The Neurochemistry of Depression: The Good, The Bad and The Ugly.

A large constellation of experimental evidence suggests that neuroinflammation is involved in the onset of depression and neurodegenerative disorders. Many studies have shown impairments in tryptophan metabolism, the major pathway for the synthesis of serotonin, the mood regulating neurotransmitter. This article reviews the various metabolites generated in the competing pathways of tryptophan metabolism including the kynurenine pathway. Increased synthesis of the neurotoxic compound quinolinic acid occurs at the expense of the synthesis of the neuroprotective metabolite kynurenic acid. This shift in equilibrium plays a critical role in the induction of oxidative stress, neuroinflammation, and neurotoxicity. Sufficient protein intake with adequate amounts of tryptophan along with dietary antioxidants and flavonoids may offer protection against major depressive and neurodegenerative disorders.

Novel Coumarin Derivatives as Potential Urease Inhibitors for Kidney Stone Prevention and Antiulcer Therapy: From Synthesis to In Vivo Evaluation.

The presence of ammonium ions in urine, along with basic pH in the presence of urease-producing bacteria, promotes the production of struvite stones. This causes renal malfunction, which is manifested by symptoms such as fever, nausea, vomiting, and blood in the urine. The involvement of urease in stone formation makes it a good target for finding urease enzyme inhibitors, which have the potential to be developed as lead drugs against kidney stones in the future. The documented ethnopharmacology of coumarin 2-one against bacterial, fungal and viral strains encouraged us to synthesize new derivatives of coumarins by reacting aromatic aldehydes with 4-aminocoumarin. The synthesized compounds (2a to 11a) were evaluated for their antimicrobial, in vitro, and in silico properties against the urease enzyme. The study also covers in vivo determination of the synthesized compounds with respect to different types of induced ulcers. The molecular docking study along with extended MD simulations (100 ns each) and MMPBSA study confirmed the potential inhibitory candidates as evident from computed ∆Gbind (3a = -11.62 and 5a = -12.08 Kcal/mol) against the urease enzyme. The in silico analyses were augmented by an enzymatic assay, which revealed that compounds 3a and 5a had strong inhibitory action, with IC50 of 0.412 µM (64.0% inhibition) and 0.322 µM (77.7% inhibition), respectively, compared to standard (Thiourea) with 82% inhibition at 0.14 µM. Moreover, the most active compound, 5a, was further tested in vivo for antiulcer activity by different types of induced ulcers, including pyloric ligation-, ethanol-, aspirin-, and histamine-induced ulcers. Compound 5a effectively reduced gastric acidity, lipid peroxidation, and ulceration in a rat model while also inhibiting gastric ATPase activity, which makes it a promising candidate for ulcer treatment. As a result of the current research, 3a and 5a may be used as new molecules for developing potent urease inhibitors. Additionally, the compound 3a showed antibacterial activity against Staphylococcus aureus and Salmonella typhimurium, with zones of inhibition of 41 ± 0.9 mm and 35 ± 0.9 mm, respectively. Compound 7a showed antibacterial activity against Staphylococcus aureus and Salmonella typhimurium, with zones of inhibition of 30 ± 0.8 mm and 42 ± 0.8 mm, respectively. These results prove that the synthesized compounds also possess good antibacterial potential against Gram-positive and Gram-negative bacterial strains.

In-vitro high-throughput library screening-Kinetics and molecular docking studies of potent inhibitors of α-glucosidase.

High throughput screening of synthetic compounds against vital enzymes is the way forward for the determination of potent enzyme inhibitors. In-vitro high throughput library screening of 258 synthetic compounds (comp. 1-258), was performed against α-glucosidase. The active compounds out of this library were investigated for their mode of inhibition and binding affinities towards α-glucosidase through kinetics as well as molecular docking studies. Out of all the compounds selected for this study, 63 compounds were found active within the IC50 range of 3.2 µM to 50.0 µM. The most potent inhibitor of α-glucosidase out of this library was the derivative of an oxadiazole (comp. 25). It showed the IC50 value of 3.23 ± 0.8 µM. Other highly active compounds were the derivatives of ethyl-thio benzimidazolyl acetohydrazide with IC50 values of 6.1 ± 0.5 µM (comp. 228), 6.84 ± 1.3 µM (comp. 212), 7.34 ± 0.3 µM (comp. 230) and 8.93 ± 1.0 µM (comp. 210). For comparison, the standard (acarbose) showed IC50 = 378.2 ± 0.12 µM. Kinetic studies of oxadiazole (comp. 25) and ethylthio benzimidazolyl acetohydrazide (comp. 228) derivatives indicated that Vmax and Km, both change with changing concentrations of inhibitors which suggests an un-competitive mode of inhibition. Molecular docking studies of these derivatives with the active site of α-glucosidase (PDB ID:1XSK), revealed that these compounds mostly interact with acidic or basic amino acid residues through conventional hydrogen bonds along with other hydrophobic interactions. The binding energy values of compounds 25, 228, and 212 were -5.6, -8.7 and -5.4 kcal.mol-1 whereas RMSD values were 0.6, 2.0, and 1.7 Å, respectively. For comparison, the co-crystallized ligand showed a binding energy value of -6.6 kcal.mol-1 along with an RMSD value of 1.1 Å. Our study predicted several series of compounds as active inhibitors of α-glucosidase including some highly potent inhibitors.

Effect of varying thickness properties of the slow release fertilizer films on morphology, biodegradability, urea release, soil health, and plant growth.

Green biomass is a renewable and biodegradable material that has the potential use to trap urea to develop a high-efficiency urea fertilizer for crops' better performance. Current work examined the morphology, chemical composition, biodegradability, urea release, soil health, and plant growth effects of the SRF films subjected to changes in the thickness of 0.27, 0.54, and 1.03 mm. The morphology was examined by Scanning Electron Microscopy, chemical composition was analyzed by Infrared Spectroscopy, and biodegradability was assessed through evolved CO2 and CH4 quantified through Gas Chromatography. The chloroform fumigation technique was used for microbial growth assessment in the soil. The soil pH and redox potential were also measured using a specific probe. CHNS analyzer was used to calculate the total carbon and total nitrogen of the soil. A plant growth experiment was conducted on the Wheat plant (Triticum sativum). The thinner the films, the more they supported the growth and penetration of the soil's microorganisms mainly the species of fungus possibly due to the presence of lignin in films. The fingerprint regions of the infrared spectrum of SRF films showed all films in soil changed in their chemical composition due to biodegradation but the increase in the thickness possibly provides resistance to the films' losses. The higher thickness of the film delayed the rate and time for biodegradation and the release of methane gas in the soil. The 1.03 mm film (47% in 56 days) and 0.54 mm film (35% in 91 days) showed the slowest biodegradability as compared to the 0.27 mm film with the highest losses (60% in 35 days). The slow urea release is more affected by the increase in thickness. The Korsymer Pappas model with release exponent value of < 0.5 explained the release from the SRF films followed the quasi-fickian diffusion and also reduced the diffusion coefficient for urea. An increase in the pH and decrease in the redox potential of the soil is correlated with higher total organic content and total nitrogen in the soil in response to amending SRF films with variable thickness. Growth of the wheat plant showed the highest average plant length, leaf area index and grain per plant in response to the increase in the film's thickness. This work developed an important knowledge to enhance the efficiency of film encapsulated urea that can better slow the urea release if the thickness is optimized.

Isoxazole Derivatives against Carbonic Anhydrase: Synthesis, Molecular Docking, MD Simulations, and Free Energy Calculations Coupled with In Vitro Studies.

Heterocyclic compounds with a five-membered ring as a core, particularly those containing more than one heteroatom, have a wide spectrum of biological functions, especially in enzyme inhibition. In this study, we present the synthesis of five-membered heterocyclic isoxazole derivatives via sonication of ethyl butyrylacetate with aromatic aldehyde in the presence of a SnII-Mont K10 catalyst. The synthesized compounds were characterized using sophisticated spectroscopic methods. In vitro testing of the compounds reveals three derivatives with significant inhibitory action against carbonic anhydrase (CA) enzyme. The compound AC2 revealed the most promising inhibitory activity against CA among the entire series, with an IC50 = 112.3 ± 1.6 µM (%inh = 79.5) followed by AC3 with an IC50 = 228.4 ± 2.3 µM (%inh = 68.7) compared to the standard with 18.6 ± 0.5 µM (%inh = 87.0). Molecular docking (MD) study coupled with extensive MD simulations (400 ns) and MMPBSA study fully supported the in vitro enzyme inhibition results, evident from the computed ΔG bind (AC2 = -13.53 and AC3 = -12.49 kcal/mol). The in vitro and in silico studies are also augmented by a fluorescence-based enzymatic assay in which compounds AC2 and AC3 showed significant fluorescence enhancement. Therefore, on the basis of the present study, it is inferred that AC2 and AC3 may serve as a new framework for designing effective CA inhibitors.

Diclofenac derivatives as concomitant inhibitors of cholinesterase, monoamine oxidase, cyclooxygenase-2 and 5-lipoxygenase for the treatment of Alzheimer's disease: synthesis, pharmacology, toxicity and docking studies.

Targeting concomitantly cholinesterase (ChEs) and monoamine oxidases (MAO-A and MAO-B) is a key strategy to treat multifactorial Alzheimer's disease (AD). Moreover, it is reported that the expression of cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) is increased significantly in the brain of AD patients. Using the triazole of diclofenac 12 as a lead compound, we synthesized a variety of analogs as multipotent inhibitors concomitantly targeting COX-2, 5-LOX, AChE, BChE, MAO-A and MAO-B. A number of compounds showed excellent in vitro inhibition of the target biological macromolecules in nanomolar concentration. Compound 39 emerged as the most potent multitarget ligand with IC50 values of 0.03 µM, 0.91 µM, 0.61 µM, 0.01 µM 0.60 µM and 0.98 µM towards AChE, BChE, MAO-A, MAO-B, COX-2 and 5-LOX respectively. All the biologically active compounds were found to be non-neurotoxic and blood-brain barrier penetrant by using PAMPA assay. In a reversibility assay, all the studied active compounds showed reversibility and thus were found to be devoid of side effects. MTT assay results on neuroblastoma SH-SY5Y cells showed that the tested compounds were non-neurotoxic. An in vivo acute toxicity study showed the safety of the synthesized compounds up to a 2000 mg kg-1 dose. In docking studies three-dimensional construction and interaction with key residues of all the studied biological macromolecules helped us to explain the experimental results.

Impaired calcium signalling and neuropsychiatric disorders in Darier disease: An exploratory review.

Darier (Darier-White) disease (DD) is an autosomal dominant skin disorder caused by pathogenic mutations in the ATP2A2 gene which encodes a calcium ATPase in the sarco-endoplasmic reticulum (SERCA2). Defects in the SERCA2 protein lead to an impairment of cellular calcium homeostasis, which in turn, triggers cell death pathways. There is a high prevalence of neuropsychiatric disorders in patients affected by this condition, namely intellectual disability, bipolar disorder, schizophrenia, and suicidality. Though these associations have been well-documented over the years, little has been discussed or investigated regarding the pathophysiological mechanisms. The goal of this article is to review the literature related to the most commonly associated neuropsychiatric disorders found in patients with DD, highlight the pathophysiological mechanisms underlying each condition, and examine potential interventions that may be of interest for future development. A literature search was performed using PubMed to access and review relevant articles published in the last 40 years. Keywords searched included Darier disease neuropsychiatric, Darier disease pathophysiology, SERCA2 central nervous system, SERCA 2 skin, ATP2A2 central nervous system, ATP2A2 skin, sphingosine-1-phosphate signalling skin, sphingosine-1-phosphate signalling central nervous system, P2X7 receptor skin, and P2X7 receptor central nervous system. Our search resulted in 2692 articles, of which 61 articles were ultimately included in this review.

It Is Time for an Oil Change: Polyunsaturated Fatty Acids and Human Health.

Polyunsaturated fatty acids (PUFAs) such as docosahexaneoic acid (DHA) and eicosapentaneoic acid (EPA), play a critical role in a variety of neuronal functions, including facilitating neuronal growth and differentiation, increasing the density of the neuritic network, modulating cell membrane fluidity, regulating intracellular signaling and gene expression, and exhibiting antioxidant characteristics. Dietary DHA is selectively enriched and actively retained in the central nervous system, mainly in synaptic membranes, dendrites, and photoreceptors. In this review, we highlight the myriad roles of PUFAs in brain function and human health. Diets rich in DHA are inversely proportional to cognitive decline and incidence of neurodegenerative disorders. Conversely, diets deficient in DHA impair the proper development of brain and the visual system in children and increase risk of brain disorders in the elderly. Finally, DHA and EPA have been shown to reduce inflammation and may prove to be beneficial in reducing the severity of the SARS-COVID infection.

Enzyme inhibition and antibacterial potential of 4-Hydroxycoumarin derivatives

The 4-Hydroxycoumarin derivatives are known to show a broad spectrum of pharmacological applications. In this paper we are reporting the synthesis of a new series of 4-Hydroxycoumarin derivatives synthesized through Knovenegal condensation; they were characterized by using UV-Vis, FT-IR, NMR spectroscopies. The synthesized compounds were evaluated for antibacterial activity against Staphylococcus aureus and Salmonella typhimurium strains. The compounds (2), (3) and (8) showed favorable antibacterial activity with zone of inhibitions 26.5± 0.84, 26.0 ± 0.56 and 26.0 ± 0.26 against Staphylococcus aureus (Gram-positive) respectively. However, the compounds (5) and (9) were found more active with 19.5 ± 0.59 and 19.5 ± 0.32 zone of inhibitions against Salmonella typhimurium (Gram-negative). Whereas, in urease inhibition assay, none of the synthesized derivatives showed significant anti-urease activity; although, in carbonic anhydrase-II inhibition assay, the compound (2) and (6) showed enzyme inhibition activity with IC50 values 263±0.3 and 456±0.1, respectively.

Bacterial Expression and Characterization of Recombinant ß-Xylosidase from the Thermophilic Xylanolytic Bacterium Bacillus sp.

With the passage of time, energy sources are decreasing day by day. In order to meet the world's demand, much attention is being paid to the study of enzymes with xylanolytic activity as a potential means of generating energy. A thermophilic xylanolytic bacterium, Bacillus sp., was isolated from naturally decaying material by enrichment culture and serial dilution methods. The bacterium was grown in MH medium at 50°C and pH 7 for 10 h. The xylanolytic Bacillus sp. produced clear yellow haloes around the colonies in the presence of p-nitrophenyl beta-D-xylopyranoside (pNPX) as a substrate. After condition optimization, it was found that the organism produced the higher level of xylosidase activity after 14 h in the presence of arabinose as a carbon source and ammonium sulfate as a nitrogen source in the pH 7 medium of at 55°C. The maximum ß-xylosidase activity after optimizing the culture condition was 5.0 U/mL. Later this thermophilic Bacillus sp. was used as a donor in cloning of the ß-xylosidase gene. A genomic library of Bacillus sp. was prepared by digesting the genomic DNA of the Bacillus with the restriction endonuclease BamHI, ligating the fragments in the pUC18 cloning vector and then transforming the competent E. coli DH5α cells with the resultant chimeric plasmid. The ß-xylosidase gene was identified by screening the transformants in duplicates on LB agar plates overlaid with pNPX as a substrate. Commercial production of ß-xylosidase to be used as a methanol-producing enzyme can help to overcome fuel shortages.

Forgotten fatty acids-Surface properties supply conclusive evidence for including carotenoic acids.

The term "fatty acids" is conceptually well defined with regard to fats, whose extent of saturation or unsaturation is precisely indicated in the content description of foodstuff. In contrast, the term "fatty acid" gives no hint of being associated with "soap" (Na and K salts of fatty acids). Fatty acids in edible fats or in cleaning soaps have one thing in common: they are colorless. The prevalence of colorless fats and unadulterated white soaps has ensured that fatty acids are not associated with color. However, colored conjugated polyunsaturated fatty acids do exist, occurring abundantly in nature or manufactured at large scale. We endeavor to extricate conjugated polyenoic acids from oblivion by Based on the presented results (alkalicarotenoates have similar surface properties to alkalicarboxylates, carotenoic acids react like carboxylic acids to lipids), we argue for inclusion of conjugated carotenoic acids in fatty acid inventories and organic chemistry textbooks. Carotenoic acids and -salts have outstanding qualities by combining visibility and traceability with biological activity.

The plasma membrane calcium pumps-The old and the new.

The plasma membrane Ca2+-ATPase (PMCA) pumps play a critical role in the maintenance of calcium (Ca2+) homeostasis, crucial for optimal neuronal function and cell survival. Loss of Ca2+ homeostasis is a key precursor in neuronal dysfunction associated with brain aging and in the pathogenesis of neurodegenerative disorders. In this article, we review evidence showing age-related changes in the PMCAs in synaptic plasma membranes (SPMs) and lipid raft microdomains isolated from rat brain. Both PMCA activity and protein levels decline progressively with increasing age. However, the loss of activity is disproportionate to the reduction of protein levels suggesting the presence of dysfunctional PMCA molecules in aged brain. PMCA activity is also diminished in post-mortem human brain samples from Alzheimer's disease and Parkinson's disease patients and in cell models of these neurodegenerative disorders. Experimental reduction of the PMCAs not only alter Ca2+ homeostasis but also have diverse effects on neurons such as reduced neuritic network, impaired release of neurotransmitter and increased susceptibility to stressful stimuli, particularly to agents that elevate intracellular Ca2+ [Ca2+]i. Loss of PMCA is likely to contribute to neuronal dysfunction observed in the aging brain and in the development of age-dependent neurodegenerative disorders. Therapeutic (pharmacological and/or non-pharmacological) approaches that can enhance PMCA activity and stabilize [Ca2+]i homeostasis may be capable of preventing, slowing, and/or reversing neuronal degeneration.

Changes in thyroid function status of suicidal patients

Abstract Background This study was carried out at Punjab Institute of Mental Health and Centre for Nuclear Medicine Mayo Hospital, Lahore. It is aimed at the possible association of thyroid malfunctioning with suicide attempts of patients. Objective Determination of thyroid function status of suicidal psychiatric patients and their comparison with psychiatric patients without suicide attempt or ideation. Methods Total 54 patients with either past history of suicide attempt or current suicidal ideation were selected for analysis of their thyroid function status (age 15-55 years). Age matched 50 non-suicide psychiatric patients were included for comparison. Results Two patients with suicide attempt had overt thyroid dysfunction. Remaining patients had serum FT4, FT3 and TSH level within normal range. Suicide attempter patients had lower FT4 but increased FT3 and TSH levels compared to suicidal ideation patients. Serum FT4 and TSH levels in suicidal patients were not different from psychiatric patients. Serum FT3 in suicidal patients was lower than psychiatric patients (3.7 ± 0.8 vs. 4.3 ± 0.5; p < 0.05). Female suicidal patients had lower FT3 levels compared to male patients (3.4 ± 0.6 vs. 3.9 ± 0.8 pmol/L; p < 0.05). Discussion Local suicidal patients have higher incidence of overt thyroid disorder and lower FT3 levels compared to non-suicidal psychiatric patients.

CHLOROGENIC ACID: A PHARMACOLOGICALLY POTENT MOLECULE.

Chlorogenic acid (CGA; (IS,3R,4R.5R)-3-{[(2Z)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid) is a naturally occurring polyphenol mostly present in vegetables and fruits. CGA is a prominent component of Traditional Chinese Medicines and is known for various pharmacological activities such as antioxidant, antimicrobial, anti-inflammatory and hepatoprotective etc. This mini-review is an attempt to summarize the available literature in the last decade and to point out future perspectives in this area of research.

SYNTHESIS, CHARACTERIZATION, ANTIMICROBIAL AND PHYTOTOXIC SCREENING OF 1-AROYL-3,5-DIARYLPYRAZOLINE DERIVATIVES.

Pyrazolines are biologically and pharmaceutically very active scaffolds. Derivatives of. (3,5-diphenyl-4,5-dihydro-1H-pyrazol-1-yl)(phenyl)methanone were synthesized by the cyclization of chalcones (1a-c) with substituted benzyl hydrazides (2a-e) using a few drops of piperidine as catalyst. Structures of all the synthesized compounds were confirmed by FTIR, 1H NMR, 13C NMR and mass spectrometric analysis. All the pyrazolines were subjected to antimicrobial and phytotoxic assays. Compound 3a and 3c showed maximum antimicrobial activities while all the synthesized compounds were active acc. to their phytotoxic assays.

Novel cationic polyene glycol phospholipids as DNA transfer reagents--lack of a structure-activity relationship due to uncontrolled self-assembling processes.

Cationic glycol phospholipids were synthesized introducing chromophoric, rigid polyenoic C20:5 and C30:9 chains next to saturated flexible alkyl chains of variable lengths C6-20:0. Surface properties and liposome formation of the amphiphilic compounds were determined, the properties of liposome/DNA complexes (lipoplexes) were established using three formulations (no co-lipid, DOPE as a co-lipid, or cholesterol as a co-lipid), and the microstructure of the best transfecting compounds inspected using small angle X-ray diffraction to explore details of the partially ordered structures of the systems that constitute the series. Transfection and cytotoxicity of the lipoplexes were evaluated by DNA delivery to Chinese hamster ovary (CHO-K1) cells using the cationic glycerol phospholipid 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) as a reference compound. The uncontrollable self-association of the molecules in water resulted in aggregates and liposomes of quite different sizes without a structure-property relationship. Likewise, adding DNA to the liposomes gave rise to unpredictable sized lipoplexes, which, again, transfected without a structure-activity relationship. Nevertheless, one compound among the novel lipids (C30:9 chain paired with a C20:0 chain) exhibited comparable transfection efficiency and toxicity to the control cationic lipid EPC. Thus, the presence of a rigid polyene chain in this best performing achiral glycol lipid did not have an influence on transfection compared with the chiral glycerolipid reference ethyl phosphocholine EPC with two flexible saturated C14 chains.

Effects of gangliosides on the activity of the plasma membrane Ca2+-ATPase.

Control of intracellular calcium concentrations ([Ca(2+)]i) is essential for neuronal function, and the plasma membrane Ca(2+)-ATPase (PMCA) is crucial for the maintenance of low [Ca(2+)]i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca(2+) homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by d-PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca(2+) transporter.

Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology.

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+-Cl- co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease.

Plasma membrane Ca-ATPases: Targets of oxidative stress in brain aging and neurodegeneration.

The plasma membrane Ca(2+)-ATPase (PMCA) pumps play an important role in the maintenance of precise levels of intracellular Ca(2+) [Ca(2+)](i), essential to the functioning of neurons. In this article, we review evidence showing age-related changes of the PMCAs in synaptic plasma membranes (SPMs). PMCA activity and protein levels in SPMs diminish progressively with increasing age. The PMCAs are very sensitive to oxidative stress and undergo functional and structural changes when exposed to oxidants of physiological relevance. The major signatures of oxidative modification in the PMCAs are rapid inactivation, conformational changes, aggregation, internalization from the plasma membrane and proteolytic degradation. PMCA proteolysis appears to be mediated by both calpains and caspases. The predominance of one proteolytic pathway vs the other, the ensuing pattern of PMCA degradation and its consequence on pump activity depends largely on the type of insult, its intensity and duration. Experimental reduction of PMCA expression not only alters the dynamics of cellular Ca(2+) handling but also has a myriad of downstream consequences on various aspects of cell function, indicating a broad role of these pumps. Age- and oxidation-related down-regulation of the PMCAs may play an important role in compromised neuronal function in the aging brain and its several-fold increased susceptibility to neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and stroke. Therapeutic approaches that protect the PMCAs and stabilize [Ca(2+)](i) homeostasis may be capable of slowing and/or preventing neuronal degeneration. The PMCAs are therefore emerging as a new class of drug targets for therapeutic interventions in various chronic degenerative disorders.

Age-associated changes in synaptic lipid raft proteins revealed by two-dimensional fluorescence difference gel electrophoresis.

Brain aging is associated with a progressive decline in cognitive function though the molecular mechanisms remain unknown. Functional changes in brain neurons could be due to age-related alterations in levels of specific proteins critical for information processing. Specialized membrane microdomains known as 'lipid rafts' contain protein complexes involved in many signal transduction processes. This study was undertaken to determine if two-dimensional fluorescence difference gel electrophoresis (2D DIGE) analysis of proteins in synaptic membrane lipid rafts revealed age-dependent alterations in levels of raft proteins. Five pairs of young and aged rat synaptic membrane rafts were subjected to DIGE separation, followed by image analysis and identification of significantly altered proteins. Of 1046 matched spots on DIGE gels, 94 showed statistically significant differences in levels between old and young rafts, and 87 of these were decreased in aged rafts. The 41 most significantly altered (p<0.03) proteins included several synaptic proteins involved in energy metabolism, redox homeostasis, and cytoskeletal structure. This may indicate a disruption in bioenergetic balance and redox homeostasis in synaptic rafts with brain aging. Differential levels of representative identified proteins were confirmed by immunoblot analysis. Our findings provide novel pathways in investigations of mechanisms that may contribute to altered neuronal function in aging brain.