Thermodynamic analysis of protein kinase A Iα activation

Thermodynamic analysis of protein kinase A (PKA) Iα activation was performed using Quantum 3.3.0 docking software and a Gaussian 03W quantum mechanical computational package. Expected stacking interactions between adenine of 3′:5′-AMP and aromatic moieties of amino acids were taken into account by means of MP2/6-31G(d) IPCM (iso-density polarizable continuum model) computations (ɛ = 4.0). It is demonstrated that thermodynamically favorable agonist-induced PKA Iα activation is mediated by two processes. First, 3′:5′-AMP binding is accompanied by structural changes leading to a thermodynamically favorable regulatory subunit conformation, which is hardly realized in the absence of the ligand (ΔG_R^o = −23.9 ± 8.2 kJ/mol). Second, 3′:5′-AMP affinity to the regulatory subunit conformation observed after agonist-induced PKA Iα activation is higher than that to inactive holoenzyme complex (ΔG_{3′:5′−AMP}^o = −28.1 ± 9.7 kJ/mol). ATP is capable of docking into the 3′:5′-AMP-binding site B of the regulatory subunit complexed with the catalytic one, resulting in inhibition of kinase activation. True constants of 3′:5′-AMP binding to PKA Iα holoenzyme were found to be 60 and 57 μM for the regulatory subunit domains A and B, respectively. These constants, unlike the binding equilibrium constant determined using established experimental techniques and ranging from 15 nM to 2.9 μM, are proved to be direct measures of 3′:5′-AMP-PKA Iα binding affinity. Their values are in a reasonable agreement with the changes in 3′:5′-AMP concentration in the cell (2-55 μM) and account for PKA Iα activation in response to adequate stimuli.     

Organo-vanadium compounds are potent activators of the protein kinase B signaling pathway and protein tyrosine phosphorylation: mechanism of insulinomimesis

Organo-vanadium compounds (OVC) have been shown to be more effective than inorganic vanadium compounds in ameliorating glucose homeostasis and insulin resistance in rodent models of diabetes mellitus. However, the precise molecular mechanism of OVC efficiency remains poorly defined. Since inorganic vanadium compounds have been found to activate several key components of the insulin signaling cascade, such as protein kinase B (PKB), the objective of the present study was to investigate if stimulation of PKB and its downstream target glycogen synthase kinase-3 (GSK-3), are responsible for the more potent insulinomimetic effects of OVC. Among several vanadium compounds tested, vanadium (IV) oxo bis (acetylacetonate) and vanadium (IV) oxo bis(maltolato) markedly induced the phosphorylation of PKB as well as GSK-3beta compared to vanadyl sulfate (VS), an inorganic vanadium salts in Chinese hamster ovary cells overexpressing the insulin receptor (IR). Furthermore, the OVC were stronger inhibitors of protein tyrosine phosphatase (PTPase) activity than VS. The higher PTPase inhibitory potential of the OVC was associated with more robust tyrosine phosphorylation of several cellular proteins, including the IRbeta subunit and insulin receptor substrate-1 (IRS-1). In addition, greater IRS-1/p85alpha interaction was elicited by the OVC than by VS. These data indicate that the higher PTPase inhibitory potential of OVC translates into greater phosphorylation of PKB and GSK-3beta, which, in turn, may contribute to a more potent effect of OVC on glucose homeostasis.     

Conventional protein kinase C isoenzymes undergo dephosphorylation in neutrophil-like HL-60 cells treated by chelerythrine or sanguinarine

The quaternary benzo[c]phenanthridine alkaloid chelerythrine is widely used as an inhibitor of protein kinase C (PKC). However, in biological systems chelerythrine interacts with an array of proteins. In this study, we examined the effects of chelerythrine and sanguinarine on conventional PKCs (cPKCs) and PKC upstream kinase, phosphoinositide-dependent protein kinase 1 (PDK1), under complete inhibition conditions of PKC-dependent oxidative burst. In neutrophil-like HL-60 cells, sanguinarine and chelerythrine inhibited N-formyl-Met-Leu-Phe, phorbol 12-myristate 13-acetate (PMA)-, and A23187-induced oxidative burst with IC₅₀ values not exceeding 4.6 μmol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold higher alkaloid concentrations. At concentrations below 10 μmol/L, sanguinarine and chelerythrine prevented phosphorylation of ∼80 kDa protein and sequestered ∼60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired PMA-stimulated translocation of autophosphorylated PKCα/βII isoenzymes, but both alkaloids induced dephosphorylation of the turn motif in PKCα/βII. The dephosphorylation did not occur in unstimulated cells and it was not accompanied by PKC degradation. Furthermore, cell treatment with sanguinarine or chelerythrine resulted in phosphorylation of ∼70 kDa protein by PDK1. We conclude that PKC-dependent cellular events are affected by chelerythrine primarily by multiple protein interactions rather than by inhibition of PKC activity.     

Inhibition protein tyrosine phosphatases by an oxovanadium glutamate complex,Na<Subscript>2</Subscript>[VO(Glu)<Subscript>2</Subscript>(CH<Subscript>3</Subscript>OH)](Glu = glutamate)

The insulin-sensitizing effect of vanadium complexes has been linked to their ability to inhibit protein tyrosine phosphatases (PTPs). Considering that vanadium complexes may exchange in vivo with amino acids, forming in situ vanadium–amino acid complexes, we have synthesized and characterized an oxovanadium glutamate complex, Na₂[V(IV)O(Glu)₂(CH₃OH)]H₂O (1·H₂O). The complex showed potent inhibition against four human PTPs (PTP1B, TCPTP, HePTP, and SHP-1) with IC₅₀ in the 0.21–0.37 μM ranges. Fluorescence titration studies suggest that the complex binds to PTP1B with the formation of a 2:1 complex. Enzyme kinetics analysis using Lineweaver–Burk plots indicates a typical competitive inhibition mode.     

Interactions between tachykinins and diverse,human nicotinic acetylcholine receptor subtypes

Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter receptors and play critical roles in chemical signaling throughout the nervous system. Reports of effects of substance P (SP) on nAChR function prompted us to investigate interactions between several tachykinins and human nAChR subtypes using clonal cell lines as simple experimental models. Acute exposure to SP inhibits carbamylcholine- or nicotinestimulated function measured using⁸⁶Rb⁺ efflux assays of human ganglionic (α3β4) nAChR expressed in SH-SY5Y neuroblastoma cells (IC₅₀∼2.3 μM) or of human muscle-type (α1β1γδ) nAChR expressed in TE671/RD clonal cells (IC₅₀∼21 μM). SP also acutely blocks function of rat ganglionic nAChR expressed in PC12 pheochromocytoma cells (IC₅₀∼2.1 μM). Neurokinin A and eledoisin inhibit function (extrapolated IC₅₀ values between 60 and 160 μM) of human muscle-type or ganglionic nAChR, but neurokinin B does not, and neither human nAChR is as sensitive as PC12 cell α3β4-nAChR to eledoisin or neurokinin A inhibition. At concentrations that produce blockade of nAChR function, SP fails to affect binding of [³H]acetylcholine to human muscle-type or ganglionic nAChR. SP-mediated blockade of rat or human ganglionic nAChR function is insurmountable by increasing agonist concentrations. Collectively, these results indicate that tachykinins act noncompetitively to inhibit human nAChR function with potencies that vary across tachykinins and nAChR subtypes. They also indicate that tachykinin actions at nAChR could further contribute to complex cross-talk between nicotinic cholinergic and tachykinin signals in regulation of nervous system activity.     

Sequence preference in DNA binding: de novo designed helix–turn–helix metallopeptides recognize a family of DNA target sites

The DNA-binding behavior and target sequences of two designed metallopeptides have been investigated with an iterative electrophoresis mobility shift assay followed by PCR amplification, and by circular dichroism spectroscopy. Peptides P3W and P5b were designed based on the structural similarity of the helix–turn–helix motif of homeodomains and the EF-hand motifs of calmodulin, as previously described for P3W. Like P3W, P5b binds both Eu(III) (K _d=12.6±1.9 μM) and Ca(II) (K _d=70±8 μM) with reasonable affinity. Binding selection from a library of randomized 8-mer DNA oligonucleotide sequences identified one target family for CaP5b [5′-pur-T-pur-G-(G/C)-3′], and two target sites for CaP3W [5′-(A/T)-G-G-G-(T/C)-3′ and 5′-A-T-(G/T)-T-G-3′]. Circular dichroism studies indicate that unlike EuP3W, EuP5b is poorly folded in the absence of DNA. In the presence of DNA containing target-binding sites for both peptides, both EuP3W and EuP5b increase in helical content, in the latter case significantly. These results suggest that EuP5b binding to target DNA involves an induced-fit mechanism. These small chimeric metallopeptides have been found to bind selectively to DNA targets, analogous to natural protein–DNA interactions. This corroborates our earlier conclusions (J. Am. Chem. Soc. 125:6656, 2003) that sequence-preferential DNA cleavage by Ce(IV)P3W was due to sequence recognition. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Kinetics and molecular modelling studies of the inhibition of protein tyrosine phosphatases by N,N-dimethylhydroxylamine complexes of vanadium(V)

 Bis(N,N-dimethylhydroxamido)hydroxooxovanadate and a derivative complex formed with dithiothreitol have been shown to be excellent inhibitors of the function of two protein tyrosine phosphatases, leucocyte antigen related phosphatase (LAR) and protein tyrosine phosphatase-1B (PTP1B). Inhibition constants of 1.0±0.3 μM and 2.3±0.3 μM, respectively, were determined for the inhibition of LAR by the two complexes. The inhibition of PTP1B is not substantially different. Unlike the structurally related hydrogen peroxide complexes of vanadium, these complexes inhibit in a fully reversible manner that is consistent with a non-oxidative process. Molecular modelling studies suggest the main stabilizing interaction is a cyclic H-bonded structure involving the conserved active site aspartate. Hydrophobic stabilization interactions were also suggested. Received: 17 February 1998 / Accepted: 6 July 1998     

Expression of Cyclic Nucleotide-Gated Cation Channels in Airway Epithelial Cells

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 are inhibited by l-cis-diltiazem with an IC₅₀ of 154 μm, by 2′,4′-dichlorobenzamil with an IC₅₀ of 50 μm and by amiloride with an IC₅₀ of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC₅₀ of 87 μm, by 2′4′-dichlorobenzamil with an IC₅₀ of 38 μm and by amiloride with an IC₅₀ of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC₅₀ of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells. Received: 24 February/Revised: 28 May 1999     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vivo and in vitro inhibition of mice thioredoxin reductase by methylmercury

The thioredoxin (Trx) system, involving redox active Trxs and thioredoxin reductases (TrxRs), sustain a number of important Trx-dependent pathways. These redox active proteins support several processes crucial for cell function, cell proliferation, antioxidant defense, and redox-regulated signaling cascades. Methylmercury (MeHg) is an important environmental toxicant that has a high affinity for thiol groups and can cause oxidative stress. The Trx system is the major system responsible for maintaining the redox state of cells and this function involves thiol reduction mediated by selenol groups in TrxRs. MeHg has a great affinity to thiols and selenols, thus the potential toxic effects of MeHg on TrxR inhibition were determined in the current study. A single administration of MeHg (1, 5, and 10 mg/Kg) caused a marked inhibition of kidney TrxR activity, while significant inhibition was observed in the liver after exposure to 5 and 10 mg/Kg of MeHg. TrxR activity was determined 24 h after MeHg. In the brain, MeHg did not inhibit TrxR activity. In vitro exposure to MeHg indicated that MeHg inhibits cerebral (IC₅₀, 0.158 μM), hepatic (IC₅₀, 0.071 μM), and renal TrxR activity (IC₅₀, 0.078 μM). The results presented herein demonstrated for the first time that renal and hepatic TrxRs can serve as an in vivo target for MeHg. This study suggests that MeHg can bind to selenocysteine residues present in the catalytic site of TrxR, in turn causing enzyme inhibition that can compromise the redox state of cells.     

Vanadyl Causes Hydroxyl Radical Mediated Degradation of Deoxyribose

Vanadyl caused a time- and dose-dependent degradation of deoxyribose to carbonyl products detectable with thiobarbituric acid. This process was inhibited by catalase, ethanol or HEPES; whereas superoxide dismutase was without effect. Vanadate did not substitute for vanadyl even in the presence of a source of O₂^? plus H₂ O ₂; but it did so in the presence of reductants such as thiols or NADH. It appears that hydrogen peroxide, generated by the autoxidation of vanadyl, is reduced by vanadyl to the hydroxyl radical; which, in turn, was responsible for the degradation of deoxyribose. A similar process might contribute to the toxic and pharmacological effects of vanadium salts.     

Yeast surviving factor Svf1 as a new interacting partner, regulator and in vitro substrate of protein kinase CK2

Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant CK2α (K _m 0.35 μM) and CK2α′ (K _m 0.18 μM) as well as CK2 holoenzyme (K _m 1.1 μM). Different K _m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor Svf1. Reconstitution of α′₂ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit. This effect was not observed in the case of α₂ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments). Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K¹⁹⁹EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED²⁴⁸ of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation of cell survival pathways.     

The in vitro anti-platelet,antioxidant and cellular immunity activity of <Emphasis Type="Italic">Phellinus gilvus</Emphasis> fractional extracts

The in vitro anti-platelet and antioxidant activities of various solvent extracts from Phellinus gilvus (PG), and the effects of hot water extract from PG (PGW) on murine cellular immunity were investigated. Chloroform extract (CE), methanol extract (ME) and butanol extract (BE) from PG could significantly inhibit platelet aggregation induced by thrombin. Ethyl acetate extract (EAE), BE, ME from PG had significant 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity compared with the control, and the EAE showed the highest effect with IC₅₀ values of 13.34 μg/ml, which is higher than that of ascorbic acid (40 μg/ml). In addition, EAE displayed the inhibition of xanthine oxidase (XO) activity with IC₅₀ value of 2.45 μg/ml. As to the cellular immunity activity, PGW could enhance both the lipopolysaccharide (LPS)-induced B lymphocyte proliferation and concanavalin A (Con A)-induced T lymphocyte proliferation in vitro. The phagocytosis of both peritoneal macrophages and RAW264.7 macrophage cells were also increased by the addition of PGW. Moreover, PGW was found to inhibit the nitric oxide (NO) production of RAW264.7 macrophages induced by LPS in a concentration-dependant manner.     

Pharmacological evidence for the stimulation of NADPH oxidase by P2X7 receptors in mouse submandibular glands

ATP in the 100 μM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), a P2X₇ agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X₄ receptor activation with ivermectin did not modify the response to ATP. ATP did not increase the oxidation of DCFH in cells isolated from submandibular glands of P2X₇ knockout mice or in cells pretreated with a P2X₇ antagonist. The inhibition of protein kinase C or of mitogen-activated protein kinase (MAP kinase) or of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocked the oxidation of DCFH without affecting the increase of the intracellular concentration of calcium or the uptake of ethidium bromide in response to extracellular ATP. From these results it is concluded that the activation of the P2X₇ receptors from submandibular glands triggers an intracellular signalling cascade involving protein kinase C and MAP kinase leading to the stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species.     

Inhibition of human platelet aggregation and membrane lipid peroxidation by food spice, saffron

The inhibitory activity of saffron extract was studied on human platelets. Platelet aggregation and lipid peroxidation were evaluated with platelet rich plasma (PRP) and platelet membranes respectively obtained from blood of healthy human volunteers. Human platelets were subjected to stimulation with a variety of agonists like ADP (61 μM), epinephrine (76 μM), collagen (11 μg/ml), calcium ionophore A 23187 (6 μM) and ristocetin (1.25 μg/ml) in the presence and absence of saffron extract with IC₅₀ being 0.66, 0.35, 0.86 and 0.59 mg respectively and no inhibition with ristocetin. The inhibitory effect was dose dependent with concentrations varying between 0.16 to 0.80 mg and time dependent at IC₅₀. A significant decrease was observed in malondialdehyde (MDA) formed, one of the end products of arachidonic acid metabolism and of serotonin released from dense granules of platelets at respective IC₅₀. Lipid peroxidation in platelet membranes induced by iron-ascorbic acid system was inhibited by saffron extract significantly with IC₅₀ of 0.33 mg. Hence, it may be said that aqueous extract of saffron may have component(s), which protect platelets from aggregation and lipid peroxidation. (Mol Cell Biochem 278: 59–63, 2005)     

Antimicrobial Butyrolactone I Derivatives from the Ecuadorian Soil Fungus Aspergillus terreus Thorn. var terreus

Summary The fungus Aspergillus terreus Thorn var. terreus isolated from an Ecuador soil sample was cultured in liquid and solid media and yielded three main metabolites identified as terreic acid (1), butyrolactone I (2) and lovastatin (3). The natural products as well as three synthetic butyrolactone I derivatives were assessed for antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi as well as for seed germination and seedling growth. Furthermore, the compounds were assessed as inhibitors towards the enzymes acetylcholinesterase, β-glucosidase, and β-glucuronidase. Terreic acid, butyrolactone I, butyrolactone 4′,4′′-diacetate (2.1), and 3′-(3-methylbutyl)-butyrolactone II (2.2) were active towards the phytopathogenic bacteria Erwinia carotovora with IC₅₀ of 5 and 4–18 μg/ml, respectively. Under the same experimental conditions, the IC₅₀ of streptomycin was 1.9 μg/ml. 3′-(3-Methylbutyl)-butyrolactone II was moderately active against Pseudomonas syringae and Botrytis cinerea with IC₅₀ of 21μg/ml and MIC of 15.6 μg/ml, respectively. Butyrolactone I also inhibited germination of the dicot Lactuca sativa with an IC₅₀ of 5 × 10⁻⁵ M. The IC₅₀ of reference herbicide acetochlor was 1 × 10⁻⁵ M. The effect of 2.2 and 2.3, known as butyrolactone III on Panicum millaceum germination and growth was stronger than that of 2 and 2.1. Reduction of the double bond in the isoprenyl side chain of butyrolactone I increased the antibacterial effect against E. carotovora as well as acetylation. To our best knowledge, this is the first report on the antibacterial effect of butyrolactone derivatives towards Erwinia carotovora and the phytopathogenic fungus Botrytis cinerea. The butyrolactone I derivative 2.2 presented a moderate inhibitory effect against the enzyme acetylcholinesterase with an IC₅₀ of 47 μg/ml. Under the same experimental conditions, the reference inhibitor galanthamine had an IC₅₀ of 3 μg/ml.     

In Vitro evaluation of the cytotoxic and anti-proliferative properties of resveratrol and several of its analogs

Resveratrol (RES), a component of red wine, possesses anti-inflammatory properties. The studies described in the present work were aimed at evaluating the potential for RES and related stilbene analogs (piceatannol, PIC; pterostilbene, TPS; trans-stilbene, TS; and trans-stilbene oxide, TSO) to exhibit toxicity towards RAW 264.7 mouse macrophages. The effect of TS, TSO, RES and TPS on RAW 264.7 macrophage viability was determined by two standard methods: (a) the MTT assay and (b) the trypan blue dye exclusion test. Whereas macrophages were more sensitive to PIC (LC_{50 trypan} ∼ 1.3 μM) and to TPS (LC_{50 trypan} ∼ 4.0 μM and LC_{50 MTT} ∼ 8.3 μM) than to RES (LC_{50 trypan} ∼ 8.9 μM and LC_{50 MTT} ∼ 29.0 μM), they were relatively resistant to TSO (LC_{50 trypan} ∼ 61.0 μM and LC_{50 MTT} > 100 μM) and to TS (LC_{50 trypan} ≥ 5.0 μM and LC_{50 MTT} ≥ 5.0 μM). The ability of selected stilbenes (RES, TPS and PIC) to exhibit growth inhibitory effects was also examined. Although RES and TPS were observed to inhibit cell proliferation in macrophages (IC₅₀ ≤ 25 μM), these cells were resistant to growth inhibition by PIC (IC₅₀ ≥ 50 μM). The data obtained in the present analysis demonstrate that substituted stilbene compounds such as RES have the capacity to exhibit cytotoxic and anti-proliferative activities in macrophages.     

Biochemical controls of citrate synthase in chickpea bacteroids

Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear. The K _m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by NADH was linear competitive with respect to acetyl-CoA (K _is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K _is = 56 ± 3.8 μM and K _ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD⁺ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely to be relieved by NAD⁺ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle. Received: 14 July 1999 / Accepted: 20 September 1999     

Vanadyl bisacetylacetonate protects β cells from palmitate-induced cell death through the unfolded protein response pathway

Endoplasmic reticulum (ER) stress induced by free fatty acids (FFA) is important to β-cell loss during the development of type 2 diabetes. To test whether vanadium compounds could influence ER stress and the responses in their mechanism of antidiabetic effects, we investigated the effects and the mechanism of vanadyl bisacetylacetonate [VO(acac)₂] on β cells upon treatment with palmitate, a typical saturated FFA. The experimental results showed that VO(acac)₂ could enhance FFA-induced signaling pathways of unfolded protein responses by upregulating the prosurvival chaperone immunoglobulin heavy-chain binding protein/78-kDa glucose-regulated protein and downregulating the expression of apoptotic C/EBP homologous protein, and consequently the reduction of insulin synthesis. VO(acac)₂ also ameliorated FFA-disturbed Ca²⁺ homeostasis in β cells. Overall, VO(acac)₂ enhanced stress adaption, thus protecting β cells from palmitate-induced apoptosis. This study provides some new insights into the mechanisms of antidiabetic vanadium compounds.     

Protein kinase involvement in land snail aestivation and anoxia: Protein kinase A kinetic properties and changes in second messenger compounds during depressed metabolism

In response to environmental stress (low water, low oxygen) snails sharply suppress their metabolic rate, a process that is coordinated at the molecular level by reversible protein phosphorylation of key enzymes and functional proteins. Factors affecting protein kinase activity are, therefore, critical to metabolic suppression. Changes in the concentration of protein kinase second messenger compounds were followed over the first 24 h of aestivation and anoxia exposure in the terrestrial snail Otala lactea (Muller) (Pulmonata, Helicidae). The results showed declining concentrations of cyclic AMP over the first 24 h of anoxia exposure and aestivation in foot. Cyclic AMP concentrations in hepatopancreas transiently decreased with the lowest concentration observed at 4 h in both anoxic and aestivating animals. A transient increase in foot muscle cyclic GMP concentrations was apparent 4 h after the start of aestivation whereas a slow, steady increase was seen in anoxic foot muscle. Foot muscle 1,4,5-inositol triphosphate (IP3) concentrations decreased transiently during anoxia exposure and aestivation. Hepatopancreas IP₃ concentrations were significantly lower in 24 h anoxic snails and foot IP3 concentrations were significantly lower in 24 h aestivating snails. Kinetic characterization of purified PKA catalytic subunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg²⁺ ion but was inhibited at Mg²⁺ concentrations above 0.5 mM. Increasing concentrations of neutral salts and phosphate also inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (I₅₀ = 39 mM) was much lower than that of the neutral salts (I₅₀ 240 mM). The enzyme exhibited a broad pH optimum between pH 6.5–8.5. Arrhenius plots gave an activation energy of 13.3 kcal/mol corresponding to a Q₁₀ value of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed.Abbreviations CAMP adenosine 3:5-cyclic monophosphate - cGMP-guanosine 3:5-cyclic monophosphate - H-89N [2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCI - IP₃ d-myo-inositol 1,4,5-triphosphate - I₅₀ the concentration of inhibitor required to reduce the velocity to one half its original value - PKA cAMP dependent protein kinase - PKAc PKA catalytic subunit - PKA-I PKA inhibitor protein - PKC calcium and phospholipid-dependent protein kinase - PKC-I PKC inhibitor protein - PKG cGMP dependent protein kinase - mU nmol of phosphate transferred per minute