Sumantra Das

Single-nucleotide polymorphism (A118G) in exon 1 of OPRM1 gene causes alteration in downstream signaling by mu-opioid receptor and may contribute to the genetic risk for addiction

The opioid receptor mu1 (OPRM1) mediates the action of morphine. Although genetic background plays an important role in the susceptibility toward abuse of drugs as evident from familial, adoption and twin studies, association of specific single‐nucleotide polymorphisms of OPRM1 gene with narcotic addiction is to be established. Here, we demonstrate the involvement of A118G polymorphism of exon1 of human OPRM1 gene (hOPRM1), with heroin and alcohol addiction, in a population in eastern India. Statistical analysis exhibited a significant association of G allele with both heroin and alcohol addiction with a risk factor of Ptrend < 0.05. The functional significance of G allele in A118G single‐nucleotide polymorphisms was evaluated by studying the regulation of protein kinase A (PKA), pCREB, and pERK1/2 by morphine in Neuro 2A cells, stably transfected with either wild type or A118G mutant hOPRM1. Unlike acute morphine treatment, both chronic morphine exposure and withdrawal precipitated by naloxone were differentially regulated by A118 and G118 receptor isoforms when both PKA and pERK1/2 activities were compared. Results suggest that the association of A118G polymorphism to heroin and alcohol addiction may be because of the altered regulation of PKA and pERK1/2 during opioid and alcohol exposures.

Hypothyroidism in the developing rat brain is associated with marked oxidative stress and aberrant intraneuronal accumulation of neurofilaments

The effects of hypothyroidism on parameters of oxidative stress and on intraneuronal distribution of neurofilaments have been investigated in the developing rat brain. Progressive hypothyroidism during the first 4 weeks of postnatal development led to an increase in superoxide dismutase and catalase activity, decline in the level of glutathione and mitochondrial cytochrome c oxidase activity and increase in the level of .OH radical along with enhanced protein carbonylation and lipid peroxidation. Immunocytochemical staining of cryostat sections of normal and hypothyroid cerebella from 25 day postnatal rats with anti neurofilament (NF) light chain (L) antibody showed aberrant accumulation of neurofilaments in the perikaryon of the hypothyroid Purkinje neurons in contrast to relatively uniform distribution in the controls. The morphological and biochemical alterations in the neurons of the developing hypothyroid brain are comparable to those seen in several neurodegenerative diseases.

Macroporous silicon based simple and efficient trapping platform for electrical detection of Salmonella typhimurium pathogens

A thermally oxidized macroporous silicon substrate with simple electrode structure without interdigitated electrode configuration has been reported for the detection of Salmonella typhimurium pathogens by electrical impedance measurement using antibody-antigen binding method. Macroporous silicon which has been fabricated by anodizing silicon in HF and DMF solution is a regular network of pores of 1-2 microm diameters. This has been thermally oxidized to yield the surface hydrophilic for antibody immobilization as well as provide suitable electrical insulation of the metal contacts from the underlying conducting silicon substrate. The macroporous silicon surface has been immobilized by Salmonella specific antibody and has been tested with different concentration of S. typhimurium pathogens in phosphate buffer solution (PBS). It has been found that such macroporous silicon substrates is capable of detecting down to 10(3)CFU/ml in pure culture using a 3 mm x 1 mm electrode structure with a wide spacing of 1mm. The selectivity of the macroporous silicon substrates with reference to S. typhimurium has been tested to be satisfactory by carrying out controlled experiments with Escherichia coli O157:H7.

Thyroid hormone-induced morphological differentiation and maturation of astrocytes involves activation of protein kinase A and ERK signalling pathway

Thyroid hormone (TH) has a profound effect on astrocyte differentiation and maturation. Astrocytes cultured under TH‐deficient conditions fail to transform from flat polygonal morphology to mature, process‐bearing, stellate cells. Supplementation of physiological concentrations of TH initiate gradual transformation of the cells and the process takes ≈ 48 h to complete. The signal transduction pathways associated with TH‐mediated maturation of astrocytes have been investigated. TH treatment caused an initial activation of protein kinase A (PKA), with a peak activity at 2 h which fell back to basal level there after. Although there was no visible change in morphology of the cells during the observed activation of PKA, it was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. PKA inhibitors as well as the MEK inhibitor PD098059 attenuated the TH‐induced morphological transformation. Further studies showed that TH treatment resulted in a biphasic response on the cellular phospho‐MAP kinase (p‐MAPK or p‐ERK) level: an initial decline in the p‐ERK level followed by an induction at 18–24 h, both of which could be blocked by a PKA inhibitor. Such sustained activation of p‐ERK levels by TH at this later stage coincided with initiation of morphological differentiation of the astrocytes and appeared to be critical for the transformation of astrocytes. The nitric oxide synthase (NOS) inhibitor 7‐NI inhibited this induction of p‐ERK activity. Moreover, the induction was accompanied by a parallel increase in phospho‐CREB activity which, however, persisted at the end of the transformation of the astroglial cells.

Self-Assembling Dipeptide-Based Nontoxic Vesicles as Carriers for Drugs and Other Biologically Important Molecules

Self-assembling short peptides can offer an opportunity to make useful nano-/microstructures that find potential application in drug delivery. We report here the formation of multivesicular structures from self-assembling water-soluble synthetic amphiphilic dipeptides containing a glutamic acid residue at the C-terminus. These vesicular structures are stable over a wide range of pH (pH 2-12). However, they are sensitive towards calcium ions. This causes the rupturing of these vesicles. Interestingly, these vesicles can not only encapsulate an anticancer drug and a fluorescent dye, but also can release them in the presence of calcium ions. Moreover, these multivesicular structures have the potential to carry biologically important molecules like cyclic adenosine monophosphate (cAMP) within the cells keeping their biological functions intact. A MTT cell-survival assay suggests the almost nontoxic nature of these vesicles. Thus, these peptide vesicles can be used as biocompatible delivery vehicles for carrying drugs and other bioactive molecules.

Epistatic effects between variants of kappa-opioid receptor gene and A118G of mu-opioid receptor gene increase susceptibility to addiction in Indian population.

OBJECTIVE Unequivocal evidence suggests contribution of κ-opioid receptor (KOR) in addiction to drugs of abuse. A study was undertaken to identify the single nucleotide polymorphisms (SNP) at selective areas of kappa opioid receptor 1 (OPRK1) gene in heroin as well as in alcohol addicts and to compare them with that in control population. The potential interaction of the identified KOR SNPs with A118G of μ opioid receptor was also investigated. METHODS Two hundred control subjects, one hundred thirty heroin and one hundred ten alcohol addicts, all male and residing in Kolkata, a city in eastern India, volunteered for the study. Exons 3 and 4 of OPRK1 and the SNP, A118G of mu opioid receptor 1 (OPRM1) in the DNA samples were genotyped by sequencing and restriction fragment length polymorphism respectively. The SNPs identified in the population were analyzed by odds ratio and its corresponding 95% confidence interval was estimated using logistic regression models. SNP-SNP interactions were also investigated. RESULTS Three SNPs of OPRK1, rs16918875, rs702764 and rs963549, were identified in the population, none of which showed significant association with addiction. On the other hand, significant association was observed for A118G with heroin addiction (χ²=7.268, P=0.0264) as well as with alcoholic addition (χ²=6.626, P=0.0364). A potential SNP-SNP interaction showed that the odds of being addicted was 2.51 fold in heroin subjects [CI (95%)=1.1524 to 5.4947, P=0.0206] and 2.31 fold in alcoholics [CI (95%)=1.025 to 5.24, P=0.0433] with the OPRK1 (rs16918875) and A118G risk alleles than without either. A significant interaction was also identified between GG/AG of A118G and GG of rs702764 [O.R (95%)=2.04 (1.279 to 3.287), P=0.0029] in case of opioid population. CONCLUSION Our study suggests that set associations of polymorphisms may be important in determining the risk profile for complex diseases such as addiction.

Role of Thyroid Hormone in the Morphological Differentiation and Maturation of Astrocytes: Temporal Correlation with Synthesis and Organization of Actin

Morphological changes and the molecular mechanisms associated with the maturation of astrocytes were studied under normal and thyroid hormone‐deficient conditions using long‐term (30 days) primary cultures derived from the neonatal rat brain. Immunocytochemical staining of cells with a monoclonal antibody specific to glial fibrillary acidic protein demonstrated for the first time that, similar to their maturation in vivo, astrocytes maintained in normal serum‐containing medium can undergo complete maturation involving two distinct stages of morphological differentiation (from radial glia to flat polygonal cells with epithelioid morphology and then to mature process‐bearing cells with stellate morphology). Deficiency of thyroid hormone delays the first step and totally blocks the second stage of differentiation in the maturation process. Comparative staining of normal and thyroid hormone‐deficient astrocytes with filamentous actin‐specific fluorescein isothiocyanate‐phalloidin and quantitation of the various forms of intracellular actin using an improved DNase I assay demonstrated that maturation of astroglial cells is associated with characteristic alterations in the level of cytoskeletal and non‐cytoskeletal filamentous (F) actin. In particular, the maintenance of the epithelioid form of the hypothyroid astrocytes is associated with a progressive increase in the level of cytoskeletal F‐actin and a concomitant decline in the level of non‐cytoskeletal F‐actin. Quantitation of actin mRNA by Northern blot analysis and studies on the rate of actin synthesis at various stages of differentiation showed that the initial transformation into the epithelioid form is associated with an increase in the rate of synthesis of actin and the expression of its mRNA, while the final transformation into the mature process‐bearing form is correlated with a decline in these parameters. The results indicate that thyroid hormone plays an obligatory role in promoting the differentiation and maturation of astrocytes, and that during this process the hormone regulates the expression of actin and its intracellular organization in a way conducive to morphological differentiation.

Thyroid Hormone-Induced Morphological Differentiation and Maturation of Astrocytes Are Mediated Through the β-Adrenergic Receptor

Abstract: The molecular mechanisms associated with thyroid hormone (TH)‐induced maturation of astrocytes have been studied using primary cultures. We have previously demonstrated that unlike normal astrocyte cultures, hypothyroid cultures fail to differentiate from flat polygonal cells with epithelioid morphology into mature process‐bearing cells with stellate morphology. Addition of TH to the hypothyroid cells reverses the effect, and astrocytes transform into stellate cells. The β‐adrenergic receptor (β‐AR) agonist isoproterenol (ISP) has a similar effect, whereas simultaneous addition of the β‐adrenergic antagonist propranolol blocks the differentiation induced by TH or ISP. Addition of TH or ISP to hypothyroid cultures is also associated with a decrease in the level of filamentous cytoskeletal (Fi) actin and an increase in the level of actin mRNA. Although addition of propranolol inhibited the decline in the level of Fi actin in the TH‐ or ISP‐supplemented cells as well as the induction of actin mRNA by TH, it partially inhibited the ISP‐induced actin mRNA in these cultures. The hormone‐induced maturation appears to be selectively regulated through the β2‐AR. The overall results indicate that the β‐adrenergic system plays an obligatory role in promoting TH‐induced differentiation and maturation of astrocytes and in regulating the hormone‐induced expression of actin and its intracellular organization in a way conducive to the morphological differentiation of the cells.

Delayed but sustained induction of mitogen-activated protein kinase activity is associated with β-adrenergic receptor-mediated morphological differentiation of astrocytes

Astroglial β‐adrenergic receptors (β‐ARs) are functionally linked to regulate cellular morphology. In primary cultures, the β‐AR agonist isoproterenol (ISP) can transform flat polygonal astrocytes into process‐bearing, mature stellate cells by 48 h, an effect that can be blocked by the β‐AR antagonist, propranolol. ISP induced immediate activation of protein kinase A (PKA) which persisted up to 2 h, with no visible change in cell morphology. However, activation of PKA was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. In addition to PKA inhibitors, the mitogen‐activated protein kinase (MAPK) kinase inhibitor PD098059 also blocked ISP‐induced morphological transformation. ISP treatment resulted in a biphasic response of cellular phosphorylated MAPK (phosphorylated extracellular signal‐regulated kinase; p‐ERK) level: an initial decline in p‐ERK level followed by a sustained induction at 12–24 h, both of which were blocked by PKA inhibitor. The induction in pERK level coincided with initiation of morphological differentiation of the astrocytes and nuclear translocation of p‐ERK. A long‐lasting activation of p‐ERK activity by ISP, at a later stage, appears to be critical for the transformation of astrocytes.

Essential role of docosahexaenoic acid towards development of a smarter brain.

Evolution of the high order brain function in humans can be attributed to intake of poly unsaturated fatty acids (PUFAs) of which the ω-3 fatty acid, docosahexaenoic acid (DHA) has special significance. DHA is abundantly present in the human brain and is an essential requirement in every step of brain development like neural cell proliferation, migration, differentiation, synaptogenesis etc. The multiple double bonds and unique structure allow DHA to impart special membrane characteristics for effective cell signaling. Evidences indicate that DHA accumulate in areas of the brain associated with learning and memory. Many development disorders like dyslexia, autism spectrum disorder, attention deficit hyperactivity disorder, schizophrenia etc. are causally related to decreased level of DHA. The review discusses the various reports of DHA in these areas for a better understanding of the role of DHA in overall brain development. Studies involving laboratory animals and clinical findings in cases as well as during trials have been taken into consideration. Additionally the currently available dietary source of DHA for supplementation as nutraceutics with general caution for overuse has been examined.