Investigation of the environment surrounding iron-sulfur cluster 4 of Escherichia coli dimethylsulfoxide reductase

Iron-sulfur ([Fe-S]) clusters are common in electron transfer proteins, and their midpoint potentials (E(m) values) play a major role in defining the rate at which electrons are shuttled. The E(m) values of [Fe-S] clusters are largely dependent on the protein environment as well as solvent accessibility. The electron transfer subunit (DmsB) of Escherichia coli dimethylsulfoxide reductase contains four [4Fe-4S] clusters (FS1-FS4) with E(m) values between -50 and -330 mV. We have constructed an in silico model of DmsB and addressed the roles of a group of residues surrounding FS4 in electron transfer, menaquinol (MQH(2)) binding, and protein control of its E(m). Residues Pro80, Ser81, Cys102, and Tyr104 of DmsB are located at the DmsB-DmsC interface and are critical for the binding of the MQH(2) inhibitor analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) and the transfer of electrons from MQH(2) to FS4. Because the EPR spectrum of FS4 is complicated by spectral overlap and spin-spin interactions with the other [4Fe-4S] clusters of DmsB, we evaluated mutant effects on FS4 in double mutants (with a DmsB-C102S mutation) in which FS4 is assembled as a [3Fe-4S] cluster (FS4([3Fe)(-)(4S])). The DmsB-C102S/Y104D and DmsB-C102S/Y104E mutants dramatically lower the E(m) of FS4([3Fe)(-)(4S]) from 275 to 150 mV and from 275 to 145 mV, respectively. Mutations of positively charged residues around FS4([3Fe)(-)(4S]) lower its E(m), but mutations of negatively charged residues have negligible effects. The E(m) of FS4([3Fe)(-)(4S]) in the DmsB-C102S mutant is insensitive to HOQNO as well as to changes in pH from 5 to 7. The FS4([3Fe)(-)(4S]) E(m) of the DmsB-C102S/Y104D mutant increases in the presence of HOQNO and decreasing pH. Analyses of the mutants suggest that the maximum achievable E(m) for FS4([3Fe)(-)(4S]) of DmsB is approximately 275 mV.     

A conformational study of some adenosines by use of nuclear Overhauser effect

Conformations of 8-bromo-2′-[unk]-triisopropylbenzenesulfonyladenosine ([unk]) and its 3′-[unk]-isomer ([unk]) in solution have been determined by the use of intramolecular nuclear Overhauser effects in ¹H NMR spectroscopy. Compound [unk] has been proved to have a conformation in which the adenosine and benzene rings are intramolecularly stacked and compound [unk] an elongated non-stacked conformation in dimethylsulphoxide. The 5′-[unk]-acetyl derivative of [unk] has also been found to adopt the intramolecularly stacked conformation in dimethylsulphoxide, but a non-stacked one in chloroform. Coupling constants observed are discussed in connection with the conformation of the ribose moiety. The ¹³C NMR spectra have also been examined, but no effect which could be ascribed to the stacking phenomena was observed in the carbon chemical shifts.     

The SO4(.-)-induced chain reaction of 1,3-dimethyluracil with peroxodisulphate

The sulphate radical SO4(.-) reacts with 1,3-dimethyluracil (1,3-DMU) (k = 5 X 10(9) dm3 mol-1 s-1) thereby forming with greater than or equal to 90 per cent yield the 1,3-DMU C(5)-OH adduct radical 4 as evidenced by its absorption spectrum and its reactivity toward tetranitromethane. Pulse-conductometric experiments have shown that a 1,3-DMU-SO4(.-) aduct 3 as well as the 1,3-DMU radical cation 1, if formed, must be very short-lived (t1/2 less than or equal to 1 microsecond). The 1,3-DMU C(5)-OH adduct 4 reacts slowly with peroxodisulphate (k = 2.1 X 10(5) dm3 mol-1 s-1). It is suggested that the observed new species is the 1,3-DMU-5-OH-6-SO4(.-) radical 7. At low dose rates a chain reaction is observed. The product of this chain reaction is the cis-5,6-dihydro-5,6-dihydroxy-1,3-dimethyluracil 2. At a dose rate of 2.8 X 10(-3) Gys-1 a G value of approximately 200 was observed ([1,3-DMU] = 5 X 10(-3) mol dm-3; [S2O8(2-)] = 10(-2) mol dm-3; [t-butanol] = 10(-2) mol dm-3). The peculiarities of this chain reaction (strong effect of [1,3-DMU], smaller effect of [S2O(2-)8]) is explained by 7 being an important chain carrier. It is proposed that 7 reacts with 1,3-DMU by electron transfer, albeit more slowly (k approximately 1.2 X 10(4) dm3 mol-1 s-1) than does SO4(.-). The resulting sulphate 6 is considered to hydrolyse into 2 and sulphuric acid which is formed in amounts equivalent to those of 2. Computer simulations provide support for the proposed mechanism. The results of some SCF calculations on the electron distribution in the radical cations derived from uracil and 1-methyluracil are also presented.     

3-[Benzimidazo- and 3-[benzothiadiazoleimidazo-(1,2-c)quinazolin-5-yl]-2H-chromene-2-ones as potent antimicrobial agents

A series of 3-[benzimidazo(1,2-c)quinazolin-5-yl]-2H-chromene-2-one (6a-6f) and 3-[benzothiadiazole- imidazo(1,2-c)quinazolin-5-yl]-2H-chromene-2-one derivatives (7a-7f) that incorporate a variety of substituents at the 6- and/or 8-positions of the coumarin moieties have been synthesized utilizing cellulose sulfuric acid as an efficient catalyst under both conventional heating and microwave irradiation procedures. These analogs were evaluated for their antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes (Gram-positive bacteria), Escherichia Coli, Klebsiella pneumonia, Salmonella typhimurium (Gram-negative bacteria), and Aspergillus niger, Candida albicans, and Aspergillus flavus (Fungi). Two analogs, 6c (a 6,8-dichloro analog, MIC([SA]) = 2.5 μg/mL; MIC([ST]) = 2.5 μg/mL) and 7d (a 6,8-dibromo analog, MIC([ST]) = 2.5 μg/mL) were identified as potent antibacterial agents, and two analogs, 6b (a 6-bromo analog, MIC([AF]) = 10 μg/mL) and 6d (a 6,8-dibromo analog, MIC([AF]) = 15 μg/mL; MIC([CA]) = 15μg/mL), were identified as potent antifungal agents. Based on the MIC data, analogs 6b, 6c, 6d, and 7d were identified as the most potent antimicrobial agents in the series.     

Effect of a sudden and a slow concentration increase in plasma urea on its concentration in some tissues of the dog and rat

The urea concentration in the renal cortex ([RC]) of pentobarbital-anaesthetized dogs was 5.71 times higher than the plasma concentration ([P]); the liver ([L]) and the skeletal muscle ([SM]) concentrations were the same as ([P]). Rapid infusion of 20% urea (1 g/kg b.w. within 1 min) was followed by a sudden increase in [P]; [RC] and [L] rose to values nonsignificantly different from [P] and remained non-significantly different for the whole 4 hours of the experiment; at the end, [P] was still about 10 times higher than before infusion. Surprisingly, [SM] 2 and 6 min after infusion was significantly lower than [P]; later they were the same. The experiment thus does not testify to the existence of active transport of urea in the RC. The permeability of the skeletal muscle membrane for urea is lower than that of the RC and liver. Chronic uraemia was induced in rats by transplanting the trigonum vesicae into the peritoneal cavity. In addition to the chemical determination of urea, 14C-urea (marked*) was also measured. [RC]/[P] and [RC*]/[P*] fell as [P] rose; [L], [L*] [SM] and [SM*] never differed from [P] or [P*]. Fluid [PF] collected in the peritoneal cavity had the same chemically determined and radioactive urea concentration as P, but it was hypoosmolar and had a lower [Na+] than P. These experiments likewise did not testify to active urea transport in the RC. It is not clear what caused the osmolarity and sodium gradient between the PF and P, but the peritoneal wall definitely did not act as a simple dialyzing membrane.     

Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids

Lipase-catalyzed esterification of glucose with fatty acids in ionic liquids (ILs) mixture was investigated by using supersaturated glucose solution. The effect of ILs mixture ratio, substrate ratio, lipase content, and temperature on the activity and stability of lipase was also studied. The highest yield of sugar ester was obtained in a mixture of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim][TfO]) and 1-methyl-3-octylimidazolium bis[(trifluoromethyl)-sulfonyl]amide ([Omim][Tf₂N]) with a volume ratio of 9:1, while Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) showed the optimal stability and activity in a mixture of [Bmim][TfO] and [Omim][Tf₂N] with a 1:1 volume ratio. Reuse of lipase and ILs was successfully carried out at the optimized reaction conditions. After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained.     

Ru(phen)2DPPZ]2+ is in contact with DNA bases when it forms a luminescent complex with single-stranded oligonucleotides

The luminescence intensity of the Delta- and Lambda-enantiomer of [Ru(phen)2DPPZ]2+ ([Ru(phenanthroline)2 dipyrido[3,2-a:2',3'-c]phenazine]2+) complex enhanced upon binding to double stranded DNA, which has been known as "light switch effect". The enhancement of the luminescence required the intercalation of the large ligand between DNA base pairs. In this study, we report the enhancement in the luminescence intensity when the metal complexes bind to single stranded oligonucleotides, indicating that the "light switch effect" does not require intercalation of the large DPPZ ligand. Oligonucleotides may provide a hydrophobic cavity for the [Ru(phen)2DPPZ]2+ complex to prevent the quenching by the water molecule. In the cavity, the metal complex is in contact with DNA bases as is evidenced by the observation that the excited energy of the DNA bases transfer to the bound metal complex. However, the contact of the metal complex with DNA bases is different from the stacking of DPPZ in the intercalation pocket. In addition to the normal two luminescence lifetimes, a short lifetime in the range of 1-2 ns was found for both the delta- and lambda-enantiomer of [Ru(phen)2DPPZ]2+ when complexed with single stranded oligonucleotides, which may be assigned to the metal complex that is outside of the cavity, interacting with phosphate groups of DNA.     

Pituitary 1,25-dihydroxyvitamin D3 receptors in hyperthyroid- and hypothyroid-rats

The binding of 1 alpha,25-dihydroxy (26,27-methyl-[3H]) cholecalciferol ([3H]1,25-(OH)2D3) to its receptor in cytosol of the anterior pituitary cells was examined in hyperthyroid- and hypothyroid rats, as well as in normal rats. The binding capacity increased by 41% in L-Thyroxine-treated hyperthyroid rats and decreased by 49% in propylthiouracil-ingested hypothyroid rats as compared with normal control rats, whereas the affinity of the receptor for [3H]-1,25(OH)2D3 showed no difference among these 3 animal groups. These findings indicate that the number of 1,25(OH)2D3 receptors in the pituitary may be regulated by thyroid hormone, and further suggest that 1,25-(OH)2D3 may play some role in regulating functions of the anterior pituitary.     

Bacterial ApbC can bind and effectively transfer iron-sulfur clusters

The metabolism of iron-sulfur ([Fe-S]) clusters requires a complex set of machinery that is still being defined. Mutants of Salmonella enterica lacking apbC have nutritional and biochemical properties indicative of defects in [Fe-S] cluster metabolism. ApbC is a 40.8 kDa homodimeric ATPase and as purified contains little iron and no acid-labile sulfide. An [Fe-S] cluster was reconstituted on ApbC, generating a protein that bound 2 mol of Fe and 2 mol of S (2-) per ApbC monomer and had a UV-visible absorption spectrum similar to known [4Fe-4S] cluster proteins. Holo-ApbC could rapidly and effectively activate Saccharomyces cerevisiae apo-isopropylmalate isolomerase (Leu1) in vitro, a process known to require the transfer of a [4Fe-4S] cluster. Maximum activation was achieved with 2 mol of ApbC per 1 mol of apo-Leu1. This article describes the first biochemical activity of ApbC in the context of [Fe-S] cluster metabolism. The data herein support a model in which ApbC coordinates an [4Fe-4S] cluster across its dimer interface and can transfer this cluster to an apoprotein acting as an [Fe-S] cluster scaffold protein, a function recently deduced for its eukaryotic homologues.     

G-protein involvement in central-nervous-system muscarinic-receptor-coupled polyphosphoinositide hydrolysis.

Potentiation of muscarinic-agonist-stimulated polyphosphoinositide (PPI) hydrolysis was demonstrated in a rat cerebral-cortical membrane preparation prelabelled with myo-[3H]inositol. Accumulation of myo-[3H]inositol 1,4-bisphosphate ([3H]IP2) was used to assess brain [3H]phosphatidylinositol 4,5-bisphosphate hydrolysis as its immediate metabolite, myo-[3H]inositol 1,4,5-trisphosphate, was rapidly hydrolysed to [3H]IP2. Inclusion of ATP (100 microM) and Mg2+ (5 mM) in the assay medium was necessary to demonstrate the effect of GTP analogues on carbachol-stimulated brain [3H]PPI turnover. Carbachol (100 microM) induced only a small increment in [3H]IP2 accumulation (142% of control) in 1 min. However, its effect was markedly enhanced, to 800% and 300% of control, by 100 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) respectively. GTP[S] and p[NH]ppG also stimulated [3H]IP2 accumulation by over 500% and 200% of control, respectively. The GTP-analogue-potentiated carbachol effect was antagonized by 10 microM-atropine, whereas the GTP-analogue stimulation was unaffected. This report confirms the involvement of a G (GTP-binding) protein(s) in brain PPI metabolism and provides new evidence for the role of G protein(s) in the coupling of stimulated muscarinic receptors to PPI hydrolysis in the central nervous system.     

Biosynthesis of theobroxide and its related compounds, metabolites of Lasiodiplodia theobromae

Administration of (13)C labeled acetates ([1-(13)C], [2-(13)C] and [1,2-(13)C(2)] to Lasiodiplodia theobromae showed the tetraketide origins of both theobroxide, a potato-tuber inducing substance [1, (1S, 2R, 5S, 6R)-3-methyl-7-oxa-bicyclo[4.1.0]hept-3-en-2,5-diol]) and its carbonyldioxy derivative [2, (1S, 4R, 5S, 6R)-7,9-dioxa-3-methyl-8-oxobicyclo [4.3.0]-2-nonene-4,5-diol]. The incorporation of acetate-derived hydrogen into 1 and 2 was studied using [2-(2)H(3), 2-(13)C]acetate. Three and one deuterium atoms were incorporated at one methyl and epoxy carbons, respectively. The observed loss of deuterium atoms from the methyl group suggests a considerable amount of exchange from the methyl group of [2-(2)H(3), 2-(13)C]acetate during biosynthesis of 1 and 2. Incorporation of [1-(13)C]- and [1,2-(13)C(2)]acetates indicates the carbonyl carbon of the carbonyldioxy derivative is derived from the carboxy carbon of the precursor.     

MgATP-Dependent Transport of Phytochelatins Across the Tonoplast of Oat Roots

In Cd-exposed oat (Avena sativa) roots Cd was found to be associated primarily with the phytochelatin ([gamma]-glutamylcysteinyl)3-glutamic acid [([gamma]EC)3G], with a peptide to Cd ratio of 1:3 (cysteine to Cd ratio of 1:1), even though both ([gamma]EC)2G and ([gamma]EC)3G were present in the roots. Phytochelatins are known to accumulate in the vacuoles of plant cells on exposure to Cd, but the mechanism is not clear. Here we present evidence for the transport of the phytochelatins ([gamma]EC)3G and ([gamma]EC)2G as well as the Cd complex Cd-([gamma]EC)3G across the tonoplast of oat roots. Transport of ([gamma]EC)3G had a Km, for MgATP of 0.18 mM and a Vmax of 0.7 to 1 nmol mg-1 protein min-1. Transport of ([gamma]EC)3G was also energized by MgGTP and to a lesser extent MgUTP and was highly sensitive to orthovanadate, with a 50%-inhibitory concentration of 0.9 [mu]M. The Cd complex Cd-([gamma]EC)3G and ([gamma]EC)2G were also transported in a MgATP-dependent, vanadate-sensitive manner. Therefore, this process is a candidate for the transport of both phytochelatins, and Cd as its peptide complex, from the cytoplasm into the vacuole.     

Studies on the mechanism of enkephalin receptor down regulation

The association of [3H] [D-Ala2, D-Leu5] enkephalin ([3H]DADLE]) with mouse neuroblastoma cells (N4TG1) was investigated. Under identical conditions the time course, dose response curve and temperature dependence for ligand uptake were similar to those for ligand-induced receptor loss (down regulation). Uptake of [3H]DADLE was inhibited by opiate ligands as well as by the metabolic inhibitors sodium azide and 2,4 dinitrophenol. Comparison of the effects of these inhibitors on receptor binding, ligand uptake and receptor loss indicated that these cells accumulate [3H]DADLE in excess of their surface receptor number. The data suggest that receptor recycling occurs and that ligand is internalized via receptor mediated endocytosis.     

Cyclic retro-inverso dipeptides with two aromatic side chains. II. Conformational analysis.

The conformations of cis and trans cyclic retro-inverso dipeptides--2-[(4-hydroxy)benzyl]-5-benzyl-4,6(1H,2H,3H,5H)-pyrimidinedi one (c[mTyr-gPhe]), and 2-benzyl-5-amino-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[mTyr-gPhe]), and 2-benzyl-5-amino-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[(alpha-amino)mTyr-gPhe])--and the parent cyclic dipeptides--c[tyrosyl-phenylalanine] (cis-c[L-Tyr-L-Phe]) and c[tyrosyl-D-phenylalanine] (trans-c[L-Tyr-D-Phe])--were studied by using 1H-nmr spectroscopy and semiempirical energy calculations. In the cis compounds of all the cyclic retro-inverso and parent dipeptides, the most stable conformer has both aromatic side chains sharing the space over the backbone ring in a "face-to-face" fashion. All the trans compounds predominantly assume a "sandwich" conformation in which the two aromatic rings are folded back over the backbone ring on opposite sides. However, different conformational preferences were observed for the backbones between the retro-inverso and parent cyclic dipeptides. The parent cyclic dipeptide trans-c[L-Tyr-D-Phe] adopts two types of boat structures with different side-chain orientations in almost equal amounts: one with the Tyr side chain in a pseudoaxial position and the Phe side chain in a pseudoequatorial position, the other with the Tyr side chain in a pseudoequatorial position and the Phe side chain in a pseudoaxial position. On the other hand, the cyclic retro-inverso dipeptides trans-c[mPhe-gTyr] and trans c[mTyr-gPhe] assume only one type of boat structure in which the malonyl side chain is in a pseudoequatorial and the gem-diamino side chain is in a pseudoaxial position. In addition to the preferred conformations, the conformational energies of the C alpha--C beta bonds in the malonyl and gem-diamino residues were estimated from the temperature variation of vicinal 1H--1H coupling constants for the H--C alpha--C beta--H groupings observed for the trans isomers of cyclic retro-inverso dipeptides. The energies were evaluated to be 1.1 and 1.8 kcal mol-1 for the malonyl and gem-diamino residues, respectively. Applying these energies to the parent cyclic dipeptide trans-c[L-Tyr-D-Phe], the observed fractions of three side-chain conformations are reasonably reproduced. The conformational energies as well as conformational properties of the molecules estimated in this investigation may be useful to refine force constants for both parent and retro-inverso peptides with aromatic side chains.     

Bicarbonate buffering of lactic acid generated during exercise

The pattern of decrease in arterial bicarbonate concentration ([HCO3-]) during progressive incremental exercise was compared with that of the rise in arterial lactate ([La-]) to determine the degree of buffering of lactic acid by bicarbonate. A mathematical model was derived for the change in [HCO3-] beyond the lactate threshold. This was based on a log-log transformation of the data, a model previously found to provide a very good fit to the [La-]-O2 consumption (VO2) relationship. The results of the analysis of incremental exercise data from 10 subjects show that the decrease in [HCO3-] very nearly matches the increase in [La-]. However, it was found by comparing regression models that the correspondence between [HCO3-] and [La-] could be improved by assuming that the [HCO3-] decrease was delayed until the arterial lactate level had increased by approximately 0.4 meq/l. This result is compatible with the existence of buffering mechanisms in the cell which buffer the initial increase of lactic acid. Beyond this initial buffering, lactic acid appears to be buffered almost entirely by the bicarbonate buffer system.     

Cytokinin oxidase from Zea mays: purification, cDNA cloning and expression in moss protoplasts

Cytokinins are degraded by cytokinin oxidases (CKOs) which catalyse cleavage of the N6-(isopent-2-enyl)-side chain resulting in formation of adenine-type compounds. CKO activity has been recorded in many plants and is thought to play a key role in controlling cytokinin levels in plants. Several partially purified CKOs have been characterised but no genes have been isolated yet. CKO activity is known to be inhibited by phenylureas, cytokinin agonists. We used 1-(2-azido-6-chloropyrid-4-yl)-3-(4-[3H])phenylurea ([3H]-azidoCPPU) to photolabel a glycosylated CKO from maize kernels. This enabled us to purify the enzyme. Peptide sequences were determined and the corresponding cDNA was cloned. The deduced amino acid sequence shares homology domains with FAD-dependent oxidases. An original assay based on transient expression of the enzyme in moss protoplasts allowed the functionality of the recombinant enzyme to be demonstrated.     

Activation of phospholipase C associated with isolated rabbit platelet membranes by guanosine 5''-[gamma-thio]triphosphate and by thrombin in the presence of GTP.

Rabbit platelets were labelled with [3H]inositol and a membrane fraction was isolated in the presence of ATP, MgCl2 and EGTA. Incubation of samples for 10 min with 0.1 microM-Ca2+free released [3H]inositol phosphates equivalent to about 2.0% of the membrane [3H]phosphoinositides. Addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) caused an additional formation of [3H]inositol phosphates equivalent to 6.6% of the [3H]phosphoinositides. A half-maximal effect was observed with 0.4 microM-GTP[S]. The [3H]inositol phosphates that accumulated consisted of 10% [3H]inositol monophosphate, 88% [3H]inositol bisphosphate ([3H]IP2) and 2% [3H]inositol trisphosphate ([3H]IP3). Omission of ATP and MgCl2 led to depletion of membrane [3H]polyphosphoinositides and marked decreases in the formation of [3H]inositol phosphates. Thrombin (2 units/ml) or GTP (4-100 microM) alone weakly stimulated [3H]IP2 formation, but together they acted synergistically to exert an effect comparable with that of 10 microM-GTP[S]. The action of thrombin was also potentiated by 0.1 microM-GTP[S]. Guanosine 5'-[beta-thio]diphosphate not only inhibited the effects of GTP[S], GTP and GTP with thrombin, but also blocked the action of thrombin alone, suggesting that this depended on residual GTP. Incubation with either GTP[S] or thrombin and GTP decreased membrane [3H]phosphatidylinositol 4-phosphate ([H]PIP) and prevented an increase in [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) observed in controls. Addition of unlabelled IP3 to trap [3H]IP3 before it was degraded to [3H]IP2 showed that only about 20% of the additional [3H]inositol phosphates that accumulated with GTP[S] or thrombin and GTP were derived from the action of phospholipase C on [3H]PIP2. The results provide further evidence that guanine-nucleotide-binding protein mediates signal transduction between the thrombin receptor and phospholipase C, and suggest that PIP may be a major substrate of this enzyme in the platelet.     

Synthesis of biologically stable gold nanoparticles using imidazolium-based amino acid ionic liquids

A novel double-step reduction procedure for the synthesis of gold nanoparticles (AuNPs) using amino acid ionic liquids has been employed. 1-Dodecyl-3-methyl imidazolium tryptophan ([C(12)mim]Trp) and 1-ethyl-3-methyl imidazolium tryptophan ([C(2)mim]Trp) were used for this synthesis. The synthesized AuNPs were characterized by UV-vis spectroscopy, transmission electron microscopy and dynamic light scattering. The behavior of these AuNPs were also probed in a biological media. It was proven that AuNPs synthesized at [C(12)mim]Trp have more stability than AuNPs synthesized at [C(2)mim]Trp due to the longer alkyl chain of the imidazolium moiety. The solubility test shows that the resultant AuNPs have a hydrophilic nature. Finally, it was seen that due to the presence of a biomolecule, namely Trp, in the structure of AuNPs protecting shell, higher stability and biocompatibility was achieved in the biological media.     

Glycerol-3-phospho-D-myo-inositol 4-phosphate (Gro-PIP) is an inhibitor of phosphoinositide-specific phospholipase C

The deacylated forms of the phosphoinositides were used to determine whether the guinea pig uterus phosphoinositide-specific phospholipase C (PI-PLC I, Mr 60,000) required fatty acids at the sn-1 and sn-2 positions for the hydrolysis of the sn-3 phosphodiester bond. L-alpha-Glycerophospho-D-myo-inositol 4-phosphate (Gro-PIP), but not glycerol 3-phosphate (Gro-3-P), L-alpha-glycerophospho-D-myo-inositol (Gro-PI), or L-alpha-glycerophospho-D-myo-inositol 4,5-bisphosphate (Gro-PIP2), inhibited PI-PLC I in a concentration-dependent manner. Assays performed with 10 microM [3H]phosphatidylinositol ([3H]PI), 10 microM [3H]phosphatidylinositol 4-phosphate ([3H]PIP) or 10 microM [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) as substrates, with increasing [Gro-PIP] revealed an IC50 = 380 microM. Kinetic studies with increasing [3H]PI substrate concentrations in the presence of 100 microM and 300 microM Gro-PIP demonstrated that Gro-PIP exhibited competitive inhibition; Kis = 40 microM. Ca2+ concentrations over the range 1.1 microM to 1 mM did not effect inhibition, suggesting that Gro-PIP inhibition of [3H]PI hydrolysis was calcium-independent. To determine whether Gro-PIP was a substrate, 20 microM and 500 microM [3H]Gro-PIP were incubated with PI-PLC I. Anion-exchange HPLC analysis revealed no [3H]IP2 product formation, indicating that [3H]Gro-PIP was not hydrolyzed. Assays performed with [3H]PI and [3H]PIP substrates in the presence of 500 microM [3H]Gro-PIP revealed approx. 75% less [3H]inositol 1-phosphate ([3H]IP1) and [3H]inositol 1,4-bisphosphate ([3H]IP2) product formation, respectively, indicating that [3H]Gro-PIP inhibited the hydrolysis of the substrates by PI-PLC I. These data suggest that Gro-PIP does not serve as a substrate, and that it inhibits PI-PLC I by competitive inhibition in a Ca2(+)-independent fashion.     

The stereochemistry of Co(III)-amine complexes. The use of nOe and COSY H NMR spectroscopy to determine structure in solution

It is shown how 1D nOe and 2D COSY ¹H NMR spectroscopy can be used to assign the stereochemistry of Co(III) amine complexes. By using d₆-DMSO as solvent together with a small quantity of DCl all non-equivalent N---H hydrogens can be distinguished at 300 MHz. Through-space (nOe), and through-bond (COSY), associations with other N---H and C---H hydrogens can then be determined. This leads to a complete assignment of structure in solution. The technique is applied to the complexes syn(N), anti(N)-[Co(cyclen) (NH₃)₂] (ClO₄)₃, syn(N), anti(Cl)-[Co(cyclen) (NH₃)Cl] (ClO₄)₂, anti(N), syn(Cl)-[Co(cyclen) (NH₃)Cl](ClO₄)₂, syn(N), anti(O)-[Co(Mecyclen)-(GlyO)](ClO₄)₂ and Δ-cis-[Co(δ-en)₂(NO₂)₂](NO₂).