Sugato Banerjee

Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding.

BackgroundCalcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide whose biological activity has potential therapeutic value for many vascular related diseases. CGRP is a 37 amino acid neuropeptide that signals through a G protein-coupled receptor belonging to the secretin receptor family. Previous studies on the calcitonin-like receptor (CLR), which requires co-expression of the receptor-activity-modifying protein-1 (RAMP1) to function as a CGRP receptor, have shown an 18 amino acid N-terminus sequence important for binding CGRP. Moreover, several investigations have recognized the C-terminal amidated phenylalanine (F37) of CGRP as essential for docking to the mature receptor. Therefore, we hypothesize that hydrophobic amino acids within the previously characterized 18 amino acid CLR N-terminus domain are important binding contacts for the C-terminal phenylalaninamide of CGRP.ResultsTwo leucine residues within this previously characterized CLR N-terminus domain, when mutated to alanine and expressed on HEK293T cells stably transfected with RAMP1, demonstrated a significantly decreased binding affinity for CGRP compared to wild type receptor. Additional decreases in binding affinity for CGRP were not found when both leucine mutations were expressed in the same CLR construct. Decreased binding characteristic of these leucine mutant receptors was observed for all CGRP ligands tested that contained the necessary amidated phenylalanine at their C-terminus. However, there was no difference in the potency of CGRP to increase cAMP production by these leucine mutant receptors when compared to wild type CLR, consistent with the notion that the neuropeptide C-terminal F37 is important for docking but not activation of the receptor. This observation was conserved when modified CGRP ligands lacking the amidated F37 demonstrated similar potencies to generate cAMP at both wild type and mutant CLRs. Furthermore, these modified CGRP ligands displayed a significant but similar loss of binding for all leucine mutant and wild type CLR because the important receptor contact on the neuropeptide was missing in all experimental situations.ConclusionThese results are consistent with previous structure-function investigations of the neuropeptide and are the first to propose specific CLR binding contacts for the amidated F37 of CGRP that are important for docking but not activation of the mature CGRP receptor.

Isobaric tagging-based quantification by mass spectrometry of differentially regulated proteins in synaptosomes of HIV/gp120 transgenic mice: implications for HIV-associated neurodegeneration.

HIV/gp120 transgenic mice manifest neuropathological features similar to HIV-associated neurocognitive disorders (HAND) in humans, including astrogliosis, microglia activation, and decreased neuronal synapses. Here, proteomic screening of synaptosomes from HIV/gp120 transgenic mice was conducted to determine potential neuronal markers and drug targets associated with HAND. Synaptosomes from 13 month-old wild-type (wt) and HIV/gp120 transgenic mouse cortex were subjected to tandem mass tag (TMT) labeling and subsequent analysis using an LTQ-Orbitrap mass spectrometer in pulsed-Q dissociation (PQD) mode for tandem mass spectrometry (MS/MS). A total of 1301 proteins were identified in both wt and HIV/gp120 transgenic mice. Three of the most differentially-regulated proteins were validated by immunoblotting. To elucidate putative pathways associated with the proteomic profile, 107 proteins manifesting a ≥1.5 fold change in expression were analyzed using a bioinformatics pathway analysis tool. This analysis revealed direct or indirect involvement of the phosphotidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, a well-known neuronal survival pathway. Immunoblots confirmed a lower phospho (p)Akt/Akt ratio in synaptosomes from HIV/gp120 transgenic animals compared to wt, suggesting that this neuroprotective pathway was inactivated in the HIV/gp120 transgenic brain. Based on this information, we then compared immunoblots of pAkt/Akt in the forebrains of these mice as well as in human postmortem brain. We observed a significant decrease in the pAkt/Akt ratio in synaptosomes and forebrain of HIV/gp120 transgenic compared to wt mice, and a similar decrease in human forebrain from HAND patients compared to neurologically unimpaired HIV+ and HIV- controls. Moreover, mechanistic insight into an additional pathway for decreased Akt activity in HIV/gp120 mouse brains and human HAND brains was shown to occur via S-nitrosylation of Akt protein, a posttranslational modification known to inhibit Akt activity and contribute to neuronal cell injury and death. Thus, MS proteomic profiling in the HIV/gp120 transgenic mouse predicted dysregulation of the PI3K/Akt pathway observed in human brains with HAND, providing evidence that this mouse is a useful disease model and that the Akt pathway may provide multiple drug targets for the treatment of HIV-related dementias.

Memory enhancement by Tamoxifen on amyloidosis mouse model

Tamoxifen (TMX) is a selective estrogen receptor modulator (SERM) used in the treatment of breast cancer. Earlier studies show its neuroprotection via regulating apoptosis, microglial functions, and synaptic plasticity. TMX also showed memory enhancement in ovariectomized mice, and protection from amyloid induced damage in hippocampal cell line. These reports encouraged us to explore the role of TMX in relevance to Alzheimers disease (AD). We report here, the effect of TMX treatment a) on memory, and b) levels of neurotransmitters (acetylcholine (ACh) and dopamine (DA)) in breeding-retired-female mice injected with beta amyloid1-42 (Aβ1-42). Mice were treated with TMX (10mg/kg, i.p.) for 15 days. In Morris water maze test, the TMX treated mice escape latency decreased during training trials. They also spent longer time in the platform quadrant on probe trial, compared to controls. In Passive avoidance test, TMX treated mice avoided stepping on the shock chamber. This suggests that TMX protects memory from Aβ induced toxicity. In frontal cortex, ACh was moderately increased, with TMX treatment. In striatum, dopamine was significantly increased, 3,4-dihydroxyphenylacetic acid (DOPAC) level and DOPAC/DA ratio was decreased post TMX treatment. Therefore, TMX enhances spatial and contextual memory by reducing dopamine metabolism and increasing ACh level in Aβ1-42 injected-breeding-retired-female mice.

Abiraterone acetate in the treatment of prostate cancer

Among all cancer-related death, prostate cancer accounts for the second prominent reason for cancer-associated death in men. Despite the castration mediated reduction in testosterone synthesis, adrenal glands, as well as tissues of prostate cancer, continue to produce androgens, which ultimately lead to the growth of prostate cancer. This phase is referred as metastatic castration-resistant prostate cancer, which throws an obstacle to treatment. Androgen antagonists, in addition to deprivation of hormone, is being used for reducing the level of prostate-specific antigen but has not successfully come in front as a choice for prolonging the life of patients suffering from prostate cancer. In this prevailing scenario, abiraterone acetate (AA) has proved to be a boon for patients suffering from prostate cancer. AA selectively inhibits the actions of enzymes C17, 20-lyase and 17α-hydroxylase on cytochrome P450 (CYP) 17 when administered orally. The signaling of androgen receptor, being important for primary to metastatic phases of prostate cancer, CYP17 is essential for the synthesis of androgen. Herein, the in-detail pharmacological profile of AA, including androgen signaling, mechanism of action of AA, mechanism of AA resistance, pharmacokinetics, latest clinical findings, predictive markers, optimal treatment sequence, toxicity, and food interaction profiles have been reviewed.

Compounds of Natural Origin and Acupuncture for the Treatment of Diseases Caused by Estrogen Deficiency

A predominant number of diseases affecting women are related to female hormones. In most of the cases, these diseases are reported to be associated with menstrual problems. These diseases affect female reproductive organs such as the breast, uterus, and ovaries. Estrogen is the main hormone responsible for the menstrual cycle, so irregular menstruation is primarily due to a disturbance in estrogen levels. Estrogen imbalance leads to various pathological conditions in premenopausal women, such as endometriosis, breast cancer, colorectal cancer, prostate cancer, poly cysts, intrahepatic cholestasis of pregnancy, osteoporosis, cardiovascular diseases, obesity, etc. In this review, we discuss common drug targets and therapeutic strategies, including acupuncture and compounds of natural origin, for the treatment of diseases caused by estrogen deficiency.

Farnesyl Transferase Inhibitors as Potential Anticancer Agents

Farnesyl Transferase is a hetero-dimer transferase that targets Ras proteins and attaches a farnesyl group to it. This Ras protein, on localization to the cell membrane, has the ability to induce activation of various growth and proliferation pathways of the cell. Over-activation of mutated Ras may lead to the development of cancer. Farnesyl Transferase catalyses the initial step in the posttranslational modification of normal as well as mutated Ras gene, thus facilitating its tethering to the cell membrane. Inhibition of Farnesyl Transferase is the main step in restricting the activity of mutant Ras protein. Thus the above enzyme has emerged as a novel target for anti-cancer agents. Here we review the role of Farnesyl Transferase in tumorigenesis and various compounds of synthetic and natural origin acting as Farnesyl Transferase inhibitors as potential anti-cancer agents.

Effect of Exercise on Type 2 Diabetes associated Cognitive Impairment in rats

Abstract OBJECTIVES: Type 2 diabetes (T2D)-associated cognitive impairment is highly prevalent especially among the geriatric population. Here, we investigate the role of exercise in T2D-associated cognitive decline in rats. METHODS: T2D was induced using high-fat diet (15 days) followed by low-dose STZ (25mg/kg). The T2D animals were subjected to aerobic exercise on running wheel for 6 weeks. Effect of aerobic exercise on cognitive performance of T2D animals was measured using step-down and transfer latency tests. This was followed by the measurement of reduced glutathione levels in hippocampal homogenates. We also measured hippocampal AchE activity and levels of neuroinflammatory markers such as IL-1 β, TNF-α and MCP-1. Morphology and density of hippocampal neurons were also determined by histopathological studies. RESULTS: Exercise led to the following changes in T2D animals. It led to decrease in fasting blood glucose level (<250 mg/kg) and HbA1c (8.5 ± 0.23) compared to diabetic (11.73 ± 0.14) animals and improved insulin resistance. There was an increase in step-down latency (p < 0.001) and a decrease in transfer latency (p < 0.01) suggesting improved cognitive function. A significant increase in GSH levels (1.828 ± 0.024) compared to diabetic group (1.52 ± 0.03; p < 0.001) and decrease in AchE activity (1.4 ± 0.05) compared to diabetic group (1.65 ± 0.03; p < 0.05) were also observed. It reduced the levels of neuroinflammatory markers such as IL-1β, TNF-α and MCP-1 (p < 0.01). Hippocampal sections showed higher CA1 and CA3 neuronal density (p < 0.001) than T2D group. CONCLUSION: We may conclude that aerobic exercise could partially reverse diabetes-associated cognitive decline by reducing oxidative stress and inflammatory milieu in T2D animal brain.

Effect of Metabolic Syndrome on Anxiety in Mice

Metabolic syndrome is a combination of obesity, dyslipidemia, insulin resistance and hypertension. Clinical evidence indicated the coexistence of metabolic syndrome and depression. However, relatively few studies have been attempted to determine the pathogenesis of metabolic syndrome-associated anxiety. In the present study, the role of metabolic syndrome in the development of anxiety and the role of neurotransmitters in metabolic syndrome-associated anxiety were evaluated in Swiss albino mice. A high fat and high carbohydrate diet was used to develop the metabolic syndrome in mice while monitoring elevated fasting blood glucose, hyperlipidemia, and hypertension. Anxiety levels were measured using elevated plus maze and marble burying test with corresponding determination of serum corticosterone levels. The role of γ-aminobutyric acid and serotonin in metabolic syndrome-associated anxiety were also evaluated. The high fat and high carbohydrate fed animals developed metabolic syndrome, characterized by significant high fasting blood glucose and insulin resistance compared to controls. These animals also had significant increase in body weight and waist circumference, low-density lipoprotein and triglyceride levels, reduced high-density lipoprotein content and high blood pressure. Both metabolic syndrome and water avoidance-anxiety groups spent significantly lower time and showed fewer entries into the open arm of elevated plus maze. They also buried more marbles, thus showing clear signs of anxiety when compared to controls. The corticosterone level in metabolic syndrome and anxiety-induced animals were higher than controls. GABA agonists showed a dose-dependent reduction in metabolic syndrome-associated anxiety as revealed by more time spent on the open arm of plus maze with a corresponding decrease in plasma corticosterone levels. Metabolic syndrome animals spontaneously developed anxiety-like behavior. GABA agonists partially reversed the metabolic syndrome-associated anxiety, suggesting a role for GABAergic pathway.

Effect of shankhpushpi on alcohol addiction in mice

Alcohol addiction is a worldwide problem. It has mainly two components: dependence and withdrawal. Characteristic properties of most anti-addictive compounds include anti-anxiety, anticonvulsant, antidepressant, and nootropic actions. Shankhpushpi (Convolvulus pluricaulis. Convolvulaceae), known ethnopharmacologically as brain tonic, possess all the properties mentioned above. Here, we screen shankhpushpi for possible anti-addictive potential. Effect of shankhpushpi churna was measured on ethanol withdrawal anxiety using elevated plus maze. The role of shankhpushpi on chronic ethanol consumption (21 days) was measured using two bottle choice protocol of voluntary drinking. We also measured the effect of the above herb on cortico-hippocampal GABA levels. Shankhpushpi was found to reduce alcohol withdrawal anxiety in a dose-dependent manner. The herb also decreased ethanol intake and increased water intake significantly (P < 0.001) after 4 days of administration. Both these effects were blocked (P < 0.001) by GABAA antagonist suggesting the role of GABAA receptor. Chronic administration of shankhpushpi also significantly (P < 0.01) increased cortico-hippocampal GABA levels in mice. Shankhpushpi reduced both alcohol dependence and withdrawal in a GABAA-dependent manner, thus showing anti-addictive potential. Abbreviations used: GABA: Gamma-Aminobutyric Acid, HIV: Human Immunodeficiency Virus, CNS: Central Nervous System

Effect of Metabolic Syndrome on Depression in Mice

Metabolic syndrome (MetS) is associated with high blood glucose, insulin resistance, dyslipidemia, central obesity, and hypertension. There is clinical evidence of the coexistence of depression and MetS, however, pathways associating these diseases are far from clear. In the present study, we evaluate and determine the pathogenesis of depression in MetS animals. Methods: Diet induced (High-fat diet long with 20% fructose water; HFHC diet for 4 weeks) MetS was developed in swiss albino mice. Fasting blood glucose levels, Lipids and blood pressure (BP) was measured in these animals. Development of depression in these animals was determined using forced swim and tail suspension tests. This was followed by measurement of GABA, dopamine, serotonin and norepinephrine levels in these animals. We also evaluated the effect of various antidepressants, on MetS associated depression. Results: MetS was induced using high fat and high carbohydrate (HFHC) diet in Swiss albino mice with high fasting blood glucose levels (>250 mg/dl), significantly increased LDL (p<0.001) and triglyceride (p<0.01) and reduced HDL levels (p<0.05) and significant increase in systolic BP; p<0.001) compared to normal controls. MetS animals showed signs of depression with significantly higher (p<0.001) immobility time in forced swim and tail suspension tests. These animals showed significantly lower corticohippocampal norepinephrine (NE) levels (p<0.01) compared to controls. Nortryptaline, showed a dose dependent decrease in immobility time in MetS animals (p<0.001) in both forced swim and tail suspension tests thus reversing MetS induced depression. Conclusion: The above results suggest that MetS may lead to depression in mice which is primarily mediated by NE system.