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To determine experimentally how the multiple folding pathways of a protein differ, in the order in which the structural parts are assembled, has been a long-standing challenge. To resolve whether structure formation during folding can progress in multiple ways, the complex folding landscape of monellin has been characterized, structurally and temporally, using the multisite time-resolved FRET methodology. After an initial heterogeneous polypeptide chain collapse, structure formation proceeds on parallel pathways. Kinetic analysis of the population evolution data across various protein segments provides a clear structural distinction between the parallel pathways. The analysis leads to a phenomenological model that describes how and when discrete segments acquire structure independently of each other in different subensembles of protein molecules. When averaged over all molecules, structure formation is seen to progress as α-helix formation, followed by core consolidation, then β-sheet formation, and last end-to-end distance compaction. Parts of the protein that are closer in the primary sequence acquire structure before parts separated by longer sequence.

α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson's disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid-liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson's disease mutations, also promote α-Syn liquid-liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson's disease pathogenesis.

The main objective of this work was to synthesize, characterize and evaluate the anticancer activity of novel pyrimidine derivatives. The present investigation was undertaken to synthesize pyrimidine derivatives containing pyrrole nucleus The compounds 4-[aminomethyl]-N-(4-methyl-3-{[4-(1H-pyrrol-2-yl) pyrimidin-2-yl]amino}phenyl) benzamide (13f) and 4-[(propylamino) methyl]-N-(4-methyl-3-{ [4-(1H- pyrrol-2-yl) pyrimidin-2 -yl] amino} phenyl) benzamide (13h) was prepared by conventional method. All the synthesized compounds were characterized by spectral (IR, NMR and MS) methods. The synthesized compounds were evaluated for their in vitro anticancer activity against non-small cell lung cancer A549 cell line by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. All the synthesized compounds showed characteristic peaks in FTIR, 1H NMR and Mass spectral analysis. In vitro anticancer activity revealed that 13f and 13h showed more potent anticancer activity as compared to the standard drug sunitinib. We designed and synthesized novel pyrimidine derivatives by conventional method. The anticancer activity determined by MTT assay showed compound 13f and 13h can be developed as a potential anticancer agent.

Liquid-liquid phase separation (LLPS) and subsequent liquid-to-solid transition is implicated in membraneless organelles formation as well as disease associated protein aggregation. However, how liquid-to-solid transition is initiated inside a liquid droplet remains unclear. Here, using studies at single droplet resolution, we show that liquid-to-solid transition of α-synuclein (α-Syn) liquid droplets is associated with significant changes in the local microenvironment as well as secondary structure of the protein, which is prominently observed at the center of the liquid droplets. With the ageing of liquid droplets, the “structured” core at the center gradually expands and propagates over entire droplets. Further, during droplet fusion, smaller, homogeneous droplets progressively dissolve and supply proteins to the larger, heterogeneous droplets containing solid-like core at their center. The present study will significantly help to understand the physical mechanism of LLPS and liquid-to-solid transition in biological compartmentalization as well as in protein aggregation associated with human neurodegenerative disorders.Abstract Figure

Fluorescent nucleic acid base mimics serve as excellent site-specific and real-time reporters of the local and global dynamics. In this work, using the fluorescent guanine mimic 6-methylisoxanthopterin (6-MI), we unravel the differential dynamics of replication fork barrier/terminator sequences (RFB1 and RFB3) mediated by fork blocking protein (Fob1). By strategic and site-specific incorporation of this probe, we show that 6-MI is able to capture the changes in global dynamics exhibited by Fob1 and aids in distinguishing between varied architectural forms like double-stranded DNA versus Holliday junctions (HJs). This is important as these barriers are hotspots for recombination. Fluorescence lifetime and anisotropy decay studies further revealed that Fob1 strongly dampens the dynamics in double-stranded RFB1, and the sequence inherently possesses lesser flexibility in comparison to RFB3. We show that 6-MI can probe the differential oligomeric status of Fob1 in response to various architectures, that is, double-stranded versus HJs. This work highlights the unique advantages of 6-MI as a probe when incorporated in nucleic acid frameworks.

Understanding the properties of the unfolded state under folding conditions is of fundamental importance for gaining mechanistic insight into folding as well as misfolding reactions. Toward achieving this objective, the folding reaction of a small protein, monellin, has been resolved structurally and temporally, with the use of the multisite time-resolved FRET methodology. The present study establishes that the initial polypeptide chain collapse is not only heterogeneous but also structurally asymmetric and nonuniform. The population-averaged size for the segments spanning parts of the β-sheet decreases much more than that for the α-helix. Multisite measurements enabled specific and nonspecific components of the initial chain collapse to be discerned. The expanded and compact intermediate subensembles have the properties of a nonspecifically collapsed (hence, random-coil-like) and specifically collapsed (hence, globular) polymer, respectively. During subsequent folding, both the subensembles underwent contraction to varying extents at the four monitored segments, which was close to gradual in nature. The expanded intermediate subensemble exhibited an additional very slow contraction, suggestive of the presence of non-native interactions that result in a higher effective viscosity slowing down intrachain motions under folding conditions.

Flexibility and dynamics in polypeptides and proteins give rise to conformational and structural heterogeneity. Quantification of the level of heterogeneity is essential for providing deeper understanding of molecular mechanisms in protein folding. Fluorescence lifetime distribution generated by the maximum entropy method (MEM) provides an unbiased estimate of intramolecular distance distribution. This article discusses several examples where MEM-generated lifetime distribution brings out site-specific conformational and structural heterogeneity in proteins. Use of the information on intamolecular distance distribution in revealing molecular mechanisms of protein folding is also discussed.

Protein folding/unfolding transitions between the native (N) and unfolded (U) states are usually describable as two-state, only because of the dominant presence of the N and/or U states, because of which high energy intermediates remain undetected. Delineation of the cooperativity underlying these transitions, and characterization of high energy intermediates that are populated sparsely, have been difficult challenges, especially under equilibrium conditions, and require the use of a sensitive probe that reports on both the structures and population distributions of the partially unfolded intermediates. In this study, the use of multisite time-resolved FRET to monitor structural change in five specific segments of the small protein monellin, has brought out local deviations from two-state behavior during unfolding. It is shown that in some segments of the protein structure, denaturant-induced unfolding proceeds first by gradual expansion of the N state, then by an all-or-none transition from the N state ensemble to the U state ensemble, followed finally by expansion of the U state. Segments encompassing the sole helix appear, however, to unfold completely through a gradual transition from the N to U states. Finally, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the β-sheet.

A low molecular weight organogelator containing 3,5-substituted-1,3,4-oxadiazole and tetrazole units was synthesized and characterized. This compound is only soluble in DMSO and forms a stable gel. The solution and gel exhibit a blue light emission. The gel was characterized by atomic force microscopy, field-emission scanning electron microscopy, 1H NMR and fluorescence measurements. The gel to solution interconversion was reversible for many cycles of heating and cooling. The compound in solution exhibited a high selectivity for the detection of picric acid, a common explosive and water pollutant. Fluorimetric titration studies with nitro explosive compounds revealed that the emission of the compound was red shifted in response to the addition of picric acid, and exhibited a shifting of fluorescence from blue to green. Theoretical and experimental studies revealed that the sensing is due to the complexation of the picrate anion with the protonated fluorophore. The shifting of emission in response to picric acid in the visible region is ideal for the naked eye detection of explosives and therefore it is promising in comparison to the detection methods based on fluorescence quenching.

Chitosan was fabricated via gelation method using CaBr2.xH2O/methanol solution and was studied as a potential adsorbent (MCh) in adsorbing anionic synthetic dyes like Bromophenol blue (BB), Direct blue 6 (DB) and Congo red (CR) from single (one dye species at a time) and multi (having two dyes; binary and all three dyes; tertiary) adsorptive systems. Physico-chemical modifications of MCh surface prior and post modification and dye adsorption were evaluated using scanning electron microscopy, Energy-dispersive X-ray spectroscopy, powder X-ray diffraction analysis, surface area analysis and Fourier-transformed infrared spectroscopy. Influential parameters influencing the adsorption process viz. initial pH of dye solution, MCh dosage, adsorption temperature and initial concentration of dye species were optimised. Adsorptive studies involving single adsorptive setups verified formation of sorbate's (dye species) monolayer over the sorbent's (MCh) surface via chemisorption; as established by Langmuir isotherm and pseudo-second order kinetics model analysis. Theoretical maximum adsorption capacities of MCh for BB, DB and CR was found to be 81.301 mg/g, 163.934 mg/g and 75.758 mg/g, respectively. Meanwhile, for all multi-adsorptive systems, competitive Langmuir isotherm model verified antagonistic behaviour of an individual dye over other dye adsorption over MCh surface in their respective adsorptive systems. Thermodynamics of the sorbate-sorbent interaction was exothermic, spontaneous, with elevated degree of disorderedness; concluding the interaction as thermodynamically favourable. Co-existing metal cations and anionic salts had minimal effect on MCh's adsorption efficiency. Phytotoxicity assay via germination of Vigna mungo seeds verified the efficacy of the adsorbent in eliminating the dye species from single and multi-adsorptive systems.

A highly stable, spontaneous, and reversible α-helical-structure formation in recombinant and chemically modified α-synuclein protein is demonstrated for the first time in a water-less (1.5 % w/w H2 O) polymer surfactant environment. Using a combination of circular dichroism and ATR-FTIR spectroscopy, we show that whilst native α-synuclein in aqueous solution shows a predominant unordered conformation (≈64 %), mixing with polyethylene glycol based anionic polymer surfactant (PS) and removing water reveals a 25 % unordered, 25 % α-helical, and 27 % β-sheet structure. Interestingly, bioconjugation of native α-synuclein with a diamine molecule, to increase the positive charge on the protein chain, and subsequent electrostatic coupling with the PS forms a conjugate with a retained unordered structure. Removal of water from this system provides a highly stable α-helical (≈74 %) water-less liquid system. Surprisingly, the α-helical-to-unordered state transition is completely reversible and is achieved at ≈25-30 w/w% of water in the system. Moreover, the α-helix shows an extraordinary temporal stability (>6 months) in a waterless environment.

SUMMARYα-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson’s disease (PD) pathogenesis. However, the early events involved in this process remain unclear. Here, using in vitro reconstitution and cellular model, we show that liquid-liquid phase separation (LLPS) of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form amyloid-hydrogel containing oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation such as low pH, phosphomimic substitution, and familial PD mutation also promote α-Syn LLPS and its subsequent maturation. We further demonstrate α-Syn liquid droplet formation in cells, under oxidative stress. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. The present work provides detailed insights into the phase separation behavior of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in PD pathogenesis.

A series of 1-(3-benzyl-4-oxo-3 H- quinazolin-2-yl)-4-(substituted) thiosemicarbazides ( AS1–AS10 ) were obtained by the reaction of 2-hydrazino-3-benzyl quinazolin-4(3 H) -one ( 6 ) with different dithiocarbamic acid methyl ester derivatives. The key intermediate, 3-benzyl-2-thioxo-2,3-dihydro-1 H -quinazolin-4-one ( 4 ), was obtained by the reaction of benzyl amine ( 1 ) with carbon disulphide and sodium hydroxide in dimethyl sulphoxide to give sodium dithiocarbamate, which was methylated with dimethyl sulphate to yield the dithiocarbamic acid methyl ester ( 2 ) and condensation with methyl anthranilate ( 3 ) in ethanol yielded the desired compound ( 4 ) via the thiourea intermediate. The SH group of compound ( 4 ) was methylated in the favourable nucleophilic displacement reaction with hydrazine hydrate, which afforded 2-hydrazino-3-benzyl-3 H- quinazolin-4-one ( 6 ). The IR, and 1H- and 13C-NMR spectra of these compounds showed the presence of peaks due to thiosemicarbazides, carbonyl (C=O), NH and aryl groups. The molecular ion peaks of the quinazolin-4-one moiety ( m / z 144) were observed in all the mass spectra of the compounds AS1–AS10 . Elemental (C, H, N) analysis satisfactorily confirmed purity and elemental composition of the synthesized compounds. All the synthesized compounds were screened for their antimicrobial activity against selective gram positive and gram negative bacteria by agar dilution method. In the present study, compounds AS8 and AS9 emerged as the most active compounds of the series.

In recent years, nanoparticles (NPs) based on biopolymers or peptides are gaining popularity for the encapsulation and release of drug molecules, especially for cancer therapy, due to their ability for targeted and controlled release. The use of collagen peptide (CP) for the preparation of chitosan (CN) NPs is especially interesting as it results in NPs that are stable under physiological conditions. In this work, mono-dispersed pH responsive CPCN NPs of about 100 nm were prepared via ionic gelation method by simple and mild co-precipitation of CN and CP. Investigation of NPs with Fourier transform infra-red (FTIR) spectroscopy and dynamic light scattering (DLS) measurements reveals that hydrogen bonding and electrostatic interactions are believed to be major driving forces for NP formation and drug encapsulation, respectively. Scanning electron microscopic (SEM) investigations show that hard and fine CPCN NPs transform to soft and bigger gel like particles as a function of collagen concentration. The unique ``polymeric gel'' structure of NPs showed high encapsulation efficiency towards doxorubicin hydrochloride (DOX) as well as pH controlled release. Anti-proliferative and cell viability analysis revealed that DOX loaded NPs showed excellent anti-proliferative characteristics against HeLa cells with favorable biocompatibility against normal cells. Such NPs have high potential for use as smart drug delivery carriers in advanced cancer therapy. (C) 2016 Elsevier B.V. All rights reserved.

In the present study, a greener approach to tanning process based on Gallic acid (GA: Trihydroxy- benzoic acid) assisted chrome tanning has been attempted. The exhaustion, the thermal stability, mechanical strength and organoleptic properties of tanned leather as well as the evaluation of eco-friendly characteristics were investi- gated. The microshrinkage, differential scanning calori- metric and thermo mechanical analyses show the shrinkage temperature, denaturation temperature and % elongation, respectively, of GA-chrome-tanned leather are more than that of chrome alone. Chrome exhaustion greater than 93 % has been achieved. This high exhaust aid offers fullness and softness to leather compared to chrome alone. The environmental impact assessment reveals that the developed high exhaust chrome tanning process is benefi- cial as significant reduction in total solids content (TSC) such as dissolved solids and suspended solids in the effluent is achieved when compared to tanning with chrome alone. The GA could bring about the enhancement of chromium(III) uptake, significant reduction in TSC resulting in improved environmental, economic and social positive impact.

Aggregation of alpha-Synuclein (alpha-Syn) into amyloid fibrils is known to be associated with the pathogenesis of Parkinson's disease (PD). Several missense mutations of the a-Syn gene have been associated with rare, early onset familial forms of PD. Despite several studies done so far, the local/residue-level structure and dynamics of alpha-Syn in its soluble and aggregated fibril form and how these are affected by the familial PD associated mutations are still not clearly understood. Here, we review studies performed by our group as well as other research groups, where time-resolved fluorescence spectroscopy has been used to understand the site-specific structure and dynamics of alpha-Syn under physiological conditions as well as under conditions that alter the aggregation properties of the protein such as low pH, high temperature, presence of membrane mimics and familial PD associated mutations. These studies have provided important insights into the critical structural properties of alpha-Syn that may govern its aggregation. The review also highlights time-resolved fluorescence as a promising tool to study the critical conformational transitions associated with early oligomerization of alpha-Syn, which are otherwise not accessible using other commonly used techniques such as thioflavin T (ThT) binding assay.

Polychlorinated biphenyls (PCBs) comprise a ubiquitous class of toxic substances associated with carcinogenic and tumor-promoting effects as well as neurotoxic properties. Reactive oxygen species, which is produced from PCBs, alters blood–brain barrier (BBB) integrity, which is paralleled by cytoskeletal rearrangements and redistribution and disappearance of tight junction proteins (TJPs) like claudin-5 and occludin. Quercetin, a potent antioxidant present in onion and other vegetables, appears to protect brain cells against oxidative stress, a tissue-damaging process associated with Alzheimer’s and other neurodegenerative disorders. The aim of this study is to analyze the role of quercetin on oxidative stress markers and transcription of transmembrane and cytoplasmic accessory TJPs on cerebrum, cerebellum and hippocampus of female rats exposed to PCBs. Rats were divided into the following four groups. Group I: received only vehicle (corn oil) intraperitoneally (i.p.); group II: received Aroclor 1254 at a dose of 2 mg/kg body weight (bwt)/day (i.p); group III: received Aroclor 1254 (i.p.) and simultaneously quercetin 50 mg/kg bwt/day through gavage and group IV: received quercetin alone gavage. From the experiment, the levels of hydrogen peroxide, lipid peroxidation and thiobarbituric acid reactive substances were observed to increase significantly in cerebrum, cerebellum and hippocampus as 50%, 25% and 20%, respectively, after exposure to PCB, and the messenger RNA expression of TJP in rats exposed to PCBs is decreased and is retrieved to the normal level simultaneously in quercetin-treated rats. Hence, quercetin can be used as a preventive medicine to PCBs exposure and prevents neurodegenerative disorders.

The aim is to study the effect of Tartrazine (E102) - synthetic food colour - on the antioxidant status of testis of Wistar rats. Twelve male Wistar rats were grouped into 2 groups of six each - Control and Tartrazine-treated groups. Control group was orally administered with water alone while the experimental group was orally administered with tartrazine dissolved in water. The treatment was carried out for 60 days and the activities of antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) and the levels of their cofactors were subsequently determined in the testis, along with histological studies. Activities of the 4 enzymes showed a common decrease with corresponding alterations in their cofactor levels. The colour orally administered to the experimental animals probably would have generated reactive oxygen species (ROS) and H2O2, thereby disrupting the enzymatic antioxidant defense of their testes. Tartrazine is capable of producing free radicals, which in turn cause damage to the cellular compartment system of rat testis.

The protective role of lycopene against various toxicants on various organs and its association with decreased oxidative stress seems to be well established. Sertoli cell function in the adult testis determines daily sperm production. Increasing evidence suggests that environmental pollutants including polychlorinated biphenyls (PCBs) are male infertility, which is associated with oxidative stress. Hence, the present study was designed to find out whether PCBs disrupt Sertoli cell function by inducing oxidative stress or endocrine disruption? Whether lycopene attenuates PCBs-induced disruption in Sertoli cells or not? Adult male rats were divided into four (vehicle control, lycopene, PCBs and PCBs + lycopene treated) groups. After the experimental period (30 days), the animals were decapitated for the blood collection and testes were removed for the isolation of Sertoli cells. In PCBs exposure, serum hormones (FSH, testosterone and estradiol), Sertoli cellular receptors (FSHR and AR) were significantly decreased whereas oxidative stress markers (H 2 O 2 and LPO) and ER-s expression in Sertoli cells were significantly increased. Sertoli cell functional proteins such as connexin 43, transferrin and androgen-binding protein expressions and the level of lactate were significantly decreased. However, simultaneous supplementation of lycopene to PCBs-exposed rats significantly decreased the endocrine disruption and oxidative stress in Sertoli cells. The findings point out that lycopene protects the Sertoli cell function by preventing PCBs-induced oxidative stress and endocrine disruption.

Prostate cancer is a leading cause of death among the aging men. Surgical or radiotherapy is effective when the cancer is confined to the prostate gland but once the cancer spreads beyond the pelvis even chemotherapy and hormonal ablation therapy fails in curing this disease. Our previous studies have shown that diallyl disulfide (DADS) induces cell cycle arrest and also induces apoptosis in PC-3 cells. And now the present study is focused to see whether there is an activation of caspase cascade pathway. Hence, in the present study the apoptotic effect of DADS is studied by Western blot analysis of caspase-3, -9, -10 and Bcl-2, Bad, and Bax protein. The Apoptotic cells were assessed by Hoechst 33342 staining with 25 and 40 microM concentrations of DADS for 24 h. The results have shown that DADS at 25 and 40 microM concentrations has induced the activation of caspases. There is a significant increase in the expression of caspases (3, 9, and 10). The proapoptotic protein Bax has significantly increased at 40 microM of DADS treatment and there is significant increase of Bad protein at both the concentration. Bcl-2 protein has significantly decreased in DADS treated cells. Therefore, the present investigation serves as evidence that DADS may be a therapeutic drug in the treatment of prostate cancer.

The central role of collagen as the major structural fibrous protein in the mammalian extracellular matrix has motivated a significant effort toward the determi- nation of its mechanical properties at all levels, ranging from single monomers and long-chain polymers to a structural element within a biological tissue. However, the stabiliza- tion of collagen against collagenolytic degradation finds sig- nificance in biomedical and industrial applications. Tannins are plant-derived polyphenols that have the ability to inhibit the collagenase activity at minimum concentration. The in- hibitory effect of wattle (Acacia mollissima) and myrobalan (Terminalia chebula) on the action of collagenase against colla- gen was probed in this study. The kinetics of the inhibition of collagenase by wattle and myrobalan was deduced from the extent of hydrolysis of 2-furanacryloyl-L-leucyl-glycyl- L-prolyl-L-alanine. Both wattle and myrobalan tannin exhib- ited competitive modes of inhibition against collagenase. Circular dichroism studies of collagenase on treatment with wattle and myrobalan revealed changes in the secondary structure of collagenase. These results suggest that the tan- nins of A. mollissima and T. chebula extracts facilitated colla- gen stabilization through collagenase inhibition. 2007

Inhibitory effect of green tea polyphenols viz., catechin and epigallocatechin gallate (EGCG) on the action of collagenase against collagen has been probed in this study. Catechin and EGCG treated collagen exhibited 56 and 95% resistance, respectively, against collagenolytic hydrolysis by collagenase. Whereas direct interaction of catechin and EGCG with collagenase exhibited 70 and 88% inhibition, respectively, to collagenolytic activity of collagenase against collagen and the inhibition was found to be concentration dependent. The kinetics of inhibition of collagenase by catechin and EGCG has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. Both catechin and EGCG exhibited competitive mode of inhibition against collagenase. The change in the secondary structure of collagenase on treatment with catechin and EGCG has been monitored using circular dichroism spectropolarimeter. CD spectral studies showed significant changes in the secondary structure of collagenase on treatment with higher concentration of catechin and EGCG. Higher inhibition of EGCG compared to catechin has been attributed to the ability of EGCG to exhibit better hydrogen bonding and hydrophobic interaction with collagenase.

Various epidemiological survey suggests that the central nervous system is the target for many environmental contaminants. One among them is Aroclor 1254, a mixture of polychlorinated biphenyls (PCBs) which explore a spectrum of biochemical and neurotoxic responses in humans and laboratory animals. Learning and motor coordination deficits are the profound effects of PCBs which may be related to cerebral dysfunction. The aim of the study is to elicit the protective effect of melatonin (Mel), a potent, blood brain permeable antioxidant against the effect of Aroclor 1254 on the signaling of glutamate-principal excitatory neurotransmitter and brain derived neurotrophic factor (BDNF) in the cerebral cortex of adult rats which plays a key role in brain functions. Adult male Wistar rats were grouped into four and treated intraperitonealy (i.p) Group I with corn oil (Control), Group II with PCBs (2 mg/kg/bwt), Group III with PCBs + Mel (2 mg/kg/bwt + 5 mg/kg/bwt) and Group IV with Mel (5 mg/kg/bwt). The protein expression of glutamate signaling molecules and mRNA expressions of GLAST, BDNF signaling molecules were analyzed. The results suggest that simultaneous melatonin treatment significantly attenuated the NMDA receptor mediated glutamate excitotoxicity and protects the inhibition of BDNF signaling caused by PCBs exposure in cerebral cortex of adult male rats. Schematic pathway illustrating the proposed mechanism by which melatonin protects against A1254 mediated glutamate induced neurodegeneration in the cerebral cortex of adult male rats. PCBs induced neurodegeneration is caused by the overactivation of NMDAR, followed by the activation of voltage dependent calcium channels leading to the increase in intracellular Ca(2+) that stimulates calpain. Calpain inturn inhibits the PKA α and neurtrophin BDNF, its receptor and downstream signaling MAPK pathway leading to neurodegeneration. Melatonin had scavenged the ROS produced by PCBS and decreased the NMDAR expression which inturn protected the cells from neurodegeneration.

The spectral characteristics of 2-(4'-N,N-dimethylaminophenyl)-pyrido[3,4-d]imidazole (DMAPPI) have been studied in a TritonX-100 (TX-100)/n-hexanol/water reverse micelle in cyclohexane as a function of water (w(0)), surfactant, cosurfactant, pH, and trifluoroacetic acid. Under the neutral conditions, dual fluorescence (normal and twiste intramolecular charge transfer) is observed, even at w(0)=0, suggesting that the TICT state is stabilized by the hydrogen bonding from n-hexanol. These studies indicate that DMAPPI molecules are present near the interface of the water pool and the micellar phase toward the micellar side, and the changes observed in the spectral characteristics with change of w(0) are due to the formation of the reverse micelles and the alignment of cosurfactant around DMAPPI. Variation of pH in the range 3-10 has no effect on the spectral characteristics of DMAPPI, suggesting that the protons do not penetrate the reverse micelles, whereas the trifluoroacetic acid protonates DMAPPI to form monocations (MCs). At w(0)=0, MC2 and MC3 (see Scheme 1) are the MCs present both in the S(0) and S(1) states, whereas with an increase in w(0), the MC2 shifts toward MC1. Biprotonic phototautomerism is observed in MC1, which leads to the formation of MC2 in the S(1) state. Copyright 2000 Academic Press.

The fluorescence quenching of 2-aminofluorene by cetylpyridinium chloride (CPy), iodide ion (I − ) and acrylamide (AC) has been studied in different concentrations of Tween-20, Tween-40, Tween-60 and Tween-80 surfactants. The results show that CPy and I − ion bind as well as get partitioned, whereas AC only gets partitioned. The high values of binding constants for CPy in comparison to I − ion are due to the long hydrocarbon chain length. The binding and partitioning coefficients of CPy increase, whereas those of I − ion decrease with the increase in the Tween number. The values of partitioning coefficient of AC also decrease with increase in the Tween number. On the other hand, quenching rate constants for each quencher in each Tween decrease in going from Tween-20 to Tween-80.

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

Background: d-Serine is a coagonist for the glycine-binding site of the N-methyl-d-aspartate receptors and has been implicated in various neuropsychiatric functions such as learning, memory, and nociception, as well as schizophrenia and Alzheimer disease. We developed an HPLC method for d- and l-serine in cerebrospinal fluid (CSF). Methods: The dabsylated racemic serine peak, automatically collected using a previously reported HPLC separation process for CSF amino acids, was desalted and subjected to a chiral resolution HPLC step with a Sumichiral column using an ultraviolet-visible detector. Results: The limits of quantification (signal-to-noise ratio = 10) for d- and l-serine were 0.8 and 1.3 μmol/L, respectively. The mean imprecision values (CVs) for within-day measurements of d- and l-serine were 2.1% and 1.8%, respectively, and for between-day were 6.2% and 6.6%. Mean recovery of CSF serine (sum of d-serine + l-serine) applied to the Sumichiral column was 87%. The mean (SD) d-serine concentrations in 45 CSF samples obtained from 16 patients with chronic pain due to degenerative osteoarthritis of the knees, 16 with postherpetic neuralgia, and 13 with no pain were, respectively, 3.97 (0.44), 1.85 (0.21), and 2.72 (0.32) μmol/L. Conclusion: d- and l-serine can be quantified with ultraviolet-visible detection of dabsyl derivatives. The dabsyl derivatives are stable and allow duplicate analysis of CSF samples in multisample runs.

Macromolecular crowding is a distinguishing property of intracellular media. Knowledge on the structure and dynamics of a protein in a crowded environment is essential for a complete understanding of its function. Reduction in intermolecular space could cause structural and functional alterations. Here, we have studied a model protein barstar to see how polyethylene glycol (PEG)-induced crowding affects its various structural states (native, unfolded and molten-globule-like) with different extents of change in conformational heterogeneity. Intramolecular distances and distance distributions were determined by time-resolved Forster resonance energy transfer from Trp53 to several acceptor sites by analysis of fluorescence decay kinetics using the Maximum Entropy Method. We observed PEG-induced narrowing of population distributions along with shifting of populations towards more compact states. Structural compactness also resulted in the slowing down of internal dynamics of the protein as revealed by fluorescence anisotropy decay kinetics of the fluorophore IAEDANS attached at several sites.

Spectral characteristics of 2-(4'N,N-dimethylaminophenyl)benzimidazole have been studied as a function of surfactant concentration and as a function of acid concentration in three surfactants. Dual fluorescence is observed in all the micelles. Fluorescence intensities of the local emission (B band) and twisted intramolecular charge transfer (A band, TICT) increase by an 17-30% and 38 to 64% respectively. When dissolved in micelles lifetimes of both the states also increase in the presence of micelles. The increase in the fluorescence intensities is attributed to the decrease in the non-radiative decay constant. cmc of the surfactants can be determined from the variation in the fluorescence intensity and the lifetime data. The equilibrium constants are determined for the prototropic reactions of the fluorophore in all the micelles in S0 and S1 states and the values obtained are discussed. Copyright 1999 Academic Press.

Splittings coming from the hyperfine interaction with the next neighbors 13C and 31P in the ESR spectra of a number of cupric and vanadyl mixed ligand complexes have been measured in chloroform solutions at 25°C. The ligands diethyldithiocarbamate (dtc−), didthyldithiophosphate (dtp−), diphenyldithiophosphinate (dtpi−), N-benzoyl-N-diethyl-thiocarbamide (btc−) and xanthate (X−) are used in the present study. Using isotopically enriched dtc it was observed that the spin density on the 13C atoms of dtc− is increased at the expense of the spin density on the 31P atoms of dtp− when the mixed ligand complex Cu(dtc)(dtp) is formed. The percentage changes in the 31P splitting in the mixed ligand complexes of dtp and dtpi are equal. It was concluded that the transfer of spin to the next neighbor in vanadyl complexes is due to the direct overlap of the dxy orbital on the metal with the orbital on the next neighbor and that in the cupric complexes is due to configuration interaction. The differences in the 31p splittings observed in M(dtp)2 and M(dtpi)2 (M = Cu2+, VO2+) have been attributed to the differences in the s character of the hybrid orbital on the phosphorus.

The ESR spectra of semiquinones in frozen DMSO show motional averaging of anisotropic ESR parameters. The following observations support the liquidlike pocket model for the environment of semiquinones giving such spectra in these systems even up to 50° below the freezing points of the solutions: (i) The temperature dependence of linewidths and spin lattice relaxation times T1 of semiquinones in frozen DMSO are characterized by an activation energy ΔE?3.2 kcal mol−1. This value is close to the ΔE associated with the viscosity η[ = η0 exp (ΔE/RT)] of DMSO in the liquid state. (ii) The dependences of linewidths and T1 on the sizes of semiquinones are significantly different. Also, T1 calculated from linewidth data using the Hubbard relation do not agree with the experimentally observed values of T1. These behaviors are similar to that seen for semiquinones in liquid alcohols. (iii) There is good agreement between the η0 estimated in this model from the linewidth data or T1 data of semiquinones of different s...

Application of a temperature jump (2.5 degrees C) to a suspension of liposomes, having phosphate (delta pK/delta T approximately 0.005) as the internal buffer and tris(hydroxymethyl)aminomethane (delta pK/delta T approximately 0.031) as the external buffer, created a delta pH (pHin - pHout) of positive sign in ca. 5 microseconds. Decay of this delta pH was monitored by using the fluorescent pH indicator 8-hydroxy-1,3,6-pyrenetrisulfonic acid entrapped inside the liposome. This technique is useful to study transmembrane proton movement in the time range 5 microseconds-10 s at physiological pH values. The kinetics of proton transport aided by ion carriers such as nigericin, monensin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and valinomycin were studied by our method. The electrogenic nature of transport by CCCP and valinomycin and electroneutral ion transport by nigericin and monensin were shown. From the kinetics of proton transport aided by gramicidin, the time-averaged single-channel conductance of gramicidin channels was estimated to be (2.1 +/- 0.5) X 10(-16) S for H+ at pH 7.5.

Spectrophotometric techniques have been employed to study the binding of bromophenol red (BPR) to hen egg white lysozyme and the consequent inhibition of enzyme activity. Experimental evidence is given from the dye binding studies in the presence of hexasaccharide and from the studies on activity that BPR binds at a site outside the proposed cleft region (A–F) in such a way that it inhibits the lytic activity towards cell walls but does not inhibit the activity towards hexasaccharide. These observations are consistent with the kinetics of binding [studied using temperature-jump (T-jump)] in the presence of Co++ or chitotriose in large concentrations and the experiments with acetylated lysozyme which suggest that the binding site of BPR is closer to a lysine residue near the cleft. It is suggested that the binding site of BPR could be important in positioning the peptide segment of the cell walls, which are cleaved in the cleft. Evidence for the statement that this binding takes place at least by a two-step process, in which the bimolecular step is followed by a slower monomolecular step, is given from the observations of two types of 1:1 complexes at 24°C in equilibrium studies and from the concentration dependence of the relaxation observed at 605 nm in the T-jump experiments. The binding process is examined by analyzing the T-jump data obtained between 18 and 33°C in the pH range 5.2–9.2 and ionic strength 0.01–01. The ionic strength and pH dependences of the equilibrium constant associated with the bimolecular step k2/k1 and the forward rate constant associated with monomolecular step k3 have been given as evidence for the suggestion that a Coulombic interaction is involved in the first step of binding. However, the final state of binding is hydrophobic in nature. The enthalpy of activation ΔH and the entropy of activation ΔS associated with kf[= k3(k1/k2)] showed compensation behavior with pH variation, with maxima around pH ∼ 7.5 in H2O. This has been interpreted as a maximal disordering of water structure in a region of the enzyme at this pH during the monomolecular step. However, the binding of chitotriose or Co++ in the cleft reduces the ΔH and ΔS associated with the monomolecular step of BPR binding, probably by disordering the structured water during their binding in the cleft. The differences in the kinetic parameters obtained in H2O and in D2O probably arise due to subtle differences in the conformation of the enzyme in the two solvents and apart from isotope effects. The correlation between the pH (or pD) dependence of the “intrinsic activity” towards cell walls and ΔH or ΔS indicates that ordered water structure could be playing a role in controlling the catalytic activity. It is also suggested that this factor is associated with the rate constant k3s of the monomolecular step leading to the formation of the final bound state of the substrate in cell lysis, which is also a factor controlling kcat.

Spectrophotometric observations have been used to study equilibria and temperature jump relaxation associated with formation of Fe(III) complexes in aqueous DMSO in the presence of SCN − . The [SCN − ] dependent relaxation observed at 315 nm could be assigned to the formation of Fe DMSO 3+ , Fe DMSO OH 2+ , Fe DMSO SCN 2+ and Fe DMSO SCN OH + from Fe 2+ and Fe OH 2+ . The relaxation observed at 450 nm could be assigned to the formation of Fe SCN 2+ by paths similar to that in aqueous solutions and a pH dependent path in which the rate determining step is the displacement of DMSO from Fe DMSO SCN OH + by H 2 O. The rate constants suggest that coordination by a DMSO instead of a H 2 O has the effect of labilising the other H 2 O coordinated to Fe OH 2+ . The observed decrease in relaxation rates with increase in [DMSO] have been explained as dominantly due to the reduction in the acid dissociation constant K H associated with the aquocomplex of Fe(III).

A large number of mixed ligand complexes of copper with di-i-propyldiselenophosphate as one of the ligands have been prepared by ligand exchange reactions and have been identified using electron spin resonance. From the measured ESR parameters in chloroform solutions it was concluded that (i) the measuredgo (and notACu) could be used to identify the mixed ligand complexes (in addition to ligand hyperfine splittings) (ii) the changes in spin transfer to77Se and31P on the same ligand dSeP are uncorrelated suggesting that the dominant mechanism of spin transfer to near neighbours77Se is different from that to the next neighbour31P. (iii) Observations on31P splittings show that on formation of mixed ligand complex, the spin density on one ligand increases at the expense of other.

Electron transfer activities and steady state reduction levels of Fe-S centers of NADH-Q oxidoreductase were measured in mitochondria, submitochondrial particles (ETPH), and complex I after treatment with various reagents. p-Chloromercuribenzenesulfonate destroyed the signal from center N-4 (gx = 1.88) in ETPH but not in mitochondria, showing that N-4 is accessible only from the matrix side of the inner membrane. N-Bromosuccinimide also destroyed the signal from N-4 but without inhibiting rotenone-sensitive electron transfer to quinone, suggesting a branched pathway for electron transfer. Diethylpyrocarbonate caused oxidation of N-3 and N-4 in the steady state without changing N-1, suggesting N-1 is before N-3 and N-4. Difluorodinitrobenzene and dicyclohexylcarbodiimide inhibited oxidation of all Fe-S centers and tetranitromethane inhibited reduction of all Fe-S centers. Titrations of the rate of superoxide (O2-) generation in rotenone-treated submitochondrial particles were similar with the ratio [NADH]/[NAD] and that of 3-acetyl pyridine adenine nucleotide in spite of different midpoint potentials of the two couples. On reaction with inhibitors the inhibition of O2- formation was similar to that of ferricyanide reductase rather than quinone reductase. The rate of O2- formation during ATP-driven reverse electron transfer was 16% of the rate observed with NADH. The presence of NAD increased the rate to 83%. The results suggest that bound, reduced nucleotide, probably E-NAD., is the main source of O2- in NADH dehydrogenase. The effect of ATP on the reduction levels of Fe-S centers in well-coupled ETPH was measured by equilibrating with either NADH/NAD or succinate/fumarate redox couples. With NADH/NAD none of the Fe-S centers showed ATP induced changes, but with succinate/fumarate all centers showed ATP-driven reduction with or without NAD present. The effect on N-2 was smaller than that on N-1, N-3, and N-4. These observations indicate that the major coupling interaction is between N-2 and the low potential centers, N-1, N-3, and N-4. Possible schemes of coupling in this segment are discussed.

The g values of 1,4-benzosemiquinone and durosemiquinone anions in methanol, ethanol, and n -butanol have been measured at temperatures T , above the freezing point of the solvents. The rapid variation of g with T in the high-viscosity region has been assigned to slow tumbling effects. The gradual increase of g with an increase in T has been assigned to an equilibrium between the hydrogen-bonded and non-hydrogen-bonded semiquinones. This has been confirmed from a study of g of DSQ in ethanol-DME, ethanol-acetonitrile, and ethanol-DMSO mixtures. Combining the results of the earlier electron spin relaxation studies with the estimated fraction of the time for which the semiquinone is not hydrogen-bonded and the enthalpy change associated with the above-mentioned reaction (∼1 kcal mole −1 ), it was concluded that in alcohols (i) in the temperature range studied there is a greater probability of finding the semiquinone in the hydrogen-bonded state; (ii) it can go to a non-hydrogen-bonded state without requiring energies of the order of hydrogen-bonding energies; (iii) only a fraction of the non-hydrogen-bonded sate corresponds to a state of free rotation; (iv) this free rotation is different from the large-step motion associated with the reorientation; (v) the descriptions of the hindered rotation of the semiquinone in alcohols in the liquid state and the frozen state are likely to be similar. The value 1 kcal mole −1 corresponds to the energy difference between the two hydrogen-bonded states of an alcohol molecule in the neighborhood of the semiquinone. In the first hydrogen-bonded state it is bonded to the semiquinone, while in the second hydrogen-bonded state it is bonded to neighboring alcohol molecules, forming a polymer but leaving the semiquinone nonhydrogen-bonded.

The formation of FeL2+ (L−=N 3 − , SCN−) in H2O-dimethylsulphoxide (DMSO) mixtures was studied by equilibrium, stopped flow and temperature jump observations. The acid dissociation constants associated with LH and Fe(H2O) 6 3+ and the formation constants associated with FeL2+ were determined at various solvent compositions. In kinetic studies, the solvent composition dependence of the overall equilibration rate was explained in terms of equations predicted by a reaction scheme which includes DMSO coordinated species. Analysis of results on the basis of these equations identified Fe·OH2+·HN3 and Fe3+·SCN− as the dominant transition states contributing to pH-independent paths of formation of FeL2+ in the cases of N 3 − and SCN− respectively. Thus, the results which support the Eigen-Tamm-Wilkins mechanism have resolved the mechanistic ambiguity in the pH-independent paths of formation of FeL2+. It is also shown that we cannot neglect species such as FeOH·HN 3 2+ , Fe·OH·N 3 + and Fe·HN 3 3+ while accounting for the kinetic behaviour.