Memory impairment is one of the greatest concerns when it comes to long-term CNS-affecting drug administration. Drugs like gabapentin, pregabalin and baclofen are administered in a long-term period in conditions such as epilepsy, neuropathic pain, spasticity associated with spinal cord injury or multiple sclerosis. Despite their wide spread use, few data are available on the effects of these drugs on cognitive functions, such as learning memory. In the present study, the effects of long-term administration of gabapentin, pregabalin and baclofen on memory were investigated in a comparative manner. Male Wistar rats received intraperitoneal (i.p.) injection of gabapentin (30 mg/kg), pregabalin (30 mg/kg), baclofen (3 mg/kg), combination of gabapentin/baclofen (30/3 mg/kg) and combination of pregabalin/baclofen (30/3 mg/kg) once a day for 3 weeks respective to their groups. After the end of treatments, rat memories were assessed using the object-recognition task. The discrimination and recognition indices (RI and DI) in the T2 trials were used as the memory indicating factors. The results showed that daily i.p. administrations of pregabalin but not gabapentin or baclofen significantly decreased DI and RI compared to saline group. In combination groups, either gabapentin or pregabalin impaired discrimination between new and familiar objects. Our findings suggested that pregabalin alone or in combination with baclofen significantly caused cognitive deficits.
Potassium bromide (KBr), an old antiepileptic agent, is illegally used in pharmaceutical or food industries to improve the product appearance. KBr has been proven to influence several pathways which are important in memory formation. Therefore, the present study aimed to evaluate the effect of KBr on spatial working memory using object recognition task (ORT). Rats received a single dose of KBr (50, 100 or 150 mg/kg), per oral, in acute treatment. KBr long term effects were also studied in animals receiving 50 mg/kg/day of KBr for 28 consecutive days. At the end of treatments, animals underwent two trials of ORT, five min each. In the first trial (T1), animals encountered with two identical objects for exploration. After 1 h, the animals were exposed to a familiar and an unfamiliar object (T2). The exploration times for discrimination (D) and recognition (R) as well as the frequency of exploration for any objects were determined. Acute administration of 150 mg/kg of KBr significantly decreased the discrimination and recognition indices (RI and DI) (P < 0.01) compared to the control. However, lower doses failed to influence the animals' performance in the test. In addition, long term administration of KBr remarkably diminished the DI and RI and the frequency of exploration (P < 0.05). The results of this study indicate that acute doses of KBr as high as 150 mg/kg are required to hamper memory function in ORT. However, cognitive impairment occured with lower doses of KBr when the duration of treatment is extended.
Tramadol hydrochloride, a synthetic opioid, acts via a multiple mechanism of action. Tramadol can potentially change the behavioral phenomena. The present study evaluates the effect of tramadol after single or multiple dose/s on the spatial memory of rat using object recognition task (ORT). Tramadol, 20 mg/kg, was injected intraperitoneally (i.p) as a single dose or once a day for 21 successive days considered as acute or chronic treatment respectively. After treatment, animals underwent two trials in the ORT. In the first trial (T1), animals encountered with two identical objects for exploration in a five-minute period. After 1 h, in the T2 trial, the animals were exposed to a familiar and a nonfamiliar object. The exploration times and frequency of the exploration for any objects were recorded. The results showed that tramadol decreased the exploration times for the nonfamiliar object in the T2 trial when administered either as a single dose (P<0.001) or as the multiple dose (P<0.05) compared to the respective control groups. Both acute and chronic tramadol administration eliminated the different frequency of exploration between the familiar and nonfamiliar objects. Our findings revealed that tramadol impaired memory when administered acutely or chronically. Single dose administration of tramadol showed more destructive effect than multiple doses of tramadol on the memory. The observed data can be explained by the inhibitory effects of tramadol on the wide range of neurotransmitters and receptors including muscarinic, N-methyl D-aspartate, AMPA as well as some second messenger like cAMP and cGMP or its stimulatory effect on the opioid, gama amino butyric acid, dopamine or serotonin in the brain.
International Journal of Radiation Research (23223243)(3)
Background: Diarrhea is a well-recognized side effect associated with pelvic radiation; however, there is not any effective common treatment for radiation-induced diarrhea. A popular alternative is probiotics, which have been used in several gastrointestinal disorders. Probiotics are live microbial food supplements. Furthermore, honey is a putative nutritional with a variety of health effects, including antibacterial, antioxidant, anti-inflammatory and prebiotic. The present study evaluated the effects of probiotic with or without honey on radiation-induced diarrhea. Materials and Methods: Sixty-seven adult patients with pelvic cancer underwent radiotherapy for four weeks. They randomized to receive probiotic (n = 22), probiotic plus honey (n = 21) or placebo (n = 24) from one week before radiotherapy for five weeks. Diarrhea grade and stool consistency score were recorded weekly according to the Common Toxicity Criteria system and the Bristol scales, respectively. Results: The results showed a decrease in the daily number of bowel movements (p = 0.003 and 0.006), diarrhea grade (p = 0.001 and 0.001) and the need for antidiarrheal medication (p = 0.021 and 0.041) also an increase in the stool consistency (p = 0.004 and 0.005) in patients who either used probiotic or probiotic plus honey (respectively), these were significant in weeks 4 and 5 of treatment. Conclusion: Probiotics with or without honey can reduce the incidence of radiation-induced diarrhea and the need for antidiarrheal medication.
Moosavirad, Saeedeh Alsadat, Rabbani khorasgani, M., Sharifzadeh, Mohammad, Hosseini-sharifabad, A.
Publication Date: 2016
Research in Pharmaceutical Sciences (17355362)(5)
Lead belongs to the heavy metal group and is considered as an environmental contaminant. Acute or chronic contact to lead can change the physiological function of human organs. One of the most important disorders following the lead exposure is neurotoxicity. Lead neurotoxicity consists of the neurobehavioral disturbances like cognitive impairment. The aim of the current study is to evaluate the possible protective effect of vitamin C (Vit C), vitamin B12 (Vit B12), omega 3 (ω-3), or their combination on the lead-induced memory disorder. Adult wistar rats were orally administered Vit C (120 mg/kg/day) or Vit B12 (1 mg/kg/day) or ω-3 (1000 mg/kg/day) or their combination for 3 weeks in groups of 7 animals each. Then lead acetate (15 mg/kg/day) was injected intraperitoneally for one week to all pretreated animals. The control group received normal saline as a vehicle while the positive control for cognitive impairment received just lead acetate. At the end of treatments animal memories were evaluated in Object Recognition Task. The results showed, although 15 mg/kg lead acetate significantly declines the memory-evaluating parameters, pretreatment with Vit C, Vit B12, ω-3, or their combination considerably inverted the lead induced reduction in discrimination (d2) index (P < 0.001) and recognition (R) index (P < 0.001, P < 0.05, P < 0.05, and P < 0.001, respectively). Our findings indicate while lead acetate impairs spatial memory in rat, administration of Vit C, Vit B12, ω-3, or their combination prior to the lead exposure inhibits the lead induced cognitive loss. There was no remarkable difference in this effect between the used supplements.
We have previously evaluated the effect of nimodipine, L-type calcium channel blocker, on memory loss during spontaneous morphine withdrawal. In the present study the effect of nimodipine on memory loss in naloxone-induced morphine withdrawal mice was investigated. Mice were made dependent by increasing doses of morphine for three days. Object recognition task that was used for evaluation of memory performance comprised of three sections: 15 min habitation, 12 min first trial and 5 min test trial. Naloxone was injected 3 h after the administration of the last dose of morphine. Recognition index was evaluated 20 min after naloxone injection. Nimodipine was administrated in repeated form (1, 5 and 10 mg/kg) with daily doses of morphine or as a single injection (5 and 10 mg/kg) on the last day. Both acute and repeated treatments with nimodipine prevented the memory impairment in naloxone-induced morphine withdrawal mice (P<0.05 comparison of acute and repeated treatment data with their corresponding control values). Corticosterone concentration was significantly increased in the brain and blood of the mice during withdrawal. Pretreatment with nimodipine, however, decreased the corticosterone concentration in both brain and blood. The present study showed that nimodipine prevents intense memory loss following naloxoneinduced morphine withdrawal.
Apoptosis has a critical role in the pathogenesis of bleomycin induced-pulmonary fibrosis. The severity of fibrosis varies among different strains of mice. Recent studies have indicated that expression of apoptotic regulatory genes may be specific in different cell types in various strains. In this study, bleomycin-induced pulmonary apoptosis in NMRI (Naval Medical Research Institute, USA) albino mice were compared with C57BL/6 black mice. Pulmonary fibrosis induced by single intratracheal administration of bleomycin (3 U/kg). Control mice were instilled with the same volume of saline. After 2 weeks, fibrotic responses were studied by biochemical measurement of collagen deposition and histological examination of pathological lung changes. Apoptosis was detected and quantitated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Bleomycin significantly (P<0.05) increased lung collagen content and also induced fibrotic histological changes in both strains. Apoptosis was detected in the bronchiolar and alveolar epithelial cells after bleomycin instillation. TUNEL-positive alveolar epithelial cells in bleomycintreated lungs of C57BL/6 and NMRI mice (19.5% ± 2.7 and 17% ± 2.0, respectively) were significantly (P<0.05) higher than that of saline-treated lungs (1.5% ± 0.5) with no significant difference between two strains of mice (P>0.05). Despite some murine strain variation in the expression of apoptotic regulatory genes in bleomycin-induced pulmonary fibrosis, the results of the present study revealed no significant differences in alveolar epithelial apoptosis between NMRI and C57BL/6 black mice. However, these results confirm the role of apoptosis in the pathogenesis of pulmonary fibrosis and suitability of both strains as experimental models of lung fibrosis.
Background: Insomnia, which is difficulty in initiating and maintaining sleep, is a very common experience for many people. Considering the increasing interest in medicinal plants in the past decade, many plants such as Coriandrum sativum, Salvia leriifolia, Salvia reuterana and Stachys lavanduli folia have been used in Iranian traditional medicine to abate insomnia. Objective: The present study was designed to investigate hypnotic effect of Salvia reuterana on male mice. Methods: Ethanolic extract of S. reuterana was prepared. Five groups of 6 animals each were pretreated with vehicle, Salvia extract (50, 100 and 250 mg/kg; i.p.) or diazepam (0.5mg/kg; i.p.) 30 minutes before ketamine injection (100 mg/kg, i.p.). Results: The latency and total sleeping times were recorded to determine the hypnotic effect of the extract. The results indicated that ethanolic extract of S. reuterana, reduced the latency time and induced the total sleeping time in a dose dependent manner, compared to saline group. Conclusion: The present study suggests that S. reuterana produces hypnotic effect which can be evaluated clinically.
Background: This study was aimed to clone and express the reteplase complementary deoxyribonucleic acid (cDNA), a thrombolytic agent used for the treatment of acute myocardial infarction and stroke, in Escherichia coli using tac promoter. Methods: Reteplase cDNA was amplified using polymerase chain reaction (PCR) with designed primers. The product was then cloned into pTZ57 plasmid. The cloned DNA was digested out and ligated into pGEX-5x-1 expression vector. The presence of the insert was confirmed by restriction digestion and determination of the nucleotide sequence. By using 0.2, 0.5, and 1 mM isopropyl beta-D thiogalactopyranoside (IPTG), reteplase was induced in escherichia coli TOP10 cells and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Findings: Electrophoresis of PCR product as well as double digested recombinant pTZ57 plasmid showed a 1068 base pair band of reteplase. SDS-PAGE analysis showed a 60 kilodalton band of protein product induced by IPTG. Conclusion: In the present study, reteplase cDNA was successfully cloned and expressed using tac promoter. This vector was used for the optimization of the expression of reteplase in escherichia coli.
Abrupt cessation of morphine leads to withdrawal signs and cognitive deficits. Endocannabinoid system is activated during withdrawal; therefore, the aim of the present study was to assess the effects of AM281, cannabinoid antagonist/inverse agonist, on memory deficit following spontaneous morphine withdrawal. Cognition was evaluated by using the object recognition task. The novel object recognition task was tested in a square wooden open-field box using objects. The test was consisting of three sections: 15 min exploration, first trial for 12 min and second one for 5 min. In the second trial the difference in exploration between a previously seen object and a novel one, was considered as an index of memory performance (recognition index - RI). Male mice were made dependent by increasing doses of morphine (30-90 mg/kg) subcutaneously twice daily for 3 days. AM281 (0.62, 1.25 and 2.5 mg/kg) were used in chronic form concurrent with morphine i.p. or acutely (2.5, 5 and 10 mg/kg) on the last day. RI was evaluated on the third day 4 h after the last dose of morphine. Chronic administration of AM281 at 2.5 mg/kg improved RI to the 22.1 ± 4.8 and single dose of AM281 at 5 mg/kg improved the memory impairment to the 8.5 ± 4, as compared with vehicle-treated which was 4.8 ± 2.5. The results suggested that administration of AM281 at a dose of 2.5 mg/kg in chronic form and 5 mg/kg in acute dose improved memory.
Iranian Journal of Basic Medical Sciences (20083874)(5)
Objective(s) Cannabinoids have been implicated in memory deficit. We examined the effect of AM281, cannabinoid antagonist/inverse agonist in prevention of scopolamine-induced cognitive deficit. Materials and Methods Object recognition task was used to evaluate memory in mice. Exploration time in the first and the second trial was recorded. The differences in exploration between a previously seen object and a novel object in second trial were taken as an index of memory. Scopolamine and AM281 were administrated at the same time, 40 min before second trial in the treatment group. Results Object discrimination was impaired after scopolamine (2 mg/kg; IP) administration. AM281 (2.5, 5 mg/kg; IP) significantly restored object recognition ability in mice treated with scopolamine by 75%. Conclusion This study extends earlier findings, suggesting the interaction of cannabinoid and cholinergic system in memory. Additionally cannabinoid antagonists seem to show variable pharmacological properties.
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the intestinal tract whose etiology has not yet been fully elucidated. Available medicines for treatment of IBD are not universally effective and result in marked deleterious effects. This challenge has thus height-ened the need for research in order to adopt new therapeutic approaches for the treatment of IBD. 5-HT 3 receptor antagonists have shown analgesic and anti-inflammatory properties in vitro and in vivo. Our aim was to investigate the effect of ondansetron, 5-HT 3 receptor antagonist, in an im-mune-based animal model of IBD, trinitrobenzene sulfonic acid (TNBS)-induced rat colitis and probable involvement of 5-HT 3 receptors. Two hours after induction of colitis (instillation of 50 mg/kg of TNBS dissolved in 0.25 ml of ethanol 50 % v/v) to male Wistar rats, ondansetron (2 mg/kg), dexamethasone (1 mg/kg), meta-chlorophenylbiguanide (mCPBG, 5 mg/kg), a 5-HT 3 receptor agonist, or ondansetron + mCPBG were administrated intraperitoneally (ip) and continued daily for six days. The animals were sac-rificed and distal colons were assessed macroscopically, histologically and biochemically [myeloperoxidase (MPO), tumor necrosis factor-alpha, interleukin-6 and interleukin-1 beta]. Ondansetron and dexamethasone resulted in a decrease in macroscopic and microscopic colonic damage significantly. In addition a dramatic reduction in MPO activity and colonic levels of in-flammatory cytokines were seen. The protective effects of ondansetron were antagonized by concurrent administration of mCPBG. Our data suggests that the beneficial effects of ondansetron in TNBS-induced colitis could be mediated by 5-HT 3 receptors.
The present study was conducted to investigate the effect of oral administration of calcium gluconate and magnesium acetate on morphine withdrawal syndrome. Mice were rendered dependent on morphine by subcutaneous injection of increasing doses of morphine. Mice were observed for 30 minutes for the withdrawal signs (jumping or standing events, diarrhea, piloerection, tremor and ptosis). Separate oral administration of magnesium (50, 75 and 100 mg/kg) and calcium (500, 750 and 1,000 mg/kg) significantly decreased the jumping, without affecting standing in animals withdrawn from morphine. Co-administration of magnesium (at a fixed dose of 100 mg/kg) and calcium (at a range of doses from 250 to 1,000 mg/kg) resulted in a significant reduction in jumping and standing events (P<0.05). In a similar fashion, the qualitative signs of withdrawal were also reduced when the above combination of calcium and magnesium was administered. Co-administration of calcium/magnesium at 500/50, 750/75 and 1,000/100 mg/kg significantly reduced the number of jumps in morphine-dependent animals without affecting the number of standing events. This study demonstrates the potential activity of the co-administration calcium and magnesium in preventing the signs associated with morphine withdrawal syndrome.
Background: The aim of this project was to isolate and purify the highly active recombinant Taq DNA polymerase from the strain of Escherichia coli BL21. This enzyme, with a molecular weight of about 94 kDa, is widely used in polymerase chain reaction (PCR). In PCR, the activity and fidelity of Taq polymerase can significantly influence the results. One of the active regions of Taq polymerase has been suggested to be the O-helix region. In previous studies, an expression vector containing mutated Asn 666 Glu Taq polymerase gene was designed. In order to investigate the effects of this mutation on the function of the enzyme, Taq polymerase needs to be purified first. Methods: In this study, after transformation of competent cells, enzyme expression was induced by isopropyl β D thiogalactopyranoside (IPTG) method. Modified Desai protocol with DNase and RNase, nickel-nitrilotriacetic acid (Ni-NTA) resin, trichloroacetic acid (TCA), and refolding protocols were subsequently used for purification of this enzyme. These protocols were finally compared. Findings: Using Desai protocol resulted in the production of a sharp band in the expected region (94 kDa) and several other visible bands. After further modification of Desai protocol, only the desirable band was observed. In protocols using TCA and Ni-NTA resin, the expected bands were weak. Refolding protocol caused a band in an undesirable region (66 kDa). Conclusion: From the different purification techniques that were used in this study, the modified method of Desai containing RNase and DNase worked best. Addition of TCA can precipitate proteins that had not been affected by heat. Using Ni-NTA resin resulted in elimination of unwanted bands. However, the amount of Taq polymerase was also decreased. The extra band that was observed in refolding protocol was probably due to the presence of proteases that were isolated with inclusion body and could digest Taq polymerase.
Motavallian, Azadeh, Andalib, Sasan, Rabbani khorasgani, M., Mahzouni P., Afsharipour M.
Publication Date: 2012
Research in Pharmaceutical Sciences (17359414)(3)
Trinitrobenzene sulfonic acid (TNBS)-induced colitis is one of the most common methods for studying inflammatory bowel disease in animal models. Several factors may, however, affect its reproducibility, rate of animal mortality, and macroscopic and histopathological outcomes. Our aim was to validate the main contributing factors to this method and compare the effects of different reference drugs upon remission of resultant colon injuries. TNBS was dissolved in 0.25 ml of ethanol (50% v/v) and instilled (25, 50, 100 and 150 mg/kg) intracolonically to the male Wistar rats. After determination of optimum dose of TNBS in male rats and assessment of this dose in female rats, they were treated with reference drugs including dexamethasone [1 mg/kg, intraperitoneally (i.p.) and 2 mg/kg, orally (p.o.)], Asacol (mesalazine, 100 mg/kg, p.o.; 150 mg/kg, enema) and hydrocortisone acetate (20 mg/kg, i.p.; 20 mg/kg, enema) which started 2 h after colitis induction and continued daily for 6 consecutive days. Thereafter, macroscopic and microscopic parameters and clinical features were assessed and compared in different groups. We found that the optimum dose of TNBS for the reproducibility of colonic damage with the least mortality rate was 50 mg/kg. Amongst studied reference drugs, hydrocortisone acetate (i.p.), dexamethasone (i.p. and p.o.) and Asacol (p.o.) significantly diminished the severity of macroscopic and microscopic injuries and could be considered effective for experimental colitis studies in rats. Our findings suggest that optimization of TNBS dose is essential for induction of colitis under the laboratory conditions; and gender exerts no impact upon macroscopic and histological characteristics of TNBS-induced colitis in rats. Furthermore, the enema forms of hydrocortisone and Asacol are not appropriate reference drugs.
The anxiolytic effect of the flower of Echium. amoenum was shown in several experimental studies in mice. The present study was aimed to determine whether tolerance develops to anxiolytic action of E. amoenum in mice. NMRI male mice were injected intraperitoneal with hydroalcoholic extract (12.5, 25 and 50 mg/kg) or saline once each day (8 am) for period of 7 days and then tested on light/dark box model. Anxiolytic effect was determined by light/dark box and elevated plus-maze. According to the results, hydroalcohoic extract of E. amoenum when given both acutely and chronically (7 days) at 25 and 50 mg/kg, significantly increased the time in the illuminated zone. The number of transitions in the light/dark apparatus, however, was not significantly altered by the tested doses of the plant. Diazepam at 0.5 and 1 mg/kg produced anxiolytic effect in both model of anxiety, namely, the light/dark box and elevated plus-maze. No tolerance was developed to the anxiolytic effect of E. amoenum extract after 7 days of treatment. Our results suggest that one week treatment with extract of the E. amoenum does not produce tolerance to its anxiolytic action. Longer period of treatment using implant procedure is probably necessary to cause molecular changes in order to induce tolerance.
Genetic Testing and Molecular Biomarkers (19450257)(1-2)
The estrogen receptor β (ERβ) mediates the action of estrogen on metabolism of lipids and lipoprotein. Therefore, its gene is a promising candidate gene for cardiovascular disease. The aim of the present study was to investigate whether the ERβ A1730G polymorphism modifies the metabolic response to hormone replacement therapy (HRT) in postmenopausal women. The population included 60 normolipidemic postmenopausal women with equal numbers of each A1730G genotype followed during a 90-day experimental period. All subjects received oral estrogen together with a progestin therapy during the HRT. ABCA1 gene expression and serum lipid and lipoprotein concentrations were measured at the beginning and end of the HRT trial. At baseline, ABCA1 gene expression, lipid or lipoprotein concentrations were not significantly different among the ERβ A1730G genotype groups. After HRT, however, subjects with GG genotype had a greater increase in ABCA1 gene expression (p=0.002) and a trend toward greater increase in apoA-I (p=0.058) than subjects carrying the A allele. An interaction effect between genotype and HRT effect was observed on ABCA1 gene expression. In conclusion, the positive changes of ABCA1 gene expression and apoA-I were affected by the ERβ A1730G polymorphism in women taking estrogen-progesterone therapy. Copyright 2011, Mary Ann Liebert, Inc.
Reteplase is a segment of tissue plasminogen activator used for the removal of thrombi in blood vessels. In the present study the cloned reteplase gene was used for its expression in competent E. coli. The recombinant plasmid, pET15b/reteplase (rpET-BL21), was transformed into competent E.coli strain BL21 (DE3) cells. Overnight culture of the transformed bacteria was induced by the addition of isopropylthio-ß-Dgalactoside (IPTG) to the final concentrations of 0.25, 0.5, 1 and 1.5 mM. Also, the effects of different temperatures(25, 30, 37 and 39°C), shaking speeds (100, 170 and 190 rpm), and various glucose concentrations (0.25, 0.5, 0.75 and 1 mM) on the expression of reteplase were examined. Samples were analyzed by SDS-PAGE. Maximum amount of protein production was obtained by the addition of 1 mM IPTG at 37°C, 100 rpm of shaking speed in the absence of glucose.
Anxiety disorders are amongst the most popular diseases which interfere with normal life. Benzodiazepines are used as a first line of treatment, but difficulties with pharmacotherapy of anxiety disorders such as dependence and low response rate, encourage researchers to find new approaches. From the past, the role of medicinal plants have been a subject of intense interest. In this respect, Citrus aurantium, Coriandrum sativum, Crocus sativus, Echium amoenum, Nepeta persica Boiss, Stachys lavandulifolia and Salix aegyptiaca are widely used by Iranian population. This review summarized the information on Iranian plant species that have been explored for their potential anti-anxiety profile using validated animal models, doses and possible mechanism.
The present study was undertaken to screen the soil samples collected in Iran for the presence of the Bacillus subtilis lipase A gene. The bacterial colonies obtained from the collected soil samples were examined by physical appearance, biochemical tests and the polymerase chain reaction (PCR). Only four colonies were identified as putative B. subtilis strains and all contained the lipase A gene. However, the intensities of the DNA bands were different and correlated with the differences obtained from the biochemical tests. Polymorphism of the lipase gene was also determined in samples using SSCP assay. In conclusion, this study demonstrates an easy and reliable method for detection of the lipase gene in B. subtilis strains. Further screening of the soil by this method will enable the detection and identification of industrially more favorable lipases.
Taq DNA polymerase is widely used in laboratories and for this reason many investigators have focused their attention on understanding the role of various regions and amino acids in this enzyme. O-helix is a part of taq polymerase suggested to play an important role in the enzyme fidelity. The influence of Asn666 in this helix on the enzyme function has never been investigated, and therefore by using nested PCR, a portion of taq DNA polymerase gene containing Asn666Glu mutation was amplified. This DNA was digested with Eco RI restriction enzyme to confirm the presence of Asn666Glu mutation. After digesting this product and the wild type taq-pET-15b plasmid with NheI and BamHI restriction enzymes, they were ligated and used for the transformation of E. coli DH5α competent cells. The obtained colonies were screened for the presence of the mutated taq polymerase gene using EcoRI, NdeI and BamHI restriction enzymes. In conclusion, with the use of the obtained recombinant plasmid it is possible to study the role of this amino acid on taq DNA polymerase function.
DARU, Journal of Pharmaceutical Sciences (15608115)(4)
Background and the purpose of the study: Vasopressin type 2 receptor (V2R), a G protein coupled receptor (GPCR), plays an important role in the regulation of renal antidiuretic function. The highly conserved DRH motif is essential for G protein signaling of V2R; however its role especially regarding the histidin residue is not fully understood. Methods: Site directed mutagenesis was performed with replacements of the histidin to isoleucine by using nested polymerase chain reaction. ELISA was performed for receptor expression assay and the adenylyl cyclase activity assay was performed for functional characterization of DRI mutation on V2R signaling.Results and major conclusion: The adenylyl cyclase activity assay in COS-7 cells showed no difference in the amount of cAMP production between the wild type and the mutant V2 receptors. The V2 receptor expression was not changed in the presence of this mutation using ELISA assay. These results suggest that the role of histidin residue is not critical in the V2 receptor function, however further mutagenesis studies are required to define the role of this motif in V2R function.
Background: Vasopressin type 2 receptor (V2R) plays an important role in the water reabsorption in the kidney collecting ducts. V2R is a G protein coupled receptor (GPCR) and the triplet of amino acids aspartatearginine- histidine (DRH) in this receptor might significantly influence its activity similar to other GPCR. However, the role of this motif has not been fully confirmed. Therefore, the present study attempted to shed some more light on the role of DRH motif in G protein coupling and V2R function with the use of sitedirected mutagenesis. Methods: Nested PCR using specific primers was used to produce DNA fragments containing aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations with replacements of the arginine to lysine and histidine to tyrosine, respectively. After digestion, these inserts were ligated into the pcDNA3 vector and transformation into E. coli HB101 was performed using heat shock method. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using diethylaminoethyl-dextran method, the adenylyl cyclase activity assay was performed for functional study. The cell surface expression was analyzed by indirect ELISA method. Results: The functional assay indicated that none of these mutations significantly altered cAMP production and cell surface expression of V2R in these cells. Conclusion: Since some substitutions in arginine residue have shown to lead to the inactive V2 receptor, further studies are required to define the role of this residue more precisely. However, it seems that the role of the histidine residue is not critical in the V2 receptor function.
Askari Badouei M., Zahraei salehi, T., Rabbani khorasgani, M., Tadjbakhsh H., Nikbakht brujeni, G.
Publication Date: 2010
Veterinary Record (00424900)(22)
The virulence properties of Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrhoeic and non-diarrhoeic calves were compared. The strains were also tested for O157:H7, O111 and O26 serotypes, using PCR and conventional serotyping methods. E coli strains isolated from 297 faecal samples, from 200 diarrhoeic and 97 non-diarrhoeic calves, were screened by multiplex PCR assay for the stx1, stx2, eae and Ehly virulence genes. STECs were recovered from 8 per cent of diarrhoeic calves and 10.3 per cent of non-diarrhoeic calves. The predominant virulence gene profile was stx1/eae/Ehly (47.3 per cent) among isolates from diarrhoeic calves and eae/Ehly (36.8 per cent) among isolates from non-diarrhoeic calves. Among three tested serogroups, the predominant serogroup was O26 (18.4 per cent), and O157:H7 was not detected. Intimin subtyping by restriction fragment length polymorphism analysis revealed only three intimin subtypes (β, γ and θ). A significant difference was observed in the distribution of Int-θ between two groups. Int-6 was present in 50 per cent of the isolates from diarrhoeic calves and in 11.1 per cent of the isolates from non-diarrhoeic calves; this difference was statistically significant (P=0.01).
The aim of present study was to assess the effects of chronic administration of calcium-magnesium soft gels on the development of morphine tolerance and dependence in mice. Tolerance was assessed using the tail-pinch test and withdrawal signs of morphine were precipitated by injecting naloxone 2 h after the final morphine injection. CalMag capsules were given in final doses of 50/25, 25/12.5 and 12.5/6.25 mg/kg based on calcium/magnesium ratio. Similar doses of Ca and/or Mg were prepared, separately. CalMag at 50/25, 25/12.5 mg/kg and the mixture of calcium and magnesium (Ca + Mg) at 50/25, 25/12.5, 12.5/6.25 mg/kg and calcium at 50, 25 mg/kg significantly reduced the number of jumps. The number of standings was only reduced after the administration of CalMag at 50/25 mg/kg and Ca + Mg at 25/12.5 mg/kg. The development of morphine tolerance was prevented in all drug-treated groups, except the one which received 6.25 mg/kg Mg. The data suggested that combination of calcium and magnesium at 50/25 and 25/12.5 mg/kg prevented the development of tolerance and dependence. It seems that other ingredients of CalMag capsules do not have an important effect on preventing tolerance and withdrawal signs. Compared to the acute effects, chronic administration of CalMag allowed the effective dose to be reduced. Unlike the acute treatment, chronic administration of calcium alone was effective in reducing morphine tolerance and dependence, and magnesium had no significant effect on withdrawal signs, suggestive of some pharmacological adaptations.
Safaeian L., Jafarian, A., Rabbani khorasgani, M., Mirmohammad, Sadeghi H., Torabinia n.,
Publication Date: 2009
Daru (15608115)(1)
Background and the purpose of the study: Recent studies have indicated the role of apoptosis and angiotensin in the pathogenesis of bleomycin induced-pulmonary fibrosis. Losartan, an angiotensin type 1 receptor (AT 1R) antagonist, has ameliorated apoptosis and fibrosis from bleomycin. In this study, alterations in the expression of apoptosis-regulatory genes (bcl-2 and bax) were investigated in different cells of lung tissue of mice treated with bleomycin in the presence of losartan. Methods: Losartan (10 mg/kg, i.p.) was given to mice two days before administration of bleomycin (3 U/kg) and throughout the test period. After two weeks, lung tissues of mice were evaluated for fibrosis by biochemical measurement of collagen deposition and semiquantitative analysis of pathological changes of the lung. The expression of bcl-2 and bax was assessed by immunohistochemical assay using biotin-streptavidin staining method on paraffin-embedded lung tissues. Results and major conclusion: Pre-treatment with losartan significantly (P < 0.05) reduced the increase in lung collagen content and also inhibited the histological changes induced by bleomycin. Immunohistochemical studies showed that losartan significantly (P < 0.05) reduced the bax/bcl-2 expression ratio in the alveolar epithelial cells, lymphocytes, macrophages and interstitial myofibroblasts. Losartan also inhibited the bcl-2 upregulation which was educed by bleomycin in neutrophils. By reduction of bax/bcl-2 ratio as a determinant of susceptibility of a cell to apoptosis, losartan exerted protective effects on the alveolar epithelial cells that may be important in the amelioration of pulmonary fibrosis. These results may help to better understanding of the role of angiotensin II and apoptosis in pulmonary fibrosis.
Bleomycin-induced pulmonary fibrosis is a widely used experimental model for human lung fibrosis. The severity of fibrosis varies among different strains of mice and investigation on different strains and finding the mechanisms of variation is important in understanding the pathogenesis of human lung fibrosis. In the present study, NMRI mice were used to investigate the severity and also time-course of bleomycin-induced pulmonary fibrosis in comparison with C57BL/6 mice. After single dose administration of intratracheal bleomycin, the fibrotic response was studied by biochemical measurement of collagen deposition and semiquantitative analysis of pathological lung changes. NMRI mice developed lung fibrosis from 1 to 4 week after bleomycin instillation, with significant increases in lung collagen content and significant morphological changes (P < 0.05). These findings indicate that NMRI mice might be suitable as an experimental model of bleomycin-induced lung fibrosis.
The present study was aimed at evaluating the acute effects of Calcium-Magnesium soft gels (CalMag) in morphine tolerant and dependent mice. Mice were rendered tolerant and dependent on morphine by subcutaneous injection of morphine over a fixed time period. Withdrawal signs were precipitated by injecting naloxone 2 h after the final injection of morphine. The tail-pinch assay was used to investigate the effects of various compounds on the development and reversal of morphine tolerance. Acute injection of CalMag (containing 50 mg/kg calcium and 25 mg/kg magnesium) significantly reduced the number of jumps, stands and fast breathing in morphine dependent mice. Co-administration of calcium (50 mg/kg) and magnesium (25 mg/kg) was also effective in preventing the development of morphine tolerance and dependence. Administration of calcium (up to 50 mg/kg) alone did not significantly block the development of tolerance and dependence. The mean latency to pain was significantly increased in animals pretreated with CalMag (containing 50 mg/kg calcium and 25 mg/kg magnesium). The mixture of calcium and magnesium at specific concentrations seem to be critical for preventing the development of morphine tolerance and dependence.
Iranian Journal Of Medical Sciences (02530716)(3-4)
Background: The cAMP signal transduction systems are known to play a critical role in the acute and chronic effects of ethanol and other drugs of abuse. Objective: To investigate the role of protein kinase C (PKC) in the action of ethanol on platelets and human erythroleukemia (HEL) cells. Methods: HEL, HEK 293 cells [transfected with AC type VII(AC7)] and platelets were used to study the effects of ethanol. cAMP accumulation assay was used to determine the percentage conversion of [3H]ATP into [3H]cAMP. Each fraction was separated through sequential chromatography and the radioactive material was then quantified using a liquid scintillation counter. Results: Ethanol enhanced the PGE1-stimulated AC activity in HEL cells. The percent enhancement of cAMP accumulation by 200 mM ethanol was similar in HEL cells, platelets and HEK 293 cells transfected with AC7. When combined with PDBu, 200 mM ethanol further enhanced the PDBu stimulation of AC activity suggesting a separate mechanism by which ethanol and PDBu exert their effects on the AC. Pretreatment of the cells with staurosporine and chelerythrine significantly blocked the PDBu-and/or ethanol-enhancement of cAMP accumulation. Conclusion: These results indicate a clear role for PKC in the action of ethanol on AC in platelets.
Chronic barbital treatment significant increased the net K+-stimulated uptake of 45Ca2+ in cerebrocortical synaptosomal preparations, 24 h after withdrawal from chronic barbital administration. Basal uptake was not significantly changed. Hippocampal synaptosomal preparations showed a similar pattern, but the increase was not significant. The synaptosomal Ca2+ uptake was not affected by incubation with the dihydropyridine Ca2+ channel antagonist, nitrendipine, in controls or after chronic barbital treatment. Acute administration of a single dose of barbital did not alter the basal or stimulated uptake of 45Ca2+ in cortical synaptosomes, when this was measured 36 h after the barbital administration. Hippocampal slices prepared 24 h after withdrawal from chronic barbital treatment showed a significant increase in K+-stimulated uptake of 45Ca2+, and the basal uptake was significantly decreased. Both changes were prevented by nitrendipine. An increase in the density of dihydropyridine-sensitive binding sites was found in the cerebral cortex. The results indicate that both dihydropyridine-sensitive and insensitive neuronal Ca2+ channels are altered by chronic barbiturate treatment. These changes may be involved in physical dependence on barbiturates. Copyright (C) 1999 Elsevier Science B.V.
Alcoholism: Clinical and Experimental Research (01456008)(1)
Ethanol is known to enhance the activity of adenylyl cyclase (AC) in a number of cells and tissues. Recent work has suggested that the various isoforms of AC show differential sensitivity to ethanol, with Type VII AC being most sensitive. However, the mechanism of action of ethanol is unclear. In the present work, we investigated the effect of ethanol on AC activity in the human erythroleukemia (HEL) cell line, platelets, and AC VII-transfected HEK 293 cells. The HEL cells contain abundant amounts of mRNA for Type VII AC. We found that both ethanol and phorbol dibutyrate (PDBu) treatment enhanced agonist (prostaglandin E1; PGE1)-stimulated AC activity in HEL cells, as well as in platelets and HEK 293 cells transfected with AC VII. Inhibitors of protein kinase C (PKC) blocked the stimulatory effects of both ethanol and PDBu. However, the effects of ethanol and PDBu on AC activity were additive, suggesting that the mechanisms of action of ethanol and PDBu were not identical. Furthermore, a 30-min exposure of HEL cells to ethanol attenuated (desensitized) the ability of ethanol, but not PDBu, to enhance agonist-activated AC activity. On the other hand, a 30-min pretreatment with PDBu attenuated the AC response to the phorbol ester, but not to ethanol; but, after a 20 hr preincubation with phorbol ester, the ability of both PDBu and ethanol to enhance prostaglandin E1-stimulated AC activity was completely eliminated. Finally, pretreatment of HEL cells with pertussis toxin blocked the effect of PDBu, but not ethanol, on AC activity. The results support the involvement of phorbol ester-sensitive PKC(s) in ethanol's enhancement of agonist-activated activity of AC in HEL cells, but suggest that the mechanism of ethanol's action is different from that of PDBu. The findings with pertussis toxin suggest that PDBu activation of PKC(s) may affect AC activity through phosphorylation of a G(i) protein, whereas ethanol may act by promoting phosphorylation of a different substrate (e.g., AC VII).
The competitive antagonists at the N‐methyl‐d‐aspartate (NMDA) receptor, CGP39551 and CGP37849, protected against the barbiturate withdrawal syndrome in mice, as measured by ratings of convulsive behaviour on handling. The effective doses of these compounds were lower than those required to prevent seizures due to NMDA in naive animals; these were in turn lower than those needed to prevent the convulsive effects of the α‐aminobutyric acid (GABA) antagonist, bicuculline. The NMDA‐receptor antagonists did not alter the increase in the incidence of convulsions due to the GABAA antagonist, bicuculline, that is seen during barbiturate withdrawal, although the latencies to these convulsions during barbital withdrawal were significantly increased after CGP39551. Barbiturate withdrawal did not affect the convulsive actions of NMDA, whether measured by the incidence of convulsions or by intravenous infusion. The Bmax for [3H]‐dizocilpine ([3H]‐MK801) binding was significantly increased by chronic barbital treatment in cerebrocortical but not in hippocampal tissues, while the Kd remained unaltered in either case. At 1 h and 24 h after administration of a single dose of barbitone, the Bmax for [3H]‐dizocilpine binding was unaltered in cerebrocortical tissue. Acute addition of barbitone in vitro did not alter [3H]‐dizocilpine binding or the displacement of binding of thienylcyclohexylpyridine. 1994 British Pharmacological Society
1. The effects of the calcium channel blocking agent, nitrendipine, were studied on seizures in mice produced during withdrawal from chronic benzodiazepine treatment and on the development of tolerance to benzodiazepines. 2. Nitrendipine produced a dose-dependent decrease in seizure incidence, when seizures were produced by the partial inverse agonist FG7142 during withdrawal from seven days treatment with flurazepam. 3. Nitrendipine did not raise the seizure thresholds in naive mice to the full inverse agonist methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), or to the γ-aminobutyric acid (GABA) antagonist, bicuculline. 4. When given concurrently with flurazepam for seven days, nitrendipine did not affect the incidence of seizures during flurazepam withdrawal. 5. When given concurrently with the benzodiazepines, nitrendipine did not prevent the development of tolerance to midazolam general anaesthesia or tolerance to the ataxic actions of flurazepam or midazolam. 6. Chronic treatment with flurazepam for seven days did not affect the K(d) or B(max) of [3H]-nimodipine binding in mouse whole brain or cerebral cortex. 7. These results with benzodiazepines are partially in contrast with those for ethanol, where nitrendipine not only decreased ethanol withdrawal seizures when given acutely, but also prevented the development of tolerance and withdrawal signs when given concurrently with ethanol. However, they do confirm the selectivity of nitrendipine for withdrawal-induced seizures.
