Alpha-amino Group Research Articles

Protein Acetylation in Archaea, Bacteria, and Eukaryotes

Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal) or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which—Alba—was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.

The Essential Escherichia coli Apolipoprotein N-Acyltransferase (Lnt) Exists as an Extracytoplasmic Thioester Acyl-Enzyme Intermediate

Escherichia coli apolipoprotein N-acyltransferase (Lnt) transfers an acyl group from sn-1-glycerophospholipid to the free alpha-amino group of the N-terminal cysteine of apolipoproteins, resulting in mature triacylated lipoprotein. Here we report that the Lnt reaction proceeds through an acyl-enzyme intermediate in which a palmitoyl group forms a thioester bond with the thiol of the active site residue C387 that was cleaved by neutral hydroxylamine. Lnt(C387S) also formed a fatty acyl intermediate that was resistant to neutral hydroxylamine treatment, consistent with formation of an oxygen-ester linkage. Lnt(C387A) did not form an acyl-enzyme intermediate and, like Lnt(C387S), did not have any detectable Lnt activity, indicating that acylation cannot occur at other positions in the catalytic domain. The existence of this thioacyl-enzyme intermediate allowed us to determine whether essential residues in the catalytic domain of Lnt affect the first step of the reaction, the formation of the acyl-enzyme intermediate, or the second step in which the acyl chain is transferred to the apolipoprotein substrate. In the catalytic triad, E267 is required for the formation of the acyl-enzyme intermediate, indicating its role in enhancing the nucleophilicity of C387. E343 is also involved in the first step but is not in close proximity to the active site. W237, Y388, and E389 play a role in the second step of the reaction since acyl-Lnt is formed but N-acylation does not occur. The data presented allow discrimination between the functions of essential Lnt residues in catalytic activity and substrate recognition.

Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma

Mistranslation from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences1-3. Throughout evolution, two editing-checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly1,2. One checkpoint is the AlaRS editing center, while the other is from widely distributed AlaXps—free-standing, genome-encoded editing proteins that clear Ser-tRNAAla. The paradox of misincorporating both a smaller (glycine) and a larger amino acid (serine) suggests a deep conflict for nature-designed AlaRS. To understand the chemical basis for this conflict, kinetic and mutational analysis, together with nine crystal structures, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the α–amino group of the bound amino acid using an acidic residue. This design, of more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently not able to find better architecture for recognition of alanine, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.

PREPARATION OF PICKLED PRODUCT FROM ANCHOVIES(STOLEPHORUS SP.)AND STUDIES ON QUALITY CHANGES DURING STORAGE

ABSTRACT In the present study, low cost anchovy pickled product has been developed using traditional technology application of different inhibitory factors to achieve a reasonable shelf life at ambient tropical temperature. Microbial safety, nutrient retention and product quality in terms of flavor and texture after rehydration have been tested. This product has been subjected to storage studies, with respect to product quality and microbial status. It can be kept well for a period of 15 weeks. The quality changes during storage are decreased in sensory scores for the overall acceptance and also for the individual attributes. Increase in nitrogenous components such as total volatile bases nitrogen, trimethylamine nitrogen and alpha amino nitrogen was obtained with the advancement of the storage period. The lipid quality of the fish showed gradual increase in preoxide value and free fatty acid accompanied by decrease in flavor and odor scores. Activity of microorganisms showed a marginal increase with increase in storage period. It is ready-to-eat product or can be lightly fried along with spices. PRACTICAL APPLICATIONS Anchovies are one of the important pelagic fish species found in the Indian Ocean, which can grow up to 20 cm (8 in), and prefer the warmer waters around the world. The limiting factors in the effective utilization of anchovies are their small size, seasonal nature of the fishery forming gluts, sensitive to physical, time and temperature changes. Anchovies are rich in omega-3 oils, calcium and iron. They can be cooked fresh, usually sold packed in salt, tinned in oil, as a paste in jars or tubes, or as a sauce. Anchovy pickled products have been developed using application of different inhibitory factors to achieve a reasonable shelf life at ambient tropical temperature. Microbial safety, nutrient retention and product quality in terms of flavor and texture after rehydration have been tested. This product has been subjected to storage studies, with respect to product quality and microbial status. It can be kept well for a period of 15 weeks. Activity of microorganisms showed a marginal increase with increase in storage period. It is a ready-to-eat product or can be lightly fried along with spices. This product is helpful in value addition and better utilization of anchovy catch.

Human Naa50p (Nat5/San) Displays Both Protein Nα- and Nϵ-Acetyltransferase Activity

Protein acetylation is a widespread modification that is mediated by site-selective acetyltransferases. KATs (lysine N(epsilon)-acetyltransferases), modify the side chain of specific lysines on histones and other proteins, a central process in regulating gene expression. N(alpha)-terminal acetylation occurs on the ribosome where the alpha amino group of nascent polypeptides is acetylated by NATs (N-terminal acetyltransferase). In yeast, three different NAT complexes were identified NatA, NatB, and NatC. NatA is composed of two main subunits, the catalytic subunit Naa10p (Ard1p) and Naa15p (Nat1p). Naa50p (Nat5) is physically associated with NatA. In man, hNaa50p was shown to have acetyltransferase activity and to be important for chromosome segregation. In this study, we used purified recombinant hNaa50p and multiple oligopeptide substrates to identify and characterize an N(alpha)-acetyltransferase activity of hNaa50p. As the preferred substrate this activity acetylates oligopeptides with N termini Met-Leu-Xxx-Pro. Furthermore, hNaa50p autoacetylates lysines 34, 37, and 140 in vitro, modulating hNaa50p substrate specificity. In addition, histone 4 was detected as a hNaa50p KAT substrate in vitro. Our findings thus provide the first experimental evidence of an enzyme having both KAT and NAT activities.

Asymmetric Hydrogenation of Enamides, α-Enol and α-Enamido Ester Phosphonates Catalyzed by IndolPhos-Rh Complexes

The scope of the IndolPhos-Rh-catalyzed asymmetric hydrogenation of enamides, alpha-enol and alpha-enamido ester phosphonates, has been investigated. In addition, Taddol-based IndolPhos ligands are introduced. High activities and good to excellent enantioselectivities up to 99% ee are obtained for a broad range of structurally diverse substrates, giving important chiral products such as alpha, beta(2), and beta(3) amino acid derivatives, arylamines, and amino and hydroxy phosphonates.

Effect of DOTA Position on Melanoma Targeting and Pharmacokinetic Properties of 111In-Labeled Lactam Bridge-Cyclized α-Melanocyte Stimulating Hormone Peptide

The purpose of this study was to examine the effect of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) position on melanoma targeting and pharmacokinetics of radiolabeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (alpha-MSH) peptide. A novel lactam bridge-cyclized alpha-MSH peptide, Ac-GluGlu-CycMSH[DOTA] {Ac-Glu-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Lys(DOTA)]}, was synthesized using standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. DOTA was directly attached to the alpha-amino group of Lys in the cyclic ring, while the N-terminus of the peptide was acetylated to generate Ac-GluGlu-CycMSH[DOTA]. The MC1 receptor binding affinity of Ac-GluGlu-CycMSH[DOTA] was determined in B16/F1 melanoma cells. Melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In were determined in B16/F1 melanoma-bearing C57 mice and compared to that of 111In-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp] (111In-DOTA-GlyGlu-CycMSH; DOTA was coupled to the N-terminus of the peptide). Ac-GluGlu-CycMSH[DOTA] displayed 0.6 nM MC1 receptor binding affinity in B16/F1 cells. Ac-GluGlu-CycMSH[DOTA]-111In was readily prepared with greater than 95% radiolabeling yield. Ac-GluGlu-CycMSH[DOTA]-111In exhibited high tumor uptake (11.42 +/- 2.20% ID/g 2 h postinjection) and prolonged tumor retention (9.42 +/- 2.41% ID/g 4 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<1.3% ID/g) except for the kidneys 2, 4, and 24 h postinjection. DOTA position exhibited profound effect on melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In, providing a new insight into the design of lactam bridge-cyclized peptide for melanoma imaging and therapy.

Identification of Apolipoprotein N-Acyltransferase (Lnt) in Mycobacteria

Lipoproteins of Gram-negative and Gram-positive bacteria carry a thioether-bound diacylglycerol but differ by a fatty acid amide bound to the alpha-amino group of the universally conserved cysteine. In Escherichia coli the N-terminal acylation is catalyzed by the N-acyltransferase Lnt. Using E. coli Lnt as a query in a BLASTp search, we identified putative lnt genes also in Gram-positive mycobacteria. The Mycobacterium tuberculosis lipoprotein LppX, heterologously expressed in Mycobacterium smegmatis, was N-acylated at the N-terminal cysteine, whereas LppX expressed in a M. smegmatis lnt::aph knock-out mutant was accessible for N-terminal sequencing. Western blot analyses of a truncated and tagged form of LppX indicated a smaller size of about 0.3 kDa in the lnt::aph mutant compared with the parental strain. Matrix-assisted laser desorption ionization time-of-flight/time-of-flight analyses of a trypsin digest of LppX proved the presence of the diacylglycerol modification in both strains, the parental strain and lnt::aph mutant. N-Acylation was found exclusively in the M. smegmatis parental strain. Complementation of the lnt::aph mutant with M. tuberculosis ppm1 restored N-acylation. The substrate for N-acylation is a C16 fatty acid, whereas the two fatty acids of the diacylglycerol residue were identified as C16 and C19:0 fatty acid, the latter most likely tuberculostearic acid. We demonstrate that mycobacterial lipoproteins are triacylated. For the first time to our knowledge, we identify Lnt activity in Gram-positive bacteria and assigned the responsible genes. In M. smegmatis and M. tuberculosis the open reading frames are annotated as MSMEG_3860 and M. tuberculosis ppm1, respectively.

The C-Terminal Active Site Cysteine ofEscherichia coliGlutaredoxin 1 Determines the Glutathione Specificity of the Second Step of Peptide Deglutathionylation

Glutaredoxins are oxidoreductases specialized in reducing glutathione-protein mixed disulfides. In the first step of deglutathionylation, glutaredoxins form a mixed disulfide with glutathione, releasing reduced peptide. The specificity of this reaction is based on the unusual amide linkage formed between the gamma-carboxylate of the N-terminal glutamic acid and the alpha-amino group of the cysteine present in glutathione. In the second step of deglutathionylation, glutathione reduces the glutaredoxin-glutathione mixed disulfide. Here we show that the specificity of this second reaction for Escherichia coli Grx1, but not for human or yeast Grx1, also is based on the unusual gamma-linkage present in glutathione. Mutating Tyr13, Thr58, and/or Asp74 to alanine in E. coli Grx1 results in the glutaredoxin-peptide mixed disulfide being thermodynamically favored over the glutaredoxin-glutathione mixed disulfide in the first step of the reaction. An increased propensity to form glutaredoxin-protein mixed disulfides was observed in vivo for these same mutants. Furthermore, we demonstrate that all mutations studied in Cys14, the C-terminal active site cysteine, abolish the specificity of E. coli Grx1 for glutathione over the corresponding tripeptide Glu-Cys-Gly, which has a normal peptide bond linking Glu-Cys instead of the gamma-linkage present in glutathione, in the second step of deglutathionylation.

Technical note: Correction of net portal absorption of nitrogen compounds for differences in methods: First step of a meta-analysis

The objective of this study was to evaluate the usefulness of correcting net portal absorption (NPA) of urea-N, ammonia, and AA-N for differences in methods before their inclusion into a meta-analysis. It was hypothesized that the difference, or portal-drained viscera (PDV) balance, between N inputs (apparently digested N plus urea-N) and outputs (ammonia plus AA-N) was 0 in the absence of measurement errors and based on the assumption that other sources of N inputs and outputs were relatively small and balanced each other. A database was built from 44 publications that reported data from 129 treatments (sheep, n = 71; beef cattle, n = 32; and dairy cows, n = 26). When necessary, NPA of urea-N (n = 38) and ammonia (n = 35) results were recalculated on a whole-blood basis, whereas NPA of AA-N (n = 87) was recalculated for all the N from AA transferred across the PDV rather than only the N from the alpha-amino group. Before corrections, PDV balance averaged 22.9% of N ingested (SD 29.0) for all treatments; after corrections, PDV balance significantly decreased to 10.2% of N ingested (SD 34.7). No difference in PDV balance was observed among species before or after corrections. Correcting NPA of urea-N, ammonia, and AA-N increased the accuracy without improving precision. Therefore, from a biological perspective, recalculating reported data seems appropriate to reduce bias due to differences in methods because this approach reduces the excess in N inputs relative to N outputs.

Charge‐derivatized amino acids facilitate model studies on protein side‐chain modifications by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The alpha-amino groups of histidine and lysine were derivatized with p-carboxylbenzyltriphenylphosphonium to form the pseudo dipeptides, PHis and PLys, which can be sensitively detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) due to the fixed positive charge of the phosphonium group. Detection limits of PHis and PLys by MALDI-TOFMS were both 30 fmol with a signal-to-noise ratio of 5:1. These pseudo dipeptides were excellent surrogates for His- or Lys-containing peptides in model reactions mimicking proteins with reactive electrophiles, prominently those generated by peroxidation of polyunsaturated fatty acids including 4-hydroxy-2(E)-nonenal (HNE), 4-oxo-2(E)-nonenal (ONE), 2(E)-octenal, and 2(E)-heptenal. An air-saturated solution of linoleic acid (d0:d5 = 1:1) was incubated in the presence of Fe(II) and ascorbate with these two pseudo dipeptides, and the reaction products were characterized by MALDI-TOFMS and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). By using PHis and PLys, the previously reported ONE-derived His-furan adduct was detected along with evidence for a cyclic alpha,beta-unsaturated ketone. A dimer formed from ONE was found to react with PHis through Michael addition. Alkenals were found to form two novel adducts with PLys. 2(E)-Octenoic acid-His Michael adduct and N(epsilon)-pentanoyllysine were identified as potential protein side-chain adducts modified by products of linoleic acid peroxidation. In addition, when PHis or PLys and AcHis or BocLys were exposed to the linoleic acid peroxidation, an epoxy-keto-ocatadecenoic acid mediated His-His cross-link was detected, along with the observation of a His-ONE/9,12-dioxo-10-dodecenoic acid-Lys derived pyrrole cross-link.

Functional Annotation of Two New Carboxypeptidases from the Amidohydrolase Superfamily of Enzymes

Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed, and purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300), while the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolated from the Sargasso Sea ( gi|44371129 ). The catalytic functions and the substrate profiles for the two enzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shown to catalyze the hydrolysis of l-Xaa-l-Xaa dipeptides in which the amino acid at the N-terminus was relatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. With Cc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, and tryptophan were hydrolyzed. The same specificity was observed with Sgx9355e, but this protein was also able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetyl and N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identified for Cc0300 were l-Ala-l-Leu with k(cat) and k(cat)/K(m) values of 37 s(-1) and 1.1 x 10(5) M(-1) s(-1), respectively, and N-formyl-l-Tyr with k(cat) and k(cat)/K(m) values of 33 s(-1) and 3.9 x 10(5) M(-1) s(-1), respectively. The best substrate identified for Sgx9355e was l-Ala-l-Phe with k(cat) and k(cat)/K(m) values of 0.41 s(-1) and 5.8 x 10(3) M(-1) s(-1). The three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 A with l-methionine bound in the active site. The alpha-carboxylate of the methionine is ion-paired to His-237 and also hydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The alpha-amino group of the bound methionine interacts with Asp-328. The structural determinants for substrate recognition were identified and compared with other enzymes in this superfamily that hydrolyze dipeptides with different specificities.

A New Family of Receptor Tyrosine Kinases with a Venus Flytrap Binding Domain in Insects and Other Invertebrates Activated by Aminoacids

BackgroundTyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor.Methods and FindingsHere we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABAB receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development.ConclusionThe identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases.

Sample stacking in CZE using dynamic thermal junctions II: Analytes with high dpKa/dT crossing a single thermal junction in a BGE with low dpH/dT

In a previous work [M. Mandaji, et al., this issue] a sample stacking method was theoretically modeled and experimentally demonstrated for analytes with low dpK(a)/dT (analytes carrying carboxylic groups) and BGEs with high dpH/dT (high pH-temperature-coefficients). In that work, buffer pH was modulated with temperature, inducing electrophoretic mobility changes in the analytes. In the present work, the opposite conditions are studied and tested, i.e. analytes with high dpK(a)/dT and BGEs that exhibit low dpH/dT. It is well known that organic bases such as amines, imidazoles, and benzimidazoles exhibit high dpK(a)/dT. Temperature variations induce instantaneous changes on the basicity of these and other basic groups. Therefore, the electrophoretic velocity of some analytes changes abruptly when temperature variations are applied along the capillary. This is true only if BGE pH remains constant or if it changes in the opposite direction of pK(a) of the analyte. The presence of hot and cold sections along the capillary also affects local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band stacking efficacy was also taken into account in the theoretical model presented. Finally, this stacking method is demonstrated for lysine partially derivatized with naphthalene-2,3-dicarboxaldehyde. In this case, the amino group of the lateral chain was left underivatized and only the alpha amino group was derivatized. Therefore, the basicity of the lateral amino group, and consequently the electrophoretic mobility, was modulated with temperature while the pH of the buffer used remained unchanged.

Expanding the Peptide β-Turn in αγ Hybrid Sequences: 12 Atom Hydrogen Bonded Helical and Hairpin Turns

Hybrid peptide segments containing contiguous alpha and gamma amino acid residues can form C(12) hydrogen bonded turns which may be considered as backbone expanded analogues of C(10) (beta-turns) found in alphaalpha segments. Exploration of the regular hydrogen bonded conformations accessible for hybrid alphagamma sequences is facilitated by the use of a stereochemically constrained gamma amino acid residue gabapentin (1-aminomethylcyclohexaneacetic acid, Gpn), in which the two torsion angles about C(gamma)-C(beta) (theta(1)) and C(beta)-C(alpha) (theta(2)) are predominantly restricted to gauche conformations. The crystal structures of the octapeptides Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (1) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (2) reveal two distinct conformations for the Aib-Gpn segment. Peptide 1 forms a continuous helix over the Aib(2)-Aib(6) segment, while the peptide 2 forms a beta-hairpin structure stabilized by four cross-strand hydrogen bonds with the Aib-Gpn segment forming a nonhelical C(12) turn. The robustness of the helix in peptide 1 in solution is demonstrated by NMR methods. Peptide 2 is conformationally fragile in solution with evidence of beta-hairpin conformations being obtained in methanol. Theoretical calculations permit delineation of the various C(12) hydrogen bonded structures which are energetically feasible in alphagamma and gammaalpha sequences.

Noninvasive imaging of tumor integrin expression using 18F-labeled RGD dimer peptide with PEG4 linkers

Various radiolabeled Arg-Gly-Asp (RGD) peptides have been previously investigated for tumor integrin alpha(v)beta(3) imaging. To further develop RGD radiotracers with enhanced tumor-targeting efficacy and improved in vivo pharmacokinetics, we designed a new RGD homodimeric peptide with two PEG(4) spacers (PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) between the two monomeric RGD motifs and one PEG(4) linker on the glutamate alpha-amino group ((18)F-labeled PEG(4)-E[PEG(4)-c(RGDfK)](2), P-PRGD2), as a promising agent for noninvasive imaging of integrin expression in mouse models. P-PRGD2 was labeled with (18)F via 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-FP) prosthetic group. In vitro and in vivo characteristics of the new dimeric RGD peptide tracer (18)F-FP-P-PRGD2 were investigated and compared with those of (18)F-FP-P-RGD2 ((18)F-labeled RGD dimer without two PEG(4) spacers between the two RGD motifs). The ability of (18)F-FP-P-PRGD2 to image tumor vascular integrin expression was evaluated in a 4T1 murine breast tumor model. With the insertion of two PEG(4) spacers between the two RGD motifs, (18)F-FP-P-PRGD2 showed enhanced integrin alpha(v)beta(3)-binding affinity, increased tumor uptake and tumor-to-nontumor background ratios compared with (18)F-FP-P-RGD2 in U87MG tumors. MicroPET imaging with (18)F-FP-P-PRGD2 revealed high tumor contrast and low background in tumor-bearing nude mice. Biodistribution studies confirmed the in vivo integrin alpha(v)beta(3)-binding specificity of (18)F-FP-P-RGD2. (18)F-FP-P-PRGD2 can specifically image integrin alpha(v)beta(3) on the activated endothelial cells of tumor neovasculature. (18)F-FP-P-PRGD2 can provide important information on integrin expression on the tumor vasculature. The high integrin binding affinity and specificity, excellent pharmacokinetic properties and metabolic stability make the new RGD dimeric tracer (18)F-FP-P-PRGD2 a promising agent for PET imaging of tumor angiogenesis and for monitoring the efficacy of antiangiogenic treatment.

Crystal Structures of Constitutive Nitric Oxide Synthases in Complex with De Novo Designed Inhibitors

New nitric oxide synthase (NOS) inhibitors were designed de novo with knowledge gathered from the studies on the nNOS-selective dipeptide inhibitors. Each of the new inhibitors consists of three fragments: an aminopyridine ring, a pyrrolidine, and a tail of various length and polarity. The in vitro inhibitory assays indicate good potency and isoform selectivity for some of the compounds. Crystal structures of these inhibitors bound to either wild type or mutant nNOS and eNOS have confirmed design expectations. The aminopyridine ring mimics the guanidinium group of L-arginine and functions as an anchor to place the compound in the NOS active site where it hydrogen bonds to a conserved Glu. The rigidity of the pyrrolidine ring places the pyrrolidine ring nitrogen between the same conserved Glu and the selective residue nNOS Asp597/eNOS Asn368, which results in similar interactions observed with the alpha-amino group of dipeptide inhibitors bound to nNOS. These structures provide additional information to help in the design of inhibitors with greater potency, physicochemical properties, and isoform selectivity.

Solid-Phase Synthesis of Chiral Bicyclic Guanidinium Oligomers

A simple solid-phase synthesis of thioether-linked chiral bicyclic guanidinium oligomers for cell internalization purposes has been developed. The approach is based on a Merrifield-like peptide synthesis on Rinkamide-p-methylbenzhydrylamine resin functionalized with Cys(methoxytrityl). A difunctionalized bicyclic guanidinium synthon, bearing both electrophile (O-mesyl) and protected nucleophile (S-methoxytrityl) group, is repeatedly grafted via a nucleophilic substitution. The sequence requires removal of methoxytrityl, reduction with 1,4-dithiothreitol to cleave any adventitious disulfides, coupling and capping with benzyl bromide. Moreover, Alloc protection of the alpha-amino group of the initial cysteine, provides a potential handle for cargo attachment after oligomer elongation to the desired internalizing agent and prior to cleaving it from the resin. Finally, a bicyclic guanidinium monomer containing an amino group and a carboxylic acid function has been evaluated as an alternative building block for novel amide-bridged oligomers or peptidomimetics.

Enantioselective Synthesis of Benzimidazolyl Quinoxalinones on Soluble Polymer Support Using Focused Microwave Irradiation

Focused microwave irradiation has been applied to a multistep synthetic sequence of reactions designed to generate benzimidazolyl quinoxalinones using a soluble polymer support. They were obtained by the ipso-fluoro (S(N)Ar) displacement of the immobilized ortho-nitro fluoro benzimidazoles with chiral alpha amino esters under microwave irradiation. Intermediate chiral organic-polymer conjugates when subjected to neutral reduction underwent a spontaneous intramolecular ring closure. Cleavage of the polymer support, at room temperature, did not cause any significant racemization resulting in the generation of a chiral molecular library with two points of structural diversity.

Nitrogen and sugar content variability in tubers of Jerusalem artichoke (Helianthus tuberosus)

Several nutritive values for tubers of 114 Jerusalem artichoke (Helianthus tuberosus) populations were evaluated during 2006. The used material is a part of wild sunflower species collection at the Institute of field and vegetable crops and it is situated in Rimski Sancevi, Novi Sad. The samples were analyzed as fresh tubers on 'Venema' automatic laboratory for alpha amino nitrogen, sodium and potassium content. Total sugar content was determined as the brix value on a refractometer. Total nitrogen was determined by the Kjeldahl method on dried samples. Significant variability was found for all analyzed traits. Total nitrogen varied from 0,695 to 2,179% dry weight (mean 1,23%), alpha amino nitrogen content 0,012 to 0,118% fresh weight (m. 0,07%), potassium 0,231 0,452% fresh weight (m. 0,403%) and sodium 0,0003 - 0,0143% fresh weight (m. 0,007%). Total sugar content varied from 13,69 - 22,94% fresh weight (m. 19,14%). Alpha amino nitrogen is an essential nutrient for animals so that it's presence in tubers of Jerusalem artichoke as food is positive. The protein content is similar to the one in potato and as such satisfactory for nutrition. The K/Na ratio is high which is useful, because an increased content of potassium in food can positively affect the reduction of Na/K ratio and lower systolic blood pressure by a significant amount in adults with mild hypertension. Inulin makes up to 80% of the total sugar content in the tubers of Jerusalem artichoke, and as a dietary fiber and a fructose polymer it positively influences digestion and sugar blood levels. The obtained results suggest that selection of cultivars and populations with inappropriate nutritive values is possible. Further research is needed to estimate the share of genetic in total variability and to determine whether the selection for new cultivars is justified.