Search

An investigation on joining of carbon fiber-reinforced thermoplastics to an aluminum alloy 6061 has been demonstrated using ultrasonic plastic welding process. A separate energy director, as requir...

N-methyl-D-aspartate receptors (NMDAR) are downregulated in schizophrenia possibly through microRNAs (miRNAs) that are differentially expressed in this condition. We screened the miRNAs that are altered in schizophrenia against the targets, Grin2A and Grin2B subunits of NMDAR using bioinformatic tools. Among the predicted miRNAs some interacted with the 3’-UTR sequences of Grin2A (miR-296, miR-148b, miR-129-2, miR-137) and Grin2B (miR-296, miR-148b, miR-129-2, miR-223) in dual luciferase assays. This was supported by downregulation of the GluN2B protein in primary hippocampal neurons upon overexpressing Grin2B targeting miRNAs. In two models of schizophrenia-pharmacological MK-801 model and neurodevelopmental methylazoxymethanol acetate (MAM) model which showed cognitive deficits - protein levels of GluN2A and GluN2B were downregulated but their transcript levels were upregulated. MiR-296-3p, miR-148b-5p and miR-137 levels showed upregulation in both models which could have interacted with Grin2A/Grin2B transcripts resulting in translational arrest. In MAM model, reciprocal changes in the expression of the 3p and 5p forms of miR-148b and miR-137 were observed. Expression of neuregulin 1 (NRG1), BDNF and CaMKIIα, genes implicated in schizophrenia, were also altered in these models. This is the first report of downregulation of GluN2A and GluN2B by miR-296, miR-148b and miR-129-2. Mining miRNAs regulating NMDA receptors might give insights into the pathophysiology of this disorder, providing avenues in therapeutics.

This investigation was focused on hard turning performed under protective gas atmosphere with Argon, since this gas provides more heat sink at the thermally mechanically affected zone (TMAZ) during the hard turning conducted with semi worn-out coated CBN insert. The innate qualities of this inert gas effectively curbed the mechanisms, which are responsible for the formation of white layer, by ensuring more heat dissipation through vaporization, regulating cooling through convection and protection against environmental reactions. The recently conducted tests and their notable results have for sure established the remarkable role of this abundantly available and harmless gas in enhancing the surface reliability and the effective elimination of white layer formation.

The complex and multifactorial nature of neuropsychiatric diseases demands multi-target drugs that can intervene with various sub-pathologies underlying disease progression. Targeting the impairments in cholinergic and glutamatergic neurotransmissions with small molecules has been suggested as one of the potential disease-modifying approaches for Alzheimer’s disease (AD). Tacrine, apotent inhibitor of acetylcholinesterase (AChE) is the first FDA approved drug for the treatment of AD. Tacrine is also a low affinity antagonist of N-methyl-D-aspartate receptor (NMDAR). However, tacrine was withdrawn from its clinical use later due to its hepato-toxicity. With an aim to develop novel high affinity multi-target directed ligands (MTDLs) against AChE and NMDAR, with reduced hepatotoxicity, we performed in silico structure-based modifications on tacrine, chemical synthesis of the derivatives and in vitro validation of their activities. Nineteen such derivatives showed inhibition with IC50 values in the range of 18.53±2.09 to 184.09±19.23 nM against AChE and 0.27±0.05 to 38.84±9.64 μM against NMDAR. Some of the selected compounds also protected rat primary cortical neurons from glutamate induced excitotoxicity. Two of the tacrine derived MTDLs, 201 and 208 exhibited in vivo efficacy in rats by protecting against behavioral impairment induced by administration of the excitotoxic agent, monosodium glutamate. Additionally, several of these synthesized compounds also exhibited promising inhibitory activities against butyrylcholinesterase and β-secretase. Given the therapeutic potential of MTDLs in disease-modifying therapy, our studies revealed several promising MTDLs of which 201 appears to be a potential candidate for immediate preclinical and clinical evaluations.

Alzheimer's disease (AD) is an obstinate and progressive neurodegenerative disorder, mainly characterized by cognitive decline. Increasing number of AD patients and the lack of promising treatment strategies demands novel therapeutic agents to combat various disease pathologies in AD. Recent progresses in understanding molecular mechanisms in AD helped researchers to streamline the various therapeutic approaches. Inhibiting acetylcholinesterase (AChE) activity has emerged as one of the potential treatment strategies. The present study discusses the identification of two potent AChE inhibitors (ZINC11709541 and ZINC11996936) from ZINC database through conventional in silico approaches and their in vitro validations. These inhibitors have strong preferences towards AChE than butyrylcholinesterase (BChE) and didn't evoke any significant reduction in the cell viability of HEK-293 cells and primary cortical neurons. Furthermore, promising neuroprotective properties has also been displayed against glutamate induced excitotoxicity in primary cortical neurons. The present study proposes two potential drug lead compounds for the treatment of AD, that can be used for further studies and preclinical evaluation.

The objective of this work is to develop a natural fiber-reinforced epoxy composite with enhanced compatibility between resin and the fiber, achieved by amino silane treatment of Luffa fiber. Amine modification on the surface of the Luffa fiber is confirmed by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Composites with different volume fractions (2%, 4%, 6%, and 8%) of amine functionalized/un-functionalized Luffa fiber and epoxy resin are fabricated. The functionalized/un-functionalized Luffa epoxy composites are subjected to various studies such as tensile, flexural, and impact in the area of the effect of amine functionalization of fiber/epoxy composites. Dynamic mechanical analysis and fatigue analysis are carried out to enable a study on the effect of amine functionalization. Variations in thermal stability of the composites are studied using TGA analysis. A maximum tensile strength value of 18.3 MPa is reached for the 6% amine functionalized composite compared to the plain epoxy of 9.4 MPa. The amine functionalized fiber-reinforced composites show improved thermal, mechanical, and morphological properties as a result of improved interaction between the fiber and matrix.

Background: Mineral insufficiencies may have far-reaching consequences both systemically and orally. The exact associations and roles of many of the elements are not unequivocally examined till now as far as chronic periodontitis (CP) is concerned. Aim: The present study was designed to evaluate and compare the serum levels of magnesium and zinc in CP cases as compared to healthy controls. Settings and Design: This study was conducted in the Department of Periodontology in MIDSR Dental College and Hospital, Latur. It was a cross-sectional study. Materials and Methods: This study included 60 individuals, 30 in each group, i.e. Group I –healthy controls and Group II – Generalized CP. Serum magnesium and serum zinc levels were analyzed using reagent kits. Statistical Analysis: The results obtained were analyzed by applying unpaired Student's t test. Results: Serum levels of magnesium and zinc were 1.30 + 0.78 mEq/L and 62.10 + 18.76 ug/dl, respectively, in CP cases and 1.89 + 0.48 mEq/L and 92.51 + 15.64 ug/dl, respectively, in healthy controls. Conclusion: The mean serum levels of magnesium and zinc were significantly altered in CP cases as compared to healthy controls.

Powder metallurgy is a popular technique to synthesize metal matrix composites with uniform distribution of the reinforcements. The present work aims to synthesize Al-B4C composites through powder metallurgy route and study their properties. The compositions of the composites are Al-5%B4C, Al-10%B4C & Al-15%B4C. Blending of powders, compaction and sintering process were carried out as per standard powder metallurgy procedure. The distributions of the reinforcements were analyzed for the sintered specimens by the help of scanning electron microscope. The effect addition of B4C in the Aluminium matrix on the properties such as density, porosity, hardness and compressive strength were discussed. Results show that addition of B4C improves the properties of Al-B4C composites.

A two-component hybrid system consisting of organic dye nanoparticles (ash) and a protein (blue) at different ratios allows for the design of narrow range tunable nanoprobes for sensing and imaging of pH variations in live cells through ratiometric signalling., Accurate monitoring of pH variations inside cells is important for the early diagnosis of diseases such as cancer. Even though a variety of different pH sensors are available, construction of a custom-made sensor array for measuring minute variations in a narrow biological pH window, using easily available constituents, is a challenge. Here we report two-component hybrid sensors derived from a protein and organic dye nanoparticles whose sensitivity range can be tuned by choosing different ratios of the components, to monitor the minute pH variations in a given system. The dye interacts noncovalently with the protein at lower pH and covalently at higher pH, triggering two distinguishable fluorescent signals at 700 and 480 nm, respectively. The pH sensitivity region of the probe can be tuned for every unit of the pH window resulting in custom-made pH sensors. These narrow range tunable pH sensors have been used to monitor pH variations in HeLa cells using the fluorescence imaging technique.

Accurate monitoring of pH variations inside cells is important for the early diagnosis of diseases such as cancer. Even though a variety of different pH sensors are available, construction of a custom-made sensor array for measuring minute variations in a narrow biological pH window, using easily available constituents, is a challenge. Here we report two-component hybrid sensors derived from a protein and organic dye nanoparticles whose sensitivity range can be tuned by choosing different ratios of the components, to monitor the minute pH variations in a given system. The dye interacts noncovalently with the protein at lower pH and covalently at higher pH, triggering two distinguishable fluorescent signals at 700 and 480 nm, respectively. The pH sensitivity region of the probe can be tuned for every unit of the pH window resulting in custom-made pH sensors. These narrow range tunable pH sensors have been used to monitor pH variations in HeLa cells using the fluorescence imaging technique.

Luffa fibers were silane functionalized using varied concentrations of 3-aminopropyl trimethoxysilane (APTMS) in methanol solution to investigate the relationship between the concentration of silanizing agent and the effective degree of silanization of luffa fibers. The aim of the present work was to identify the optimal silane concentration to adequately functionalize the luffa fibers and to investigate its influence on the properties of luffa-epoxy composites. The degree of silanization was controlled by varying the weight ratios of fiber and APTMS (1:0.5, 1:1, 1:1.5, and 1:2). The optimal degree of silanization was confirmed by testing the mechanical, thermal, and flammable properties of functionalized luffa fiber-filled epoxy matrix composites. The composite with 1:1.5 w/w silane functionalization exhibited a maximum tensile strength of 18.4 ± 0.94 MPa. The interfacial adhesion of the prepared samples is investigated through morphological studies. Thermal studies indicated that thermal stability increased with an increase in silanization. TGA analysis revealed that the silane treatment improved the thermal profile of the composites with initial degradation above 240 °C. The storage modulus of composites decreased with an increase in temperature with a 3-stage pattern of the slope. The storage modulus and Tg increased with an increase in silanization degree and the loss modulus peak shifted towards higher temperature with the increase in silanization. Flame retardant studies show that the optimized composite exhibits V-1 rating suggesting the suitability of the prepared composite for various applications.

Memory formation transpires to be by activation and persistent modification of synapses. A chain of biochemical events accompany synaptic activation and culminate in memory formation. These biochemical events are steered by interplay and modulation of various synaptic proteins, achieved by conformational changes and phosphorylation/dephosphorylation of these proteins. Calcium/calmodulin dependent protein kinase II (CaMKII) and N-methyl-d-aspartate receptors (NMDARs) are synaptic proteins whose interactions play a pivotal role in learning and memory process. Catalytic activity of CaMKII is modulated upon its interaction with the GluN2B subunit of NMDAR. The structural basis of this interaction is not clearly understood. We have investigated the role of Glu60 of α-CaMKII, a conserved residue present in the ATP binding region of kinases, in the regulation of catalysis of CaMKII by GluN2B. Mutation of Glu60 to Gly exerts different effects on the kinetic parameters of phosphorylation of GluN2B and GluN2A, of which only GluN2B binds to the T-site of CaMKII. GluN2B induced modulation of the kinetic parameters of peptide substrate was altered in the E60G mutant. The mutation almost abolished the modulation of the apparent Km value for protein substrate. However, although kinetic parameters for ATP were altered by mutating Glu60, modulation of the apparent Km value for ATP by GluN2B seen in WT was exhibited by the E60G mutant of α-CaMKII. Hence our results posit that the communication of the T-site of CaMKII with protein substrate binding region of active site is mediated through Glu60 while the communication of the T-site with the ATP binding region is not entirely dependent on Glu60.

Cellular calcium signaling events are transient. Hence they are observed in real time using fluorescence imaging or electrophysiological methods that require sophisticated instrumentation and specialized skills. For high throughput assays simple and inexpensive techniques are desirable. Many calcium channels that serve as drug targets have subtypes arising from diverse subunit combinations. These need to be targeted selectively for achieving efficacy and for avoiding side effects in therapies. This in turn increases the number of calcium channels that act as drug targets. We report a novel method for intracellular calcium sensing that utilizes the calcium dependent stable interaction between CaM kinase II (CaMKII) and its ligands such as the NMDA receptor subunit GluN2B. The CaMKII-GluN2B complex formed persists as a memory of the transient increase in calcium. In a cell-based assay system GFP-α-CaMKII expressed in the cytosol responds to calcium by translocating towards GluN2B sequence motif exogenously expressed on mitochondria or endoplasmic reticulum. The resulting punctate fluorescence pattern serves as the signal for intracellular calcium release. The pattern is stable, unaffected by sample processing and is observable without real time imaging. The activities of calcium channel proteins heterologously expressed in HEK-293 cells were detected with specificity using this technique. A calcium sensor vector and a calcium sensor cell line were developed as tools to perform this technique. This technique being simple and less expensive could significantly facilitate high throughput screening in calcium channel drug discovery.

A novel series of twenty four tacrine derivatives were designed and synthesised. Among these, thirteen were taken for the acetylcholinesterase (AChE) inhibition studies. Three compounds such as 4c, 6c and 6f were found to possess significant AChE inhibitory properties with IC50 values 12.97 ± 0.47 nM, 5.17 ± 0.24 nM and 7.14 ± 0.78 nM respectively. In silico docking studies revealed that these compounds can bind strongly in the active site of the enzyme and prevent enzyme-substrate interactions. On binding, the substituted groups were oriented either towards the peripheral anionic site (PAS) (Pocket A) or towards a hydrophobic cavity (pocket B) located near the active site. The cytotoxicity and hepatotoxicity of the compounds were tested using HEK-293 and HepG2 cell lines respectively. The compound 4c did not show any significant decrease in the cell viability even at a concentration of 300 μM indicating that its cytotoxicity and hepatotoxicity are significantly lesser compared to tacrine, due to the chemical modification. Based on the available results, it can be suggested that the compound 4c might be a potential drug lead compound with AChE inhibitory activity. However, further pharmacokinetic studies are necessary to comment on the efficacy of the compound as a drug for AD.

Calcium influx through N-methyl-D-aspartate receptors (NMDAR) and voltage-gated calcium channels (VGCC) play major roles in postsynaptic signaling mechanisms. NMDAR subunit GluN2B is phosphorylated at Ser1303. Phosphorylation at this site is a prominent event in cell culture systems as well as in vivo. However, the functional significance of phosphorylation at this site is not completely understood. In this study, we compared the effect of calcium signaling through NMDAR and VGCC on the phosphorylation status of GluN2B-Ser1303 in the rat in vivo. VGCC was activated by intraperitoneal (IP) injection of the activator, BayK8644 and NMDAR was activated by intracerebroventricular (ICV) injection of NMDA in separate experimental groups. We found that the level of phospho-GluN2B-Ser1303 in the cortex and in the hippocampus increased in response to activation of either channel. The effects could be prevented by prior ICV administration of the specific blockers of these channels such as MK-801 for NMDAR and nifedipine for VGCC. The effect was also blocked by pretreatment with ICV administration of KN-93 indicating that it is mediated through CaM kinase. Both during NMDAR activation and VGCC activation, cell survival associated signals such as phospho-AKT and phospho-CREB showed decrease, consistent with activation of cell death pathways during these treatments. We conclude that under in vivo conditions, calcium influx through either NMDAR or VGCC activates CaM kinase, which in turn phosphorylates GluN2B-Ser1303.

Calcium/calmodulin dependent protein kinase II (CaMKII) is involved in the mechanisms underlying higher order brain functions such as learning and memory. CaMKII participates in pathological glutamate signaling also, since it is activated by calcium influx through the N-methyl-d-aspartate type glutamate receptor (NMDAR). In our attempt to identify phytomodulators of CaMKII, we observed that curcumin, a constituent of turmeric and its analogs inhibit the Ca(2+)-dependent and independent kinase activities of CaMKII. We further report that a heterocyclic analog of curcumin I, (3,5-bis[β-(4-hydroxy-3-methoxyphenyl)ethenyl]pyrazole), named as pyrazole-curcumin, is a more potent inhibitor of CaMKII than curcumin. Microwave assisted, rapid synthesis of curcumin I and its heterocyclic analogues is also reported.

Gold nanoparticles of 20-100 nm diameter were synthesized within HEK-293 (human embryonic kidney), HeLa (human cervical cancer), SiHa (human cervical cancer), and SKNSH (human neuroblastoma) cells. Incubation of 1 mM tetrachloroaurate solution, prepared in phosphate buffered saline (PBS), pH 7.4, with human cells grown to ?80% confluency yielded systematic growth of nanoparticles over a period of 96 h. The cells, stained due to nanoparticle growth, were adherent to the bottom of the wells of the tissue culture plates, with their morphology preserved, indicating that the cell membrane was intact. Transmission electron microscopy of ultrathin sections showed the presence of nanoparticles within the cytoplasm and in the nucleus, the latter being much smaller in dimension. Scanning near field microscopic images confirmed the growth of large particles within the cytoplasm. Normal cells gave UV-visible signatures of higher intensity than the cancer cells. Differences in the cellular metabolism of cancer and noncancer cells were manifested, presumably in their ability to carry out the reduction process. ? 2005 American Chemical Society.

The neuronal enzyme Calcium/calmodulin dependent protein kinase type II (CaMKII) is a key molecule in biochemical events necessary for learning and memory. The alpha-subunit of CaMKII expressed in E. coli as well as in insect cells shows similar catalytic behavior [Praseeda, M., Pradeep, K. K., Krupa, A., Sri Krishna, S., Leena, S., Rajeev Kumar, R., John Cheriyan, Mayadevi, M., Srinivasan, N., and Omkumar, R. V. (2003) Biochem. J. In Press]. The association domain of the enzyme has been crystallized in its native multimeric form after expression in E. coli [Hoelz, A., Nairn, A. C. and Kuriyan, J. (2003) Molecular Cell 11, 1241]. However a major truncation product accompanies the full-length protein when expressed in E. coli. We show by epitope labeling and immunoblotting that the truncation occurs at the C-terminal half of the protein so that the N-terminal catalytic domain is complete in the truncated product. This supports the use of the preparation of alpha-CaMKII expressed in E. coli for studies on functions of the catalytic site. Our data will also be helpful in designing modified prokaryotic expression systems for CaMKII devoid of the trun-cation product, which are easier to use compared to the insect cell system.

Autoxidation of pyrogallol in alkaline medium is characterized by increases in oxygen consumption, absorbance at 440 nm, and absorbance at 600 nm. The primary products are H2O2 by reduction of O-2 and pyrogallol-ortho-quinone by oxidation of pyrogallol. About 20 % of the consumed oxygen was used for ring opening leading to the bicyclic product, purpurogallin-quinone (PPQ). The absorbance peak at 440 nm representing the quinone end-products increased throughout at a constant rate. Prolonged incubation of pyrogallol in alkali yielded a product with ESR signal. In contrast the absorbance peak at 600 nm increased to a maximum and then declined after oxygen consumption ceased. This represents quinhydrone charge-transfer complexes as similar peak instantly appeared on mixing pyrogallol with benzoquinones, and these were ESR-silent. Superoxide dismutase inhibition of pyrogallol autoxidation spared the substrates, pyrogallol, and oxygen, indicating that an early step is the target. The SOD concentration-dependent extent of decrease in the autoxidation rate remained the same regardless of higher control rates at pyrogallol concentrations above 0.2 mM. This gave the clue that SOD is catalyzing a reaction that annuls the forward electron transfer step that produces superoxide and pyrogallol-semiquinone, both oxygen radicals. By dismutating these oxygen radicals, an action it is known for, SOD can reverse autoxidation, echoing the reported proposal of superoxide:semiquinone oxidoreductase activity for SOD. The following insights emerged out of these studies. The end-product of pyrogallol autoxidation is PPQ, and not purpurogallin. The quinone products instantly form quinhydrone complexes. These decompose into undefined humic acid-like complexes as late products after cessation of oxygen consumption. SOD catalyzes reversal of autoxidation manifesting as its inhibition. SOD saves catechols from autoxidation and extends their bioavailability.

Interaction of GluN2B subunit of N-methyl-D-aspartate receptor with calcium/calmodulin dependent protein kinase II (CaMKII) is critical for the induction of long term potentiation at hippocampal CA3-CA1 synapses. We have previously reported that CaMKII binding to GluN2B increases its affinity but abolishes the cooperativity for ATP. In the present study, we demonstrate that the reduction in S(0.5) for ATP of an individual CaMKII subunit seems to be directly induced by the binding of GluN2B to the same subunit, while any GluN2B induced effects on the cooperativity and maximal velocity would additionally require the CaMKII holoenzyme structure. We measured the apparent kinetic parameters for ATP using an association domain truncated monomeric CaMKII and a heteromultimeric CaMKII (having subunits that are either GluN2B binding defective or ATP binding defective), in the presence of GluN2A or GluN2B substrates. The S(0.5) value for ATP of monomeric CaMKII is reduced ∼ 3 fold by the presence of GluN2B suggesting that the induced change in affinity for ATP is independent of the holoenzyme structure. The heteromultimeric mutant of CaMKII, did not exhibit cooperativity of ATP binding probably because of the interspersing of ATP binding defective subunits in the holoenzyme. In contrast to the wild type holoenzyme, presence of GluN2B increased the V(max) of monomeric CaMKII which resulted in an approximately 4.0 fold increase in the apparent catalytic constant (V(max)/S(0.5)) as compared to GluN2A. The kinetic parameter values of the heteromultimeric CaMKII for ATP, on the other hand, did not show any significant difference between the phosphorylation of GluN2B and GluN2A suggesting that modulation requires binding of GluN2B to the same subunit. Overall, our present study provides insights into the role of multimeric structure of CaMKII in GluN2B-mediated regulation.

The attenuation of catalytic activity that accompanies phosphorylation of Ser871 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) reflects primarily the introduction of negative charge (Omkumar, R. V., Darnay, B. G., and Rodwell, V. W. (1994) J. Biol. Chem. 269, 6810-6814). To investigate how a negative charge at position 871 attenuates activity, we phosphorylated wild-type and mutant HMG-CoA reductases and assayed reduction of the putative intermediate mevaldehyde to mevalonate. We observed attenuated activity when the phosphorylated wild-type enzyme was assayed in the presence or absence of coenzyme A, but not when assayed in the presence of desthio-CoA. These observations recall the behavior of mutant enzyme H865Q, for which coenzyme A inhibits, whereas desthio-CoA stimulates mevaldehyde reduction (Frimpong, K. F., and Rodwell, V. W. (1994) J. Biol. Chem. 269, 11478-11483). Catalysis of mevaldehyde reduction by mutant enzyme H865Q was unaffected by phosphorylation. By contrast, mutant enzymes H860Q and H868Y, in which nearby, but noncatalytic, histidines had been mutated, exhibited wild-type behavior upon phosphorylation. We conclude that the introduction of negative charge at position 871 impairs the function of His865, presumably by a specific electrostatic interaction. We propose a novel mechanism by which phosphorylation regulates activity. Phosphorylation of the terminal serine of the consensus AGxLV(K/R)SHMxxNRS motif of eukaryotic HMG-CoA reductases attenuates activity by impairing the ability of the catalytic histidine to protonate the CoAS- anion formed during the reductive deacylation of HMG-CoA to mevaldehyde.

Attenuation of Syrian hamster 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) activity by in vitro phosphorylation was studied using AMP-activated protein kinase and wild-type and mutant forms of HMG-CoA reductase. The only residue of the wild-type enzyme phosphorylated was Ser871. Substrates protected against kinase-mediated attenuation of activity, consistent with substrate-induced conformational changes at the C-terminal region. Although close to the catalytic histidine His865, Ser871 appears to play no direct role in catalysis or substrate recognition. Mutant enzymes S871A, S871H, S871N, and S871Q exhibited from 62-106% of wild-type activity and had wild-type Km values for HMG-CoA and NADPH. Replacement of Ser871 by aspartate or glutamate, but not by glutamine, asparagine, histidine, or tyrosine, severely attenuated activity. Attenuation of catalytic activity that accompanies phosphorylation thus appears to result primarily from the introduction of negative charge, not merely steric hindrance. Other than the wild-type enzyme, only mutant enzyme S871T was phosphorylated, and phosphorylation was accompanied by attenuation of activity. The AMP-activated kinase thus can also phosphorylate threonyl residues.

A concentration-dependent inactivation of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase was found on preincubation of rat liver microsomal preparations with H2O2 and at lower concentrations in the presence of KCN which inhibited the contaminating catalase. The inactivation was not affected in the presence quenchers of hydroxyl radicals and singlet oxygen and was also obtained when H2O2 was added during the reaction. HMG-CoA, but not NADPH, partially protected the enzyme from H2O2-inactivation. Even at high concentration DTT was unable to reverse this inactivation. The soluble 50 kDa-enzyme was similarly inactivated by H2O2, and the tryptic-digest of the inactivated protein indicated the presence of a disulfide-containing peptide. The results support the view that H2O2 by directly acting on the catalytic domain possibly converts an active thiol group to an inaccessible disulfide and irreversibly inactivates HMG-CoA reductase.

A novel interfacial route has been developed for the synthesis of a bright-red-emitting new subnanocluster, Au23, by the core etching of a widely explored and more stable cluster, Au25SG18 (in which SG is glutathione thiolate). A slight modification of this procedure results in the formation of two other known subnanoclusters, Au22 and Au33. Whereas Au22 and Au23 are water soluble and brightly fluorescent with quantum yields of 2.5 and 1.3%, respectively, Au33 is organic soluble and less fluorescent, with a quantum yield of 0.1%. Au23 exhibits quenching of fluorescence selectively in the presence of Cu2+ ions and it can therefore be used as a metal-ion sensor. Aqueous- to organic-phase transfer of Au23 has been carried out with fluorescence enhancement. Sol-vent dependency on the fluorescence of Au23 before and after phase transfer has been studied extensively and the quantum yield of the cluster varies with the solvent used. The temperature response of Au23 emission has been demonstrated. The inherent fluorescence of Au23 was used for imaging human hepatoma cells by employing the avidin-biotin interaction. � 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

Semiconductor quantum dots (QDs) are nanoparticles in which charge carriers are three dimensionally confined or quantum confined. The quantum confinement provides size-tunable absorption bands and emission color to QDs. Also, the photoluminescence (PL) of QDs is exceptionally bright and stable, making them potential candidates for biomedical imaging and therapeutic interventions. Although fluorescence imaging and photodynamic therapy (PDT) of cancer have many advantages over imaging using ionizing radiations and chemo and radiation therapies, advancement of PDT is limited due to the poor availability of photostable and NIR fluorophores and photosensitizing (PS) drugs. With the introduction of biocompatible and NIR QDs, fluorescence imaging and PDT of cancer have received new dimensions and drive. In this review, we summarize the prospects of QDs for imaging and PDT of cancer. Specifically, synthesis of visible and NIR QDs, targeting cancer cells with QDs, in vitro and in vivo cancer imaging, multimodality, preparation of QD-PS conjugates and their energy transfer, photosensitized production of reactive oxygen intermediates (ROI), and the prospects and remaining issues in the advancement of QD probes for imaging and PDT of cancer are summarized.

Binding of CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) to the NR2B subunit of the NMDAR (N-methyl-D-aspartate-type glutamate receptor) in the PSD (postsynaptic density) is essential for the induction of long-term potentiation. In this study, we show that binding of NR2B to the T-site (Thr(286)-autophosphorylation site binding pocket) of CaMKII regulates its catalysis as reflected in the kinetic parameters. The apparent S(0.5) (substrate concentration at half maximal velocity) and V(max) values for ATP were lower for phosphorylation of a GST (glutathione transferase)-fusion of NR2B((1271-1311)) (with the phosphorylation site Ser(1303)) when compared with phosphorylation of the analogous sequence motif from NR2A. The co-operative behaviour exhibited by the CaMKII holoenzyme towards ATP for phosphorylation of GST-NR2A was significantly altered by the interaction with GST-NR2B. Disrupting the T-site-mediated binding by mutagenesis of either NR2B or CaMKII abolished the modulation of CaMKII activity by NR2B. The active site residue of alpha-CaMKII, Glu(96), participates in effecting the modulation. The CaMKII-binding motif of the Drosophila voltage-gated potassium channel Eag interacted with the T-site of CaMKII with lower affinity and caused catalytic modulation to a lesser extent. The kinetic parameters of ATP for the Thr(286)-autophosphorylation reaction of CaMKII were also altered by NR2B in a similar manner. Interestingly, the NR2B sequence motif caused increased sensitivity of CaMKII activity to ATP, and saturation by lower concentrations of ATP, which, in effect, resulted in a constant level of activity of CaMKII over a broad range of ATP concentrations. Our findings indicate that CaMKII at the PSD may be regulated by bound NR2B in a manner that supports synaptic memories.

The interaction of calcium/calmodulin-dependent protein kinase II (CaMKII) with the NR2B subunit of N-methyl-d-aspartate-type glutamate receptor is thought to be one of the important events leading to synaptic plasticity. CaMKII binds NR2B by its catalytic site and by the autophosphorylation site binding pocket (APBP), a non-catalytic site. Mutagenesis of Glu-236, a residue in the APBP of CaMKII that is likely to be interacting with NR2B, influences phosphorylation of NR2B. The phosphorylation of syntide-2, a classical catalytic site substrate of CaMKII, is influenced to a much lesser extent by this mutation. Taken together these results indicate that interaction of NR2B at the non-catalytic site of CaMKII influences catalysis. Our data suggest that kinetic models of peptide substrate phosphorylation by CaMKII should incorporate the non-catalytic mode of binding of peptides that is dependent on the sequence of the peptide.

Theauthorsapologizeforprovidingtheincorrectchemicalstructureofthequantumdot(QD)-RoseBengaldyeconjugateinFig.9onpage210(Biju et al., 2010). The correct structure of the conjugate is provided below. Missing carbon–carbon double bonds in the original reference Tsayet al. (2007) are added here.References

Accompanying the decrease in serum cholesterol and increase in concentration of ubiquinone in liver and its microsomes, the activity, but not the protein, of HMG-CoA reductase decreased in ubiquinone-supplemented rats. A soluble 58-kDa preparation of HMG-CoA reductase was partially inhibited on addition of ubiquinone indicating a possible feedback type of action.

On repeated thawing at room temperature of frozen preparations of heavy microsomes from rat livers, HMGCoA reductase activity was solubilized due to limited proteolysis. This soluble enzyme was partially purified by fractionation with ammonium sulfate and filtration on Sephacryl S-200 column. The active enzyme was coeluted with a major 92 kDa-protein and was identified as a 58 kDa-protein after separation by SDS-PAGE and immunoblotting. Ethoxysilatrane, a hypocholesterolemic compound, which decreased the liver-microsomal activity of HMGCoA reductase on intra-peritonial treatment of animals, showed little effect on the enzyme activity with isolated microsomes or the 50 kDa-soluble enzyme when added in the assay. But it was able to inhibit the activity of the soluble 58 kDa-enzyme in a concentration-dependent, reversible manner. Cholesterol and an oxycholesterol were without effect whereas chlorophenoxyisobutyrate and ubiquinone showed small inhibition under these conditions. The extra region that links the active site domain (50 kDa protein) to the membrane, present in the 58 kDa-protein appears to be involved in mediating the inhibition by silatrane.