Morphine administration alters the profile of hippocampal postsynaptic density-associated proteins: a proteomics study focusing on endocytic proteins

Numerous studies have shown that drugs of abuse induce changes in protein expression in the brain that are thought to play a role in synaptic plasticity. Drug-induced plasticity can be mediated by changes at the synapse and more specifically at the postsynaptic density (PSD), which receives and transduces synaptic information. To date, the majority of studies examining synaptic protein profiles have focused on identifying the synaptic proteome. Only a handful of studies have examined the changes in synaptic profile by drug administration. We applied a quantitative proteomics analysis technique with the cleavable ICAT reagent to quantitate relative changes in protein levels of the hippocampal PSD in response to morphine administration. We identified a total of 102 proteins in the mouse hippocampal PSD. The majority of these were signaling, trafficking, and cytoskeletal proteins involved in synaptic plasticity, learning, and memory. Among the proteins whose levels were found to be altered by morphine administration, clathrin levels were increased to the largest extent. Immunoblotting and electron microscopy studies showed that this increase was localized to the PSD. Morphine treatment was also found to lead to a local increase in two other components of the endocytic machinery, dynamin and AP-2, suggesting a critical involvement of the endocytic machinery in the modulatory effects of morphine. Because alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are thought to undergo clathrin-mediated endocytosis, we examined the effect of morphine administration on the association of the AMPA receptor subunit, GluR1, with clathrin. We found a substantial decrease in the levels of GluR1 associated with clathrin. Taken together, these results suggest that, by causing a redistribution of endocytic proteins at the synapse, morphine modulates synaptic plasticity at hippocampal glutamatergic synapses.     

Biochemical,machine learning and molecular approaches for the differential diagnosis of Mucopolysaccharidoses

This study was aimed to construct classification and regression tree (CART) model of glycosaminoglycans (GAGs) for the differential diagnosis of Mucopolysaccharidoses (MPS). Two-dimensional electrophoresis and liquid chromatography–tandem mass spectrometry (LC–MS/MS) were used for the qualitative and quantitative analysis of GAGs. Specific enzyme assays and targeted gene sequencing were performed to confirm the diagnosis. Machine learning tools were used to develop CART model based on GAG profile. Qualitative and quantitative CART models showed 96.3% and 98.3% accuracy, respectively, in the differential diagnosis of MPS. The thresholds of different GAGs diagnostic of specific MPS types were established. In 60 MPS positive cases, 46 different mutations were identified in six specific genes. Among 31 different mutations identified in IDUA, nine were nonsense mutations and two were gross deletions while the remaining were missense mutations. In IDS gene, four missense, two frameshift, and one deletion were identified. In NAGLU gene, c.1693C?>?T and c.1914_1914insT were the most common mutations. Two ARSB, one case each of SGSH and GALNS mutations were observed. LC–MS/MS-based GAG pattern showed higher accuracy in the differential diagnosis of MPS. The mutation spectrum of MPS, specifically in IDUA and IDS genes, is highly heterogeneous among the cases studied.

     

Identification and characterization of Helicobacter pylori O‐acetylserine‐dependent cystathionine β‐synthase,a distinct member of the PLP‐II family

O‐acetylserine sulfhydrylase (OASS) and cystathionine β‐synthase (CBS) are members of the PLP‐II family, and involved in L‐cysteine production. OASS produces L‐cysteine via a de novo pathway while CBS participates in the reverse transsulfuration pathway. O‐acetylserine‐dependent CBS (OCBS) was previously identified as a new member of the PLP‐II family, which are predominantly seen in bacteria. The bacterium Helicobacter pylori possess only one OASS (hp0107) gene and we showed that the protein coded by this gene actually functions as an OCBS and utilizes L‐homocysteine and O‐acetylserine (OAS) to produce cystathionine. HpOCBS did not show CBS activity with the substrate L‐serine and required OAS exclusively. The HpOCBS structure in complex with methionine showed a closed cleft state, explaining the initial mode of substrate binding. Sequence and structural analyses showed differences between the active sites of OCBS and CBS, and explain their different substrate preferences. We identified three hydrophobic residues near the active site of OCBS, corresponding to one serine and two tyrosine residues in CBSs. Mutational studies were performed on HpOCBS and Saccharomyces cerevisiae CBS. A ScCBS double mutant (Y158F/Y226V) did not display activity with L‐serine, indicating indispensability of these polar residues for selecting substrate L‐serine, however, did show activity with OAS.     

miRNA signatures in diabetic retinopathy and nephropathy: delineating underlying mechanisms

Journal of Physiology and Biochemistry - A worldwide failure to achieve glycemic targets has led to complications associated with diabetes mellitus. In addition to genetic and other risk factors,...     

Differential response of chloride binding sites to elevated temperature: a comparative study in spinach thylakoids and PSII-enriched membranes

A study of heat effects was performed in thylakoids and photosystem II (PSII)-enriched membranes isolated from spinach in relation to Cl⁻-induced activation of PSII catalyzed oxygen evolution and the retention of Cl⁻ in the PSII complex. For this, Cl⁻-sufficient membranes and low-Cl⁻ membranes were used. The presence of Cl⁻ in the reaction medium did accelerate oxygen evolution, which remained unaffected by heat treatment up to 40°C in PSII membranes and up to 42.5°C in thylakoids. Heat resistance of Cl⁻-induced activation of oxygen evolution was found to be independent of the presence of ‘bound Cl⁻’ in the preparations. However, the functional stability of the PSII complex during heat treatment showed a marked dependence on the presence of bound Cl⁻ in PSII. Electron paramagnetic resonance study of manganese (Mn) release per reaction center/Y_D⁺ showed that there was little loss of Mn²⁺ up to 42°C in our preparations, although the PSII activity was significantly lowered. These observations together with data from steady state chlorophyll a fluorescence imply that the site of action of Cl⁻ causing direct activation of oxygen evolution was different from the site of primary heat damage. A differential response of chloride binding sites to heat stress was observed. The high-affinity (tightly bound, slow exchanging) site of chloride is affected earlier (∼37°C) while low-affinity (loosely bound, fast exchanging) site gets affected at higher temperatures (42.5°C in thylakoids and 40°C in the case of PSII-enriched membranes). Prasanna Mohanty is an INSA Honorary Scientist and Professor on Courtesy, DAVV, Indore.     

Gastroprotective effect of Terminalia arjuna bark on diclofenac sodium induced gastric ulcer

AIM: The present study was aimed to evaluate the effect of methanolic extract of Terminalia arjuna (TA) on diclofenac sodium induced gastric ulcer in experimental rats. METHODS: Animals were induced for gastric ulcer with diclofenac sodium (DIC) (80mg/kg bodyweight in water, orally) and treated orally with TA in various doses ranging from 100mg/kg bodyweight to 500mg/kg bodyweight. The effective dose was 400mg/kg bodyweight, since this dose elicited a maximum reduction in lesion index. The gastroprotective effect of TA was assessed from volume of gastric juice, pH, free and total acidity, pepsin concentration, acid output in gastric juice, the levels of non-protein sulfhydryls (NP-SH), lipid peroxide (LPO), reduced glutathione (GSH), and activities of enzymic antioxidants--super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and myeloperoxidase (MPO) in gastric mucosa. The levels of DNA, protein bound carbohydrate complexes--hexose, hexoseamine, sialic acid, fucose in gastric mucosa and gastric juice and the levels of RNA in gastric mucosa were assessed. The stomach tissues were used for adherent mucus content and also for the histological examination. RESULTS: A significant reduction in lesion index was observed in ulcer induced animals treated with TA (DIC+TA) compared to ulcerated rats (DIC). A significant increase was observed in pH, NP-SH, GSH, enzymic antioxidants, protein bound carbohydrate complexes, adherent mucus content, nucleic acids with a significant decrease in volume of gastric juice, free and total acidity, pepsin concentration, acid output, LPO levels and MPO activities in DIC+TA rats compared to DIC rats. Histological studies confirmed the gastroprotective activity of TA. CONCLUSION: From the data presented in this study it could be concluded that T. arjuna acts as an gastroprotective agent probably due to its free radical scavenging activity and cytoprotective nature.     

Lipid-protein interactions, regulation and dysfunction of brain cholesterol

The biosynthesis and metabolism of cholesterol in the brain is spatiotemporally and developmentally regulated. Brain cholesterol plays an important role in maintaining the function of neuronal receptors, which are key components in neural signal transduction. This is illustrated by the requirement of membrane cholesterol for the function of the serotonin(1A) receptor, a transmembrane neurotransmitter receptor. A crucial determinant for the function of neuronal receptors could be the availability of brain cholesterol. The Smith-Lemli-Optiz Syndrome, a metabolic disorder characterized by severe neurodegeneration leading to mental retardation, represents a condition in which the availability of brain cholesterol is limited. A comprehensive molecular analysis of lipid-protein interactions in healthy and diseased states could be crucial for a better understanding of the pathogenesis of psychiatric disorders.     

Hydrogen Cyanide-Producing Rhizobacteria Kill Subterranean Termite <Emphasis Type="Italic">Odontotermes obesus</Emphasis> (Rambur) by Cyanide Poisoning Under <Emphasis Type="Italic">In Vitro</Emphasis> Conditions

The subterranean termite Odontotermes obesus is an important pest of the Indian subcontinent, causing extensive damage to major agricultural crops and forest plantation trees. Control of termites by strategies employing their parasites has limitations because they have evolved a complex social structure, immune responses, and adaptive behavior toward pathogen-infected individuals. Nonparasitic rhizobacteria that produce harmful metabolites might facilitate the biocontrol of termites. In the present investigation, three different species of hydrogen cyanide-producing rhizobacteria were tested for their potential to kill O. obesus. The three bacterial species were found to be effective in killing the termites under in vitro conditions.     

Evidence of increased endometrial vascular permeability at the time of implantation in the short-nosed fruit bat, Cyanopterus sphinx

Early embryonic development and implantation were studied in tropical short-nosed fruit bat Cyanopterus sphinx. We report preimplantation development and embryo implantation. Different stages of cleavage were observed in embryo by direct microscopic examination of fresh embryos after retrieving them either from the oviduct or the uterus at different days, up to the day of implantation. Generally, the embryos enter the uterus at the 8-cell stage. Embryonic development continued without any delay and blastocyst were formed showing attachment to the uterine epithelium at the mesometrial side of the uterus. A distinct blue band was formed in the uterus. The site of blastocyst attachment was visualized as a blue band following intravenous injection of pontamine blue. Implantation occurred 9+/-0.7 days after mating. This study reports that bat embryonic development can be studied like other laboratory animals and that this bat shows blue dye reaction, indicating the site and exact time of implantation. This blue dye reaction can be used to accurately find post-implantational delay. We prove conclusively that this species of tropical bat does not have any type of embryonic diapause.