivo antitumor activity of F 11782 against the murine P388 leukemia (i.v. implanted) and the B16 melanoma (s.c. grafted) contrasted with the bisdioxopiperazines' general lack of activity. Overall, F 11782 and the bisdioxopiperazines appear to function as quite distinctive catalytic topoisomerase II inhibitors.     

The local anti-inflammatory action of dexamethasone in the rat carrageenin oedema model is reversed by an antiserum to lipocortin 1.

1. A local pre-injection of 1 micrograms dexamethasone sodium phosphate strongly inhibited (> 60% inhibition at 3 h; P < 0.001 at all time points) the development of carrageenin-induced paw oedema in the rat induced by a subplantar injection of 0.1 ml, 2% carrageenin. 2. Coinjection of a polyclonal rabbit antiserum raised against human 1-188 recombinant lipocortin 1, which also recognised the rat protein, reversed the inhibitory action of dexamethasone (P < 0.05 at 4 h and 5 h). At the highest volume used (40 microliters) control antisera were without any effect. 3. These data further support the concept that lipocortin 1 is involved in the anti-inflammatory mechanism of action of the glucocorticoids.     

Organ culture of young rat vas deferens as an in vitro model for the study of denervation supersensitivity

To investigate whether organ culture is a suitable in vitro model for studying the mechanisms of denervation-induced supersensitivity, we cultured 1-week-old rat vas deferens for 3 days with a basic applied tension of 20 mg. Cultured muscles showed supersensitivity to norepinephrine and methacholine with concomitant elevation of the maximal response. To compare these changes with those caused by denervation, young rats were chemically denervated by injecting 6-hydroxydopamine, and consequent sensitivity changes were investigated. Denervated muscles showed non-specific supersensitivity to norepinephrine and methacholine but the maximal response did not increase. When these denervated muscles were organ-cultured, they showed no or only a slight increase in sensitivity to norepinephrine and methacholine, but the maximal response increased greatly. These observations led to the suggestion that the increase in sensitivity may be mediated through the same mechanisms as those for denervation supersensitivity. The elevation of the maximal response was suggested to be produced by the improvement of cell-to-cell conduction as well as some other unknown factor(s) probably specific to organ culture. Thus, it was concluded that organ-cultured 1-week-old rat vas deferens is a useful model to study the mechanisms of denervation supersensitivity.     

An evaluation of the young dopamine-lesioned rat as an animal model for minimal brain dysfunction (MBD)

Three main symptoms of minimal brain dysfunction (MBD), a common disorder in children, are hyperactivity, learning disabilities, and attention deficits. Drugs like amphetamine and methylphenidate have been demonstrated to produce a significant behavioral improvement in these children. The behavioral response of young rats (3–4 weeks), with selective lesioning of the central dopaminergic system, to a novel environment was analyzed. Both the frequencies and durations of eight mutually exclusive and complementary behavioral categories were scored. By analyzing the behavior in this way it appeared that considerable hyperactivity and leaning disabilities could be demonstrated in these rats. Moreover, the bout length of some behavioral categories was somewhat shortened, which might be an indication of deficits in attention. However, treatment of the animals with amphetamine did not produce any therapeutic effect on the three symptoms. Since pharmacotherapeutic support is, in our opinion, a conditio sine qua non for the validity of the model, we do not believe that the young DA-lesioned rat is an appropriate animal model for MBD.     

Efficacy of phenyl quinoline phenol derivatives as COX-2 inhibitors; an approach to emergent the small molecules as the anti-inflammatory and analgesic therapeutics

2-(4-phenylquinoline-2-yl)phenol derivatives (4a-l) with COX-2 enzyme inhibition, analgesic, anti-inflammatory and antipyretic potentials were executed and reported. From the in vitro COX-2 enzyme inhibition assay, compounds 4 h (IC₅₀ 0.026 µM) and 4j (IC₅₀ 0.102 µM) were found as most potent COX-2 inhibitors. Consequently, to get more insight into the binding mode with COX-2, compounds 4a-l were docked into the COX-2 (PDB ID: 1CX2) active site. In the Human Red Blood Cells (HRBC) membrane stabilization assay (in vitro anti-inflammatory), compounds 4f (IC₅₀ 0.064 µM) substituted with –OH (R¹) and –3Cl (R²), 4 h (IC₅₀ 0.021 µM), 4i (IC₅₀ 0.484 µg/ml) and 4j (IC₅₀ 0.092 µM) with –CHO containing alkanol and ether group at R1 and –4F, –4Br and –OMe at R2 (C2) were showed most potent anti-inflammatory activity. Eventually, acute toxicity studies revealed that 2-(4-phenylquinoline-2-yl)phenol derivatives (4a-l) are safe up to a toleration dose limit of 100 µg/kg body weight. In the Backer’s yeast intraperitoneal injection test, compounds 4f, 4 h and 4j produced significant (p < 0.05) antipyretic activity at 1, 1.5, 2 and 2.5 h, whereas test compound 4j and the reference drug indomethacin showed significant antipyretic activity throughout the observation period up to 2.5 h. Promising in vivo results obtained were correlated with the standard non-steroidal anti-inflammatory drugs and the compounds 4f, 4 h, 4i, 4j, and 4 l were efficiently identified as therapeutically potent/fortune moieties as non-steroidal anti-inflammatory agents/analgesics. At the end, ulcerogenic study result ensured that the tested 2-(4-phenylquinoline-2-yl)phenol derivatives created no side-effect.     

Analysis of dimethyl sulfoxide immunosuppression in the rat model of collagen II autoimmune arthritis: an effect dependent upon intraperitoneal administration and associated with toxicity

The effect of the route of administration of dimethyl sulfoxide on humoral immunity and arthritis was evaluated in the rat model of collagen II autoimmune arthritis. Intraperitoneal administration of 5 g/kg/day (days 0-12) reduced serum anti-collagen II IgG levels, delayed the onset of arthritis, but induced sterile peritonitis in all of the treated animals. The same dose given subcutaneously did not alter humoral or clinical parameters. Lower intraperitoneal doses (0.04 and 0.25 g/kg/day), although non-toxic, were similarly ineffective. Subcutaneous (5 g/kg/day) or topical treatment (both hindpaws dipped twice daily into 70% dimethyl sulfoxide) of established disease (days 16-27) produced a mild anti-inflammatory effect without any immunosuppression. We suggest that the apparent suppression of autoimmunity by dimethyl sulfoxide is dependent upon intraperitoneal administration and a toxic dose of the agent.     

A comparison of haemoglobin and erythrocytes as inhibitors of smooth muscle relaxation by the NANC transmitter in the BRP and rat anococcygeus and by EDRF in the rabbit aortic strip.

1. The inhibitory effect of erythrocyte suspensions and haemoglobin solutions on the response of the bovine retractor penis muscle (BRP) and the rat anococcygeus to field stimulation of their non-adrenergic non-cholinergic (NANC) nerves has been compared. Haemoglobin 3 microM greatly reduced the relaxant response in both tissues whereas a haemoglobin-equivalent suspension of erythrocytes was without effect. 2. A similar comparison of erythrocytes and haemoglobin on the response of the rabbit aortic strip to EDRF liberated by acetylcholine (ACh) showed that both reduced EDRF-mediated relaxation, though haemoglobin was significantly more effective. 3. These results suggest that the NANC transmitter may not be as freely diffusible through the erythrocyte membrane as EDRF and may therefore not be nitric oxide.     

The involvement of oxidative stress in the mechanisms of damaging cadmium action in bone tissue: a study in a rat model of moderate and relatively high human exposure

It was investigated whether cadmium (Cd) may induce oxidative stress in the bone tissue in vivo and in this way contribute to skeleton damage. Total antioxidative status (TAS), antioxidative enzymes (glutathione peroxidase, superoxide dismutase, catalase), total oxidative status (TOS), hydrogen peroxide (H₂O₂), lipid peroxides (LPO), total thiol groups (TSH) and protein carbonyl groups (PC) as well as Cd in the bone tissue at the distal femoral epiphysis and femoral diaphysis of the male rats that received drinking water containing 0, 5, or 50 mg Cd/l for 6 months were measured. Cd, depending on the level of exposure and bone location, decreased the bone antioxidative capacity and enhanced its oxidative status resulting in oxidative stress and oxidative protein and/or lipid modification. The treatment with 5 and 50 mg Cd/l decreased TAS and activities of antioxidative enzymes as well as increased TOS and concentrations of H₂O₂ and PC at the distal femur. Moreover, at the higher exposure, the concentration of LPO increased and that of TSH decreased. The Cd-induced changes in the oxidative/antioxidative balance of the femoral diaphysis, abundant in cortical bone, were less advanced than at the distal femur, where trabecular bone predominates. The results provide evidence that, even moderate, exposure to Cd induces oxidative stress and oxidative modifications in the bone tissue. Numerous correlations noted between the indices of oxidative/antioxidative bone status, and Cd accumulation in the bone tissue as well as indices of bone turnover and bone mineral status, recently reported by us (Toxicology 2007, 237, 89-103) in these rats, allow for the hypothesis that oxidative stress is involved in the mechanisms of damaging Cd action in the skeleton. The paper is the first report from an in vivo study indicating that Cd may affect bone tissue through disorders in its oxidative/antioxidative balance resulting in oxidative stress.     

Primary culture of proximal tubular cells from normal rat kidney as an in vitro model to study mechanisms of nephrotoxicity. Toxicity of nephrotoxicants at low concentrations during prolonged exposure

The aim of this study was to set up an in vitro system to study nephrotoxicity of xenobiotics which allows exposure at low concentrations for long periods (1-5 days). A very pure preparation of isolated proximal tubular cells (PTC) from rat kidney (Boogaard et al., Toxicol Appl Pharmacol 101: 135-143, 1989) was brought into primary culture. Cells grew to confluence in 3 days and could be maintained up to 8 days in a modification of Dulbecco's modified Eagle's medium Ham F12 nutrient mixture supplemented with fetal calf serum. Fibroblast growth was completely suppressed by replacement of L-valine by D-valine and of L-arginine by L-ornithine. Polarity was retained: in cells grown on filters organic anions were transported at the basolateral membrane while D-glucose transport was located at the apical membrane. Inhibition of the latter was used to assess the functional integrity of the cells after exposure to nephrotoxins. The newly grown cells expressed gamma-glutamyltranspeptidase activity since incubation with the glutathione-conjugate of 1,1-dichloro-2,2-difluoroethylene (DCDFE) induced cytotoxicity. Both beta-lyase and acylase activities were expressed because the cysteine-S-conjugate and the corresponding mercapturate of DCDFE showed cytotoxicity. Cultured cells showed toxicity on prolonged exposure to very low concentrations of gentamicin, cephaloridine, cisplatin and the cysteine-S-conjugate of chlorotrifluoroethylene. The lowest concentrations at which toxicity can be observed are 1-3 orders of magnitude lower in primary cultures than in freshly isolated PTC in suspension. This indicates that this cell model is suitable to investigate mechanisms of nephrotoxicity in vitro, at prolonged exposure to the low concentrations that are relevant in vivo levels.     

Cholecystokinin acts as an essential factor in the exacerbation of pancreatic bile duct ligation-induced rat pancreatitis model under non-fasting condition

We examined the influence of 2 gut hormones involved in the enhancement of pancreatic exocrine secretion, secretin and cholecystokinin (CCK), in the exacerbation of pancreatitis. We also examined the role of the vagal system, which was considered to be a transmission route for these hormones. Our model of pancreatitis in the rat was prepared by pancreatic bile duct ligation (PBDL), which simultaneously ligated the pancreatic duct and the common bile duct. Serum amylase activity and histopathological changes in the pancreas were used as indices of pancreatitis. We also measured the volume of pancreatic juice, as well as the amylase activity and protein level of the pancreatic juice, as indices of increased pancreatic exocrine secretion. Two gut hormones were given 6 times at 1-h intervals. Administration of secretin (1-3 microg/kg, s.c.) did not influence serum amylase activity in rats with PBDL-induced pancreatitis. However, food stimulation and administration of CCK-8 (1 microg/kg, s.c.) increased serum amylase activity and promoted vacuolation of the pancreatic acinar cells in rats with PBDL-induced pancreatitis. Administration of atropine (3 mg/kg, s.c.) or a CCK1-receptor antagonist, Z-203 (0.1 mg/kg, i.v.), inhibited food-stimulated or CCK-8-induced (1 microg/kg, s.c.) enhancement of pancreatic exocrine secretion and exacerbation after the development of PBDL-induced pancreatitis. These results suggest that not secretin, which regulates the volume of pancreatic juice, but CCK, which regulates the secretion of pancreatic enzymes via the vagal system, plays an essential role in food-stimulated exacerbation after the development of pancreatitis.     

Characterization of anti-apoptotic action of TCDD as a defensive cellular stress response reaction against the cell damaging action of ultra-violet irradiation in an immortalized normal human mammary epithelial cell line, MCF10A

It was originally shown by Woerner and Schrenk [Woerner, W., Schrenk, D., 1998. 2,3,7,8-Tetrachlorodibenzo-p-dioxin suppresses apoptosis and leads to hyperphosphorylation of p53 in rat hepatocytes. Environ. Toxicol. Pharmacol. 6, 239-247] that TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) acts as an antagonist against the action of UV-irradiation to induce apoptosis in rat primary hepatocytes. Since prevention of apoptosis has been shown to promote carcinogenesis, we have decided to investigate this phenomenon in a human mammary gland epithelial cell line, MCF10A. We found that, in this cell line, TCDD can antagonize apoptosis that was induced by a variety of treatments, such as UV- and gamma-irradiation, growth factor starvation and trypsinization, or by the addition of H(2)O(2), TGFbeta, and staurosporine. Furthermore, other agents that are known to elicit defensive cellular responses, such as LPS, Fe(3+), nitric oxide and hypoxia could also antagonize UV induced apoptosis just as in the case of TCDD. In addition, we found that, in this cell line, such anti-apoptotic action of TCDD resembles that of exogenously added EGF or TGF alpha. To study the basic mechanism of such an action of TCDD, we tested a variety of diagnostic agents to reverse the effect of TCDD. Antagonists of TCDD which were found to be effective in this way were (a) inhibitors of c-Src kinase, such as PP-2 and CGP77675, (b) those known to block the action of TGF alpha, such as anti-TGF alpha antibody, and alpha(1)-antitrypsin, (c) PD98059, a specific inhibitor of ERK activation, but not SB202190 (an inhibitor of p38 MAPK activation) or SP600125 (a JNK inhibitor) and (d) Ah receptor antagonists, alpha-naphthoflavone and 1, 10-phenanthroline. These results support the notion that TCDD acts as an anti-apoptotic agent by mimicking the action of EGF through activation of the c-Src/ERK signaling pathway.     

N-methylation of biogenic amines. I. Characterization and properties of an N-methyltransferase in rat brain using 5-methyltetrahydrofolic acid as the methyl donor

Some properties and kinetics of a recently identified brain N-methyltransferase requiring 5-methyltetrahydrofolic acid as the methyl donor are described in this paper. In addition to the assay using dopamine as substrate, the more stable substrate, 3-hydroxy-4-methoxyphenethylamine with a rapid extraction procedure was also used. The pH optimum of the enzyme, about pH 6·4, was found to differ from that previously found with dopamine in the presence of metabisulfite. The reaction products were identified by column and thin layer chromatography and indirectly by testing various N-methyl-, dimethyl- and dimethoxy-derivatives of dopamine for their ability to be N-methylated. The reaction was a linear function of time and enzyme concentration. The K_m for 5-methyltetrahydrofolic acid was 2.5 × 10^?5 M. The kinetic experiments to determine the K_m for dopamine and 3-hydroxy-4-methoxyphenethylamine showed an anomalous behaviour of the saturation curve. When the results were plotted S/v vs S, a non-linear curve was obtained suggesting that the enzyme was behaving as an allosteric protein. Although further kinetic experiments are needed to confirm these results, a possible regulatory function can be postulated for this enzyme. In. addition to dopamine and its derivatives, amines such as tryptamine, serotonin and amphetamine were also N-methylated under the same conditions.