Caenorhabditis elegans as an in vivo Model to Assess Amphetamine Tolerance.

Amphetamine is a potent psychostimulant also used to treat attention deficit/hyperactivity disorder and narcolepsy. In vivo and in vitro data have demonstrated that amphetamine increases the amount of extra synaptic dopamine by both inhibiting reuptake and promoting efflux of dopamine through the dopamine transporter. Previous studies have shown that chronic use of amphetamine causes tolerance to the drug. Thus, since the molecular mechanisms underlying tolerance to amphetamine are still unknown, an animal model to identify the neurochemical mechanisms associated with drug tolerance is greatly needed. Here we took advantage of a unique behavior caused by amphetamine in Caenorhabditis elegans to investigate whether this simple, but powerful, genetic model develops tolerance following repeated exposure to amphetamine. We found that at least 3 treatments with 0.5 mM amphetamine were necessary to see a reduction in the amphetamine-induced behavior and, thus, to promote tolerance. Moreover, we found that, after intervals of 60/90 minutes between treatments, animals were more likely to exhibit tolerance than animals that underwent 10-minute intervals between treatments. Taken together, our results show that C. elegans is a suitable system to study tolerance to drugs of abuse such as amphetamines.

Exclusive breastfeeding practice during first six months of an infant's life in Bangladesh: a country based cross-sectional study.

BACKGROUND: Breastfeeding offers incredible health benefits to both child and mother. It is suggested by World Health Organization that an able mother should practice and maintain exclusive breastfeeding for first six months of her infant's life. The objective of this study was to determine the prevalence and factors associated with exclusive breastfeeding for first six months of an infant's life in Bangladesh. METHODS: Data was extracted from Bangladesh Demographic and Health Survey (BDHS-2014). BDHS-2014 collected data from 17,863 Bangladeshi married women in reproductive age from the entire country using two stages stratified cluster sampling. We included only mothers having at least one child currently aged not less than 6 months. Mothers who did not have child to breastfeed, some incomplete information and missing samples were excluded from the data set and consequently 3541 mothers were considered in the present study. Chi-square test, binary logistic regression models were used in this study. RESULTS: The prevalence of exclusive breastfeeding (EBF) for first six months of an infant's life in Bangladesh was 35.90%. Binary multivariable logistic regression model demonstrated that relatively less educated mothers were more likely to exclusively breastfeed their children than higher educated mothers. (AOR = 2.28, 95% CI: 1.05-4.93; p < 0.05). Housewife mothers were more likely to be EBF than their counterparts (AOR = 1.20, 95% CI: 1.02-1.42; p < 0.05). Higher rate of EBF was especially found among mothers who were living in Sylhet division, within 35-49 years old, and had access to mass media, had more than 4 children, had delivered at home and non-caesarean delivery, took breastfeeding counseling, antenatal and postnatal cares. CONCLUSIONS: Stepwise regression model exhibited that most of the important predictors were modifiable factors for exclusive breastfeeding. Authorities should provide basic education on EBF to educated mothers, and organize more general campaign on EBF.

Amphetamine potentiates the effects of ß-phenylethylamine through activation of an amine-gated chloride channel.

ß-Phenylethylamine (ßPEA) is a trace amine present in the CNS of all animals tested to date. However, its function is still not fully understood. ßPEA has been suggested to function as a neurotransmitter and/or to mimic the effect of amphetamine (Amph). In support of the latter is the observation that ßPEA and Amph produce similar but not identical behaviors. Here, we show that ßPEA, like Amph, activates the dopamine transporter and the amine-gated chloride channel LGC-55 to generate behaviors in Caenorhabditis elegans. However, although Amph-induced behaviors occurred gradually during 10 min of treatment, ßPEA induced maximal effects within 1 min. In vitro data demonstrate that ßPEA activates the LGC-55 more efficiently than Amph (Km = 9 and 152 µm, respectively) and generates saturating currents that are 10 times larger than those produced by Amph. These results suggest that activation of LGC-55 mostly accounts for the behavioral effects reached after 1 min of treatment with ßPEA. Importantly, our in vitro and in vivo data show that Amph increases the effects induced by ßPEA on the LGC-55, indicating that Amph potentiates the effects generated by the biogenic amine ßPEA. Together, our data not only identify a new target for ßPEA, but also offer a novel mechanism of action of Amph. In addition, our results highlight C. elegans as a powerful genetic model for studying the effects of biogenic and synthetic amines both at the molecular and behavioral levels.

Novel Use of Ultrasound Elastography to Quantify Muscle Tissue Changes After Dry Needling of Myofascial Trigger Points in Patients With Chronic Myofascial Pain.

OBJECTIVES: To compare a mechanical heterogeneity index derived from ultrasound vibration elastography with physical findings before and after dry-needling treatment of spontaneously painful active myofascial trigger points in the upper trapezius muscle. METHODS: Forty-eight patients with chronic myofascial pain enrolled in a prospective interventional trial of 3 weekly dry-needling treatments for active myofascial trigger points. Trigger points were evaluated at baseline and at treatment completion using palpation, the pressure-pain threshold, and the mechanical heterogeneity index. Thirty patients were reevaluated at 8 weeks. Trigger points that "responded" changed to tissue that was no longer spontaneously painful, with or without the presence of a palpable nodule. Trigger points that "resolved" changed to tissue without a palpable nodule. The mechanical heterogeneity index was defined as the proportion of the upper trapezius muscle that appeared mechanically stiffer on elastography. Statistical significance for comparisons was determined at P < .05. RESULTS: Following 3 dry needle treatments, the mechanical heterogeneity index decreased significantly for the 38 myofascial trigger points (79% of 48) that responded to treatment. Among these, the baseline mechanical heterogeneity index was significantly lower for the 13 trigger points (27% of 38) that resolved, but the decrease after 3 dry needle treatments did not reach significance. The pressure-pain threshold improved significantly for both groups. At 8 weeks, the mechanical heterogeneity index decreased significantly for the 22 trigger points (73% of 30) that responded and for the 10 (45% of 22) that resolved. The pressure-pain threshold improvement was significant for trigger points that responded but did not reach significance for resolved trigger points. CONCLUSIONS: The mechanical heterogeneity index identifies changes in muscle tissue properties that correlate with changes in the myofascial trigger point status after dry needling.

Association of CD58 rs12044852 and rs2300747 polymorphisms with the risk of multiple sclerosis: A systematic review and meta-analysis.

OBJECTIVE: Multiple sclerosis is a serious neurodegenerative disorder that causes disability in young adults. Genetic predisposition of multiple sclerosis is well documented and several single nucleotide polymorphisms (SNPs) of the CD58 were found to be associated with this disease. This systematic review and meta-analysis were done with the aim of finding the association between CD58 gene SNPs (rs12044852 and rs2300747) and the risk of multiple sclerosis (MS). METHOD: A comprehensive search was done in PubMed, Google Scholar, Embase, and MSGene.org to find the relevant data. Our search yielded 13 relevant publications which were included for meta-analysis consisting of 5194 cases and 5766 controls. All the statistical analysis was conducted using meta and metafor packages in R studio. The odds ratio (OR) along with 95 % confidence intervals and p values were determined using the fixed effects and random effects model. The I2 test was done to measure heterogeneity. Subgroup analysis was performed along with analysis for publication bias. RESULTS: We found significant association for both rs12044852 (allelic, dominant, over-dominant, heterozygous, and homozygous models) and rs2300747 (allelic, dominant, over-dominant, heterozygous models) with multiple sclerosis. Both the SNPs provided a protective effect for multiple sclerosis. Subgroup analysis indicated that rs12044852 polymorphism provided a protective effect in both Asians and Caucasians. However, for rs2300747, the Asian population showed no statistically significant association with the risk of MS. CONCLUSION: Polymorphism of rs12044852 and rs2300747 of the CD58 gene provided a protective effect for multiple sclerosis. The protective effect is more prominent in Caucasian populations compared to Asians.

Effect of cooking methods on the nutritional quality of selected vegetables at Sylhet City.

The study was carried out to analyze the impacts of boiling, steaming, and microwave cooking on the physicochemical properties, the content of bioactive compounds, and boiling effect on mineral and heavy metal content of six widely consumed vegetables in Bangladesh's north-eastern region. In comparison to raw, boiled, and microwave-cooked vegetables, those that are steam-cooked retain a higher percentage of ß-carotene with the exception of carrots. Boiling vegetables led to the most substantial reduction in ascorbic acid content (from 9.83 % to 70.88 %), with spinach experiencing the greatest decline. In contrast, microwaving had the mildest effect on ascorbic acid, preserving over 90 % of the initial content. The decrease in carotene content may be associated with color changes (decreasing greenness and increasing hue angle) in the chosen vegetables. The colorimeter shows the L* value (lightness/darkness) of all cooked vegetables significantly decreased. In terms of total polyphenol content (TPC) and total flavonoid content (TFC), boiling had a higher negative effect on most vegetables than the other two cooking methods, with losses of up to 70.3 % and 82.27 %, respectively. All cooked vegetables, with the exception of carrot and microwave pumpkin, had substantial reductions in free radicals scavenging activity, with losses ranging from 8.48 % to 56.73 %. In comparison to raw vegetables, boiled vegetables significantly lost minerals like potassium (K), magnesium (Mg), zinc (Zn), copper (Cu), and manganese (Mn). On the other hand, the calcium (Ca) and iron (Fe) content of all cooked vegetables, except for carrots and peas, exhibited an increase, ranging from 6 to 17 % and 6-12 %, respectively. The Cr concentration in all vegetables and the Zn, Fe, Mn, and Cd content in the spinach sample was higher than the FAO/WHO recommended maximum permissible level (MPL), whereas the accumulation of Cu and Ni content was lower in all vegetables. In conclusion, this study demonstrated that microwaving was the most effective method for retaining the nutritional value of vegetables, while steaming had a moderate impact.

Polymorphisms of the interleukin-6 (IL-6) gene contribute to cervical cancer susceptibility in Bangladeshi women: A case-control study.

Background and Aims: Cervical cancer is characterized by abnormal cell growth in the lining of cervix and it is the second major cause of cancer-related deaths among females in Bangladesh. Interleukin-6 (IL-6) is a multifunctional cytokine that has been heavily linked with cervical cancer. Our aim was to investigate the association of two promoter single-nucleotide polymorphisms (SNPs) of IL-6 (rs1800795 and rs1800797) with the susceptibility of cervical cancer in Bangladeshi women. Methods: DNA was extracted from venous blood samples from cervical cancer patients (n = 126) and healthy controls (n = 120). Polymerase chain reaction-restriction fragment length polymorphism was used for genotyping of the selected SNPs. Logistic regression was performed to calculate the odds ratio (OR) with 95% confidence interval (CI) and p values. Results: We found a significant association between rs1800795 and rs1800797 polymorphisms and cervical cancer. For, rs1800795 (G > C) the GC heterozygous genotype (OR = 2.80, 95% CI = 1.55-5.07, p = 0.0007) and CC mutant homozygous genotype (OR = 3.5, 95% CI = 1.29-9.51, p = 0.014) conferred an increased risk of cervical cancer. In case of rs1800797 (G > A) polymorphism, the AG heterozygous genotype (OR = 6.94, 95% CI = 3.76-12.81, p < 0.0001) and AA mutant homozygous genotype (OR = 3.88, 95% CI = 1.12-13.51, p = 0.0332) also exhibited an elevated risk of cervical cancer. Use of contraceptives was found as risk factor and patients who smoke were carriers of both the risk alleles and thus had an increased risk of cervical cancer. Conclusion: Our findings suggest that polymorphism of rs1800795 and rs1800797 of the IL-6 gene play a significant role in cervical cancer susceptibility in Bangladeshi women.

Microbial sensitivity of the common pathogens for UTIs are declining in diabetic patients compared to non-diabetic patients in Bangladesh: An institution-based retrospective study.

Background: Urinary tract infection (UTI) is one of the most recurrent infections in the community and healthcare settings. Although many studies related with microbial sensitivity (MS) of uropathogens (UPs) to antibiotics have been done in Bangladesh, no conclusive study has compared antibiotic sensitivity (AS) to UPs in diabetic and non-diabetic patients. The aim of the study is to find out whether there is a difference in AS in common UPs between diabetic and non-diabetic UTI patients. Methods: A retrospective review was conducted on 833 patients. The data was collected from different diagnostic centers located within Dhaka city in Bangladesh, and the data was analyzed using convenient statistical tools. Results: We have studied a total of 833 UTI patients. Out of 833 patients, 664 were diabetic and 169 were non-diabetic patients respectively. Among the studied population, females were found to be more inclined to have UTIs as compared to males. E. coli was found to be the leading UPs in our study. Patients within the age of 20-34 were more vulnerable to UTI in both groups. Imipenem and meropenem showed 100% sensitivity against E. coli, Staphylococcus and Klebsiella in non-diabetic patients, while both antibiotics showed lower sensitivity to the same organisms in diabetic patients. Antibiotics like nitrofurantoin (p ≤ 0.0002), ceftazidime (p ≤ 0.0124) and ceftriaxone (p ≤ 0.0168) showed less sensitivity to E. coli in diabetic UTI patients as compared to non-diabetic UTI patients. Overall sensitivity patterns elucidated that all the studied antibiotics, except ciprofloxacin and levofloxacin, showed lower sensitivity against UPs in diabetic while compared to non-diabetic UTI patients (p= <0.05 to 0.0001). Conclusion: We found significant difference in microbial sensitivity in patients with diabetes compared to non-diabetic UTI patients. Diabetes changes the pathophysiological state of the uropathogens leading to the declining sensitivity of the antibiotics in diabetic patients with UTIs.

Coenzyme Q10 ameliorates carbofuran induced hepatotoxicity and nephrotoxicity in wister rats.

Carbofuran is a widely used poisonous pesticide around the world that helps to control insects during farming. Upon oral ingestion to humans, it exaggerates oxidative stress in various organs like the liver, brain, kidney, and heart. Several studies reported that oxidative stress in the liver initiates and propagates hepatic cell necrosis, ultimately resulting in hepatotoxicity. It also reported that coenzyme Q10 (CoQ10) can neutralize oxidative stress due to its antioxidant properties. However, the hepatoprotective and nephroprotective role of CoQ10 against carbofuran toxicity has not been investigated. Therefore, the present study aimed to evaluate the hepatoprotective and nephroprotective role of CoQ10 in carbofuran-induced hepatotoxicity and nephrotoxicity in a mouse model for the first time. We determined the blood serum diagnostic markers, oxidative stress parameters, antioxidant system, and histopathological characteristics of liver and kidney tissues. The administration of 100 mg/kg of CoQ10 in carbofuran-treated rats significantly attenuated AST, ALT, ALP, serum creatinine, and BUN levels. Moreover, CoQ10 (100 mg/kg) remarkably altered the level of NO, MDA, AOPP, GSH, SOD, and CAT in both the liver and kidney. The histopathological data also unveiled that CoQ10 treatment prevented inflammatory cell infiltration in carbofuran-exposed rats. Therefore, our findings infer that CoQ10 may effectively protect liver and kidney tissues against carbofuran-induced oxidative hepatotoxicity and nephrotoxicity.

Post-market quality assessment of 22 ciprofloxacin brands by HPLC available in Bangladesh market.

Antibiotic resistance has been recognized as a public health threat in recent years, and mortality due to resistance is increasing alarmingly every year. Antibiotic resistance, among many factors, may arise due to the consumption of substandard antibiotic brands that provide subnormal levels of the drug in the blood. Post-market evaluation can provide important information in assessing pharmaceutical products in terms of quality, purity, and therapeutic aspects. Ciprofloxacin, a broad-spectrum antibiotic, has been used against a wide range of infectious diseases in Bangladesh. The present study aimed to determine the quality attributes of twenty-two commonly prescribed brands of ciprofloxacin 500 mg tablet collected from Dhaka city and the rural regions of Jessore. RP-HPLC coupled with UV-visible spectrophotometry was used to determine the potency of ciprofloxacin in tablets, and the zone of inhibition was determined using Kirby-Bauer's disc diffusion method to assess the antimicrobial efficacy against different strains of microorganisms. We found that 95.45% of brands (21 out of 22 brands) of ciprofloxacin tablets met United States Pharmacopoeia (USP) and British Pharmacopoeia (BP) specified potency, whereas one brand failed. From dissolution studies, we observed that 68.2% of brands (15 out of 22 brands) followed USP/NF dissolution test specifications, whereas 31.8% (7 out of 22 brands) failed to release 80% of the labeled amount of drug within 30 min. Drug release kinetics data showed that most brands followed the Weibull drug release kinetic model. Fit factor analysis exhibited that 8 brands out of 22 (36.4%) failed to comply similar dissolution profiles with the reference product. Minimum inhibitory concentrations, assessed against five bacterial strains, further showed good antimicrobial sensitivity by all brands.

Practical access to four stereoisomers of naftidrofuryl and their binding affinity towards 5-hydroxytryptamine 2A receptor.

Naftidrofuryl oxalate (Praxilene®, 1) has been used for the treatment of intermittent claudication for more than 30 years. It selectively blocks vascular and platelet 5-hydroxytryptamine 2 (5-HT(2)) receptors. This drug is marketed as a mixture of four stereoisomers, and so far there is no individual biological evaluation on the single isomers. The purpose of this study is to provide an improved method for the preparation of all four stereoisomers of naftidrofuryl, and more importantly, to distinguish them in terms of their binding affinity to 5-hydroxytryptamine 2A (5-HT(2A)) receptor. The bioassay results revealed that the C-2S configuration of naftidrofuryl was crucial for the binding affinity with 5-HT(2A) receptor, and the C-2' configuration was less important for binding. In conclusion, our study may pave the way to develop single naftidrofuryl isomers with C-2S configuration as inhibitors of 5-HT(2A) receptor that have clinical significance as vasodilators and CNS agents.

Mechanism of inverse agonist action of sarpogrelate at the constitutively active mutant of human 5-HT2A receptor revealed by molecular modeling.

We previously reported that sarpogrelate, a selective 5-HT2A antagonist, showed a potent inverse agonist activity to constitutively active mutant (C322K) of human 5-HT2A receptor (5-HT2AR). However, it remains to be unknown about the actual mechanism of this mutant for its constitutive activation as well as inverse agonist activity of sarpogrelate. Our model shows that mutation (C322K) of 5-HT2AR causes electronic repulsion between positively charged Arg173(3.50) and Lys322(6.34) residues resulting outward movement of the C-terminus of transmembrane helix (TMH) III. This motion of TMH III leads to a partially active structure of the receptor, which may be a key step in receptor activation. The structural model of the partially active receptor also indicates that the binding of sarpogrelate to the constitutively active receptor causes an inward swing of TMH III to an inactive receptor structure. Therefore, the present study may suggest that the electronic repulsion causing outward movement of the C-terminus of TMH III may be the key step for constitutive activation of mutant C322K of 5-HT2AR and the inward movement of TMH III causes the inverse agonist activity of sarpogrelate.

Curcumin improves D-galactose and normal-aging associated memory impairment in mice: In vivo and in silico-based studies.

Aging-induced memory impairment is closely associated with oxidative stress. D-Galactose (D-gal) evokes severe oxidative stress and mimics normal aging in animals. Curcumin, a natural flavonoid, has potent antioxidant and anti-aging properties. There are several proteins like glutathione S-transferase A1 (GSTA1), glutathione S-transferase omega-1 (GSTO1), kelch-like ECH-associated protein 1 (KEAP1), beta-secretase 1 (BACE1), and amine oxidase [flavin-containing] A (MAOA) are commonly involved in oxidative stress and aging. This study aimed to investigate the interaction of curcumin to these proteins and their subsequent effect on aging-associated memory impairment in two robust animal models: D-Gal and normal aged (NA) mice. The aging mice model was developed by administering D-gal intraperitoneally (i.p). Mice (n = 64) were divided into the eight groups (8 mice in each group): Vehicle, Curcumin-Control, D-gal (100mg/kg; i.p), Curcumin + D-gal, Astaxanthin (Ast) + D-gal, Normal Aged (NA), Curcumin (30mg/kg Orally) + NA, Ast (20mg/kg Orally) + NA. Retention and freezing memories were assessed by passive avoidance (PA) and contextual fear conditioning (CFC). Molecular docking was performed to predict curcumin binding with potential molecular targets. Curcumin significantly increased retention time (p < 0.05) and freezing response (p < 0.05) in PA and CFC, respectively. Curcumin profoundly ameliorated the levels of glutathione, superoxide dismutase, catalase, advanced oxidation protein products, nitric oxide, and lipid peroxidation in mice hippocampi. In silico studies revealed favorable binding energies of curcumin with GSTA1, GSTO1, KEAP1, BACE1, and MAOA. Curcumin improves retention and freezing memory in D-gal and nature-induced aging mice. Curcumin ameliorates the levels of oxidative stress biomarkers in mice. Anti-aging effects of curcumin could be attributed to, at least partially, the upregulation of antioxidant enzymes through binding with GSTA1, GSTO1, KEAP1, and inhibition of oxidative damage through binding with BACE1 and MAOA.

L-carnitine protects cardiac damage by reducing oxidative stress and inflammatory response via inhibition of tumor necrosis factor-alpha and interleukin-1beta against isoproterenol-induced myocardial infarction.

BRIEF INTRODUCTION: Myocardial infarction (MI) is a common manifestation of certain cardiac diseases where oxidative stress and fibrosis aggravate the condition markedly. MAIN OBJECTIVE OF THE STUDY: Investigation of L-carnitine's cardioprotective roles and mechanism of action in a rat model of MI. METHODS: To develop a MI animal model, Isoproterenol (ISO) was administered in male Long Evans rats where animals were divided into five groups (six rats/group). The oxidative stress and antioxidant enzyme activities were determined by different biochemical tests. The real-time PCR was performed to determine the expression of TNF-α and Il-1ß. Histopathological observations by hematoxylin-eosin and Masson trichrome were made to observe the tissue damage and fibrosis in heart and kidney. SIGNIFICANT FINDINGS FROM THE STUDY: The ISO-treated rats showed increased levels of troponin I and lipid peroxidation and lower antioxidant enzyme activity in heart and kidney tissues. The levels of TNF-α and IL-1ß were also increased in ISO-rats. Co-administration of L-carnitine with ISO reversed all these parameters. The elevated levels of uric acid and creatinine kinase and ALP, AST and ALT activities in ISO-rats were also significantly reduced by L-carnitine administration. L-carnitine markedly decreased the infiltration of inflammatory cells and improved the tissue architecture in heart and kidney. Control animals did not show any appreciable response upon L-carnitine administration. RELEVANT CONTRIBUTION TO KNOWLEDGE: These results suggest that L-carnitine plays a defensive role against cardiac and renal damage in ISO-treated MI rat model via suppressing oxidative stress and increasing antioxidant enzyme functions through inhibition of TNF-α and IL-1ß.

Remote Temperature-Responsive Parafilm Dermal Patch for On-Demand Topical Drug Delivery.

The development of externally controlled drug delivery systems that can rapidly trigger drug release is widely expected to change the landscape of future drug carriers. In this study, a drug delivery system was developed for on-demand therapeutic effects. The thermoresponsive paraffin film can be loaded on the basis of therapeutic need, including local anesthetic (lidocaine) or topical antibiotic (neomycin), controlled remotely by a portable mini-heater. The application of mild temperature (45 °C) to the drug-loaded paraffin film allowed a rapid stimulus response within a short time (5 min). This system exploits regular drug release and the rapid generation of mild heat to trigger a burst release of 80% within 6 h of any locally administered drug. The in vitro drug release studies and in vivo therapeutic activity were observed for local anesthesia and wound healing using a neomycin-loaded film. The studies demonstrated on-demand drug release with minimized inflammation and microbial infection. This temperature-responsive drug-loaded film can be triggered remotely to provide flexible control of dose magnitude and timing. Our preclinical studies on these remotely adjustable drug delivery systems can significantly improve patient compliance and medical practice.

Therapeutic Potentials of Colocasia affinis Leaf Extract for the Alleviation of Streptozotocin-Induced Diabetes and Diabetic Complications: In vivo and in silico-Based Studies.

INTRODUCTION: Hypoglycemia in diabetes mellitus (DM) correlates with hepatic impairment, nephropathy, lipid abnormalities, and oxidative stress and subsequently complicates the disease pathogenesis. Medicinal plants have been used for the management of diabetes since ancient times. In this study, we explored the potentials of Colocasia affinis (CA), a plant known to possess anti-allergic and anti-inflammatory activities, as a remedy for diabetes and related complications. METHODS: We induced diabetes in rats using a single intraperitoneal dose (65 mg/kg) of streptozotocin (STZ). We next treated the rats with an ethanolic extract of leaves of CA to reveal its antidiabetic and organ-protective potentials. Biomarkers of diabetes, inflammation, and oxidative stress were measured using biochemical and histopathological analysis. We also performed molecular docking for three major phytochemicals (kaempferol, myricetin, and rosmarinic acid) of CA. RESULTS: Oral administration of the CA leaves extract at 250 mg/kg and 500 mg/kg doses decreased blood glucose level significantly (p<0.05) in STZ-induced diabetic rats. The extract also considerably attenuated plasma HbA1c levels and normalized blood lipids, glycogen, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). Additionally, treatment with the extract improved kidney complications by decreasing serum creatinine and blood urea nitrogen (BUN) levels. Furthermore, CA leaves extract normalized nitric oxide (NO) and advance oxidative protein products (AOPP) in diabetic rats. The extract also showed significant improvement of the antioxidant enzymes glutathione dismutase (GSH) and superoxide dismutase (SOD) at a dose of 500 mg/kg. Besides, histological investigation demonstrated attenuation of inflammation of the vital organs, including the liver and the kidney. In silico studies revealed that three major phytochemicals (kaempferol, myricetin, and rosmarinic acid) of the ethanolic extract of leaves of CA can inhibit several molecular targets of diabetes and inflammation. CONCLUSION: Collectively, our results demonstrated the therapeutic potentials of CA for the mitigation of diabetes and diabetic complications.

Engineered mutation of some important amino acids in angiotensin II type 1 (AT1) receptor increases the binding affinity of AT1-receptor antagonists.

The present study was designed to examine the binding affinity and functional potency of selective angiotensin II type 1 (AT(1))-receptor antagonists towards specific mutants of AT(1) receptor using site-directed mutagenesis. We also compared our results with the wild-type AT(1) receptor and investigated the possible reasons behind that. Both wild-type and mutant receptors were expressed in COS-7 cells and the binding affinities of the antagonists were determined by radioligand binding assay. Inhibition of agonist-stimulated inositol phosphate accumulation by the antagonists was also done. Substitution of asparagine(235) of intracellular loop 3 of the AT(1) receptor by arginine increased the binding affinity of the antagonists 5 - 34-fold, whereas the increase in the binding affinity of the antagonists in the phenylalanine(239) mutant by arginine and tryptophan (F239R and F239W) were 3 - 19-fold and 2 - 15-fold higher, respectively, compared to the wild-type AT(1) receptor. The results suggested that substitution by a positively charged or sterically hindered amino acid in the AT(1) receptor allows it to interact with the acidic tetrazole moiety and carboxylate groups of the antagonists more strongly compared to the wild-type receptor. These findings may play an important role to change the binding affinity of the antagonists to an effective level for the pharmacological function of the drugs.

Internalization of constitutively active N111G MUTANT of AT1 receptor induced by angiotensin II-receptor antagonists candesartan, losartan, and telmisartan: comparison with valsartan.

Based on radioligand binding and signal transduction assays in our previous study, we have determined the binding pattern and functional efficacy of the constitutively active mutant N111G of angiotensin II type 1 (AT(1)) receptor. We have also shown that the N111G mutant induces homologous internalization through mediation of the AT(1)-receptor antagonist valsartan. In this study we demonstrated that other AT(1)-receptor antagonists, candesartan, losartan, and telmi-sartan, also stimulate internalization of N111G mutant receptor to the same extent. We further showed that the internalization pattern is also similar for all the AT(1)-receptor antagonists.

Constitutively active mutant N111G of angiotensin II type 1 (AT(1)) receptor induces homologous internalization through mediation of AT(1)-receptor antagonist.

The present study investigated the internalization behavior of the constitutively active mutant (CAM) N111G of angiotensin II type 1 (AT(1)) receptor and correlated the result with the mechanism of the constitutive activity of the mutant. The inverse agonist activity of valsartan, losartan, candesartan, and telmisartan was also examined by inositol phosphate (IP) accumulation study as well as receptor-internalization assay. Both wild-type (WT) and N111G mutant receptors were transiently expressed in COS-7 cells and the binding affinities towards the agonist and these four AT(1) antagonists were determined. Production of total IP was measured in the presence and absence of the compounds. The agonist-induced receptor internalization of both WT and N111G mutant receptors was also investigated. Although the mutant showed similar binding characteristics with agonist and the antagonists used as WT, the internalization of the mutant was much lower (19.56 +/- 2.87%) than that of the WT receptor (74.63 +/- 1.00%). Internalization of the mutant significantly increased (63.22 +/- 0.03%) in the presence of valsartan, which also showed significant inverse agonist activity in the N111G mutant. The results indicate that internalization of CAM N111G of the AT(1) receptor is induced by the use of valsartan, which may be an important characteristic of inverse agonist activities of AT(1) antagonists in N111G.

Levocarnitine Improves AlCl3-Induced Spatial Working Memory Impairment in Swiss albino Mice.

Background: Aluminum, a neurotoxic substance, causes oxidative stress induced-neurodegenerative diseases. Several lines of evidence suggest that levocarnitine has an antioxidant effect and also plays an important role in beta-oxidation of fatty acids. However, the role of levocarnitine in aluminum-induced neurotoxicity has not been well documented. Here we aimed to investigate the effect of levocarnitine on aluminum chloride (AlCl3)-induced oxidative stress and memory dysfunction. Methods: Male Swiss albino mice (n = 30) were treated with either control (saline) or AlCl3 or AlCl3 plus levocarnitine or levocarnitine or astaxanthin plus AlCl3 or astaxanthin alone. The spatial working memory was determined by radial arm maze (RAM). In addition, we measured the lipid peroxidation (MDA), glutathione (GSH), advanced oxidation of protein products (AOPP), nitric oxide (NO) and activity of superoxide dismutase (SOD) in the various brain regions including prefrontal cortex (PFC), striatum (ST), parietal cortex (PC), hippocampus (HIP) hypothalamus (HT) and cerebellum (CB). We used astaxanthin as a standard antioxidant to compare the antioxidant activity of levocarnitine. Results: The RAM data showed that AlCl3 treatment (50 mg/kg) for 2 weeks resulted in a significant deficit in spatial learning in mice. Moreover, aluminum exposure significantly (p < 0.05) increased the level of oxidative stress markers such as MDA, GSH, AOPP and NO in the various brain regions compared to the controls. In addition, combined administration of levocarnitine and AlCl3 significantly (p < 0.05) lowered the MDA, AOPP, GSH and NO levels in mice. Conclusion: Our results demonstrate that levocarnitine could serve as a potential therapeutic agent in the treatment of oxidative stress associated diseases as well as in memory impairment.