Synthesis of [I]iodoDPA-713: A new probe for imaging inflammation

[¹²⁵I]IodoDPA-713 [¹²⁵I]1, which targets the translocator protein (TSPO, 18 kDa), was synthesized in seven steps from methyl-4-methoxybenzoate as a tool for quantification of inflammation in preclinical models. Preliminary in vitro autoradiography and in vivo small animal imaging were performed using [¹²⁵I]1 in a neurotoxicant-treated rat and in a murine model of lung inflammation, respectively. The radiochemical yield of [¹²⁵I]1 was 44 ± 6% with a specific radioactivity of 51.8 GBq/μmol (1400 mCi/μmol) and >99% radiochemical purity. Preliminary studies showed that [¹²⁵I]1 demonstrated increased specific binding to TSPO in a neurotoxicant-treated rat and increased radiopharmaceutical uptake in the lungs of an experimental inflammation model of lung inflammation. Compound [¹²⁵I]1 is a new, convenient probe for preclinical studies of TSPO activity.     

Identification of non-specific lipid transfer protein-1 as a calmodulin-binding protein in Arabidopsis

Although non-specific lipid transfer proteins (nsLTPs) are widely present in plants, their functions and regulations have not been fully understood. In this report, Arabidopsis nsLTP1 was cloned and expressed to investigate its binding to calmodulin (CaM). Gel overlay assays revealed that recombinant nsLTP1 bound to CaM in a calcium-independent manner. The association of nsLTP1 and CaM was corroborated using CaM-Sepharose beads to specifically isolate recombinant nsLTP1 from crude bacterial lysate. The CaM-binding site was mapped in nsLTP1 to the region of 69-80 amino acids. This region is highly conserved among plant nsLTPs, implicating that nsLTPs are a new family of CaM-binding proteins whose functions may be mediated by CaM signaling.     

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development. We describe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic α-helical peptide, melittin, and selected structural analogs. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.     

Convenient synthesis of a sialylglycopeptide-thioester having an intact and homogeneous complex-type disialyl-oligosaccharide

Access to glycopeptides with C-terminal thioester functionality is essential for the synthesis of large glycopeptides and glycoproteins through the use of native chemical ligation. Toward that end, we have developed a concise method for the synthesis of a glycopeptide thioester having an intact complex-type dibranched disialyl-oligosaccharide. The synthesis employed a coupling reaction between benzylthiol and a free carboxylic acid at the C-terminus of a glycopeptide in which the peptide side chains are protected. After construction of glycopeptide on the HMPB-PEGA resin through the Fmoc-strategy, the protected glycopeptide was released upon treatment with acetic acid/trifluoroethanol and then the C-terminal carboxylic acid was coupled with benzylthiol at -20 degrees C in DMF. For this coupling, PyBOP/DIPEA was found to be the best for the formation of the thioester, while avoiding racemization. Finally, the protecting groups were removed in good yield with 95% TFA, thus affording a glycopeptide-thioester having an intact and homogeneous complex-type disialyl-oligosaccharide.     

The actin binding protein, fesselin, is a member of the synaptopodin family

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and α-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.     

NMR based structure-activity relationship analysis of an antimicrobial peptide, thanatin, engineered by site-specific chemical modification: Activity improvement and spectrum alteration

Activity improvement of an antimicrobial peptide, thanatin, has been achieved up to 4-fold higher than natural original one by site-specific chemical modifications with tert-butyl group at two cysteine residues which form an intramoleular disulfide bridge. The chemically modified thanatin (C11tBu/C18tBu) exhibited improved antimicrobial activity toward Gram-positive bacteria, Micrococcus luteus, whereas lowered activity toward Gram-negative bacteria, Escherichia coli. This finding suggests that disulfide-bridge formation is not only indispensable for exhibition of antimicrobial activity of thanatin but also closely related to the activity specificity towards bacteria. NMR analysis indicates that thanatin acts against E.coli stereospecifically by taking advantage of its C-terminal β-hairpin structure, while the activity against M. luteus does not relate to structures and correlates very well to side-chain hydrophobicity.     

Effect of sydnone SYD-1, a mesoionic compound, on energy-linked functions of rat liver mitochondria

An important antitumour effect of SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) has been shown. We now report the effects of this mesoionic compound on mitochondrial metabolism. SYD-1 (1.5 micromol mg(-1) protein) dose-dependently inhibited the respiratory rate by 65% and 40% in state 3 using sodium glutamate and succinate, respectively, as substrates. Phosphorylation efficiency was depressed by SYD-1, as evidenced by stimulation of the state 4 respiratory rate, which was more accentuated with glutamate ( approximately 180%) than with succinate ( approximately 40%), with 1.5 micromol mg(-1) protein of SYD-1. As a consequence of the effects on states 3 and 4, the RCC and ADP/O ratios were lowered by SYD-1 using both substrates, although this effect was stronger with glutamate. The formation of membrane electrical potential was inhibited by approximately 50% (1.5 micromol SYD-1mg(-1) protein). SYD-1 interfered with the permeability of the inner mitochondrial membrane, as demonstrated by assays of mitochondrial swelling in the presence of sodium acetate and valinomycin +K(+). SYD-1 (1.5 micromol mg(-1) protein) inhibited glutamate completely and succinate energized-mitochondrial swelling by 80% in preparations containing sodium acetate. The swelling of de-energized mitochondria induced by K(+) and valinomycin was inhibited by 20% at all concentrations of SYD-1. An analysis of the segments of the respiratory chain suggested that the SYD-1 inhibition site goes beyond the complex I and includes complexes III and IV. Glutamate dehydrogenase was inhibited by 20% with SYD-1 (1.5 micromol mg(-1) protein). The hydrolytic activity of complex F(1)F(o) ATPase in intact mitochondria was greatly increased ( approximately 450%) in the presence of SYD-1. Our results show that SYD-1 depresses the efficiency of electron transport and oxidative phosphorylation, suggesting that these effects may be involved in its antitumoural effect.     

Effect of the headgroup variation on the gene transfer properties of cholesterol based cationic lipids possessing ether linkage

Eight cholesterol based cationic lipids differing in the headgroup have been synthesized based on the ether linkage between the cationic headgroup and the cholesterol backbone. All the lipids formed stable suspensions in water. Transfection efficacies were examined in the absence and presence of serum using their optimized liposomal (lipid:DOPE) formulations. Our results showed that the transfection activities depend on the nature of the headgroup. Lipid bearing 4-N,N′-dimethylaminopyridine (DMAP) as headgroup showed the maximum transfection efficacy in the presence of serum. Importantly, the optimized formulation for this cationic lipid does not require DOPE, which is being used by most commercially available formulations. Cytotoxicity studies showed that the introduction of the positive charge decreases the cell viability of the cationic lipid formulations. Gel electrophoresis and Ethidium bromide exclusion assay revealed the different DNA binding abilities of formulations depending upon the headgroup of the cholesteryl lipid.     

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (gamma-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1mM CHB with 5 mM GSH at 37 degrees C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.     

A sesquiterpene lactone, costunolide, interacts with microtubule protein and inhibits the growth of MCF-7 cells

Costunolide is an active sesquiterpene lactone of medicinal herbs with anti-inflammatory and potential anti-cancer activity. Nevertheless, the pharmacological pathways of costunolide have not yet been fully elucidated. In this study we showed that costunolide exerts a dose-dependent antiproliferative activity in the human breast cancer MCF-7 cells. In addition, light microscopy observations indicated that costunolide affected nuclear organization and reorganized microtubule architecture. The antiproliferative and antimicrotubular effects of costunolide were not influenced by paclitaxel, well-known microtubule-stabilizing anticancer agent. The microtubule-interacting activity of costunolide was confirmed by in vitro studies on purified microtubular protein. In fact, costunolide demonstrated polymerizing ability, by inducing the formation of well organized microtubule polymers. Our data suggest an interaction of costunolide with microtubules, which may represent a new intracellular target for this drug.     

Lysosome destabilization by cytosolic extracts, putative involvement of Ca(2+)/phospholipase C

Lysosomal disintegration is a crucial event for living cells, but mechanisms for the event are still unclear. In this study, we established that the cytosolic extracts could enhance lysosomal osmotic sensitivity and osmotically destabilize the lysosomes. The cytosol also caused the lysosomes to become more swollen in the hypotonic sucrose medium. The results indicate that the cytosol induced an osmotic shock to the lysosomes and an influx of water into the organelle. Since the effects of cytosol on the lysosomes could be abolished by O-tricyclo[5.2.1.0(2,6)]dec-9-yl dithiocarbonate potassium salt (D609), a specific inhibitor of cytosolic phospholipase C (PLC), the PLC might play an important role in the lysosomal osmotic destabilization. The activity of cytosolic PLC and the extent of enzyme latency loss of the cytosol-treated lysosomes exhibited a similar biphasic dependence on the cytosolic Ca2+ concentration. In addition, the cytosol did not osmotically destabilize the lysosomes until the cytosolic calcium ions rose above 100 nM. It suggests that the destabilization effect of cytosol on the lysosomes is Ca(2+)-dependent.     

Mechanism of pH-triggered collapse of phosphatidylethanolamine liposomes stabilized by an ortho ester polyethyleneglycol lipid

The mechanism of pH-triggered destabilization of liposomes composed of a polyethyleneglycol-orthoester-distearoylglycerol lipid (POD) and phosphatidyl ethanolamine (PE) has been studied using an ANTS/DPX leakage and a lipid-mixing assay. We developed a kinetic model that relates POD hydrolysis to liposome collapse. This minimum-surface-shielding model describes the kinetics of the pH-triggered release of POD/PE liposomes. In the model, when acid-catalyzed hydrolysis lowers the mole percentage of POD on the liposome surface to a critical level, intervesicular lipid mixing is initiated, resulting in a burst of contents release. Two phases of content leakage are observed: a lag phase and a burst phase. During the lag phase, less than 20% of liposomal contents are released and the leakage begins to accelerate when approaching to the transition point. During the burst phase, the leakage rate is dependent on interbilayer contact. The burst phase occurs when the surface density of the PEG lipid is 2.3 +/- 0.6 mol%, regardless of the pH. Vesicles containing 4 mol% of a pH-insensitive PEG-lipid conjugate and 10% POD did not leak contents or collapse at any pH. These data are consistent with the stalk theory to describe the lamellar-to-inverted hexagonal phase transition and set a lower bound of approximately 16 PE lipids on the external monolayer as the contact site required for lipid mixing between two bilayers.     

The effect of calcium ions on subcellular localization of aldolase-FBPase complex in skeletal muscle

In skeletal muscles, FBPase-aldolase complex is located on alpha-actinin of the Z-line. In the present paper, we show evidence that stability of the complex is regulated by calcium ions. Real time interaction analysis, confocal microscopy and the protein exchange method have revealed that elevated calcium concentration decreases association constant of FBPase-aldolase and FBPase-alpha-actinin complex, causes fast dissociation of FBPase from the Z-line and slow accumulation of aldolase within the I-band and M-line. Therefore, the release of Ca2+ during muscle contraction might result, simultaneously, in the inhibition of glyconeogenesis and in the acceleration of glycolysis.     

Synthesis and cytogenetic studies of structure--biological activity relationship of esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers

The esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers have been prepared and their cytogenetic studies of structure-biological activity relationship were evaluated. The cytogenetic effects (sister chromatid exchanges (SCEs) induction and proliferation rate indices (PRIs) depression) by o-, m- and p-[N,N-bis(2-chloroethyl)amino] cinnamic acid were also investigated. Among the above compounds tested, those of the m-[N,N-bis(2-chloroethyl)amino] cinnamic acid and of the o-[N,N-bis(2-chloroethyl)amino] cinnamic acid ester of aza-homo-Hecogenin were more active in comparison to the others.     

Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

UDP (uridine diphosphate) galactopyranose mutase (UGM) is involved in the cell wall biosynthesis of many pathogenic microorganisms. UGM catalyzes the reversible conversion of UDP-α-d-galactopyranose into UDP-α-d-galactofuranose, with the latter being the precursor of galactofuranose (Galf) residues in cell walls. Glycoconjugates of Galf are essential components in the cell wall of various pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. The absence of Galf in humans and its bacterial requirement make UGM a potential target for developing novel antibacterial agents. In this article, we report the synthesis, inhibitory activity, and X-ray crystallographic studies of UDP-phosphono-galactopyranose, a nonhydrolyzable C-glycosidic phosphonate. This is the first report on the synthesis of a phosphonate analog of UDP-α-d-galactopyranose by a chemoenzymatic phosphoryl coupling method. The phosphonate was evaluated against three bacterial UGMs and showed only moderate inhibition. We determined the crystal structure of the phosphonate analog bound to Deinococcus radiodurans UGM at 2.6 Å resolution. The phosphonate analog is bound in a novel conformation not observed in UGM-substrate complex structures or in other enzyme-sugar nucleotide phosphonate complexes. This complex structure provides a structural basis for the observed micromolar inhibition towards UGM. Steric clashes, loss of electrostatic stabilization between an active-site arginine (Arg305) and the phosphonate analog, and a 180° flip of the hexose moiety account for the differences in the binding orientations of the isosteric phosphonate analog and the physiological substrate. This provides new insight into the ability of a sugar-nucleotide-binding enzyme to orient a substrate analog in an unexpected geometry and should be taken into consideration in designing such enzyme inhibitors.     

Sample-to-SNP kit: A reliable,easy and fast tool for the detection of HFE p.H63D and p.C282Y variations associated to hereditary hemochromatosis

Classical hereditary hemochromatosis involves the HFE-gene and diagnostic analysis of the DNA variants HFE p.C282Y (c.845G > A; rs1800562) and HFE p.H63D (c.187C > G; rs1799945). The affected protein alters the iron homeostasis resulting in iron overload in various tissues. The aim of this study was to validate the TaqMan-based Sample-to-SNP protocol for the analysis of the HFE-p.C282Y and p.H63D variants with regard to accuracy, usefulness and reproducibility compared to an existing SNP protocol. The Sample-to-SNP protocol uses an approach where the DNA template is made accessible from a cell lysate followed by TaqMan analysis. Besides the HFE-SNPs other eight SNPs were used as well. These SNPs were: Coagulation factor II-gene F2 c.20210G > A, Coagulation factor V-gene F5 p.R506Q (c.1517G > A; rs121917732), Mitochondria SNP: mt7028 G > A, Mitochondria SNP: mt12308 A > G, Proprotein convertase subtilisin/kexin type 9-gene PCSK9 p.R46L (c.137G > T), Plutathione S-transferase pi 1-gene GSTP1 p.I105V (c313A > G; rs1695), LXR g.-171 A > G, ZNF202 g.-118 G > T. In conclusion the Sample-to-SNP kit proved to be an accurate, reliable, robust, easy to use and rapid TaqMan-based SNP detection protocol, which could be quickly implemented in a routine diagnostic or research facility.     

Solution state NMR structure and dynamics of KpOmpA, a 210 residue transmembrane domain possessing a high potential for immunological applications

The three-dimensional structure of the outer membrane protein A from Klebsiella pneumoniae transmembrane domain was determined by NMR. This protein induces specific humoral and cytotoxic responses, and is a potent carrier protein. This is one of the largest integral membrane proteins (210 residues) for which nearly complete resonance assignment, including side chains, has been achieved so far. The methodology rested on the use of 900 MHz 3D and 4D TROSY experiments recorded on a uniformly ¹⁵N,¹³C,²H-labeled sample and on a perdeuterated methyl protonated sample. The structure was refined from 920 experimental constraints, giving an ensemble of 20 best structures with an r.m.s. deviation of 0.54 Å for the main chain atoms in the core eight-stranded β-barrel. The protein dynamics was assessed, in a residue-specific manner, by ¹H-¹⁵N NOEs (pico- to nanosecond timescale), exchange broadening (millisecond to second) and ¹H-²H chemical exchange (hour-weeks).