Christos Zikos

The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues.

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimers disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.

Spacer Site Modifications for the Improvement of the in Vitro and in Vivo Binding Properties of 99mTc-N3S-X-Bombesin[2−14] Derivatives

It has been shown that gastrin releasing peptide receptors (GRPRs) are overexpressed in various types of cancer cells. Bombesin is an analogue of the mammalian GRP that binds with high specificity and affinity to GRPRs. Significant research efforts have been lately devoted to the design of radiolabeled 8 or 14 aminoacid bombesin (BN) peptides for the detection (either with gamma or positron emitting radionuclides) and therapy (with beta(-) emitting radionuclides) of cancer. The specific aim of the present study was to further investigate the radiolabeled peptide structure and to determine whether the total absence of a linker or the use of a basic diverse amino acid linker could influence the biodistribution profile of the new compounds for specific targeting of human prostate cancer. Thus, two new derivatives with the structure Gly-Gly-Cys-X-BN[2-14], where linker X is either zero (I) or Orn-Orn-Orn (Orn: ornithine) (II) were designed and synthesized. The corresponding (99m)Tc-BN derivatives were obtained with high radiochemical yield (>98%) and had almost identical retention times in RP-HPLC with the (185/187)Re complexes, which were also characterized by ESI-MS. Metabolic stability was found to be high in human plasma, moderate in PC-3 cells, and rather low in mouse liver and kidney homogenates for both BN derivatives studied. The BN derivative without the spacer was less stable in cell culture and liver homogenates. A satisfactory binding affinity to GRPRs, in the nanomolar range, was obtained for both BN derivatives as well as for their Re complexes, with BN (II) demonstrating the highest one. In vitro internalization/externalization assays indicated that approximately 6% of BN (I) and approximately 25% of BN (II) were internalized into PC-3 cells. In vivo evaluation in normal Swiss mice and in tumor bearing SCID mice showed that BN (II) presented higher tumor and pancreas uptake than BN (I). Small animal SPECT dynamic imaging, carried out after an injection of BN (II) in mice bearing PC-3 tumors, resulted in PC-3 tumor delineation with low background activity. Overall, this study performed for two new N(3)S-X-BN[2-14] derivatives indicated that hydrophilicity and charge strongly affected the in vitro and in vivo binding properties and the biodistribution pattern. This finding is confirmed by SPECT imaging of BN (II), which is under further in vivo evaluation for detecting cancer-positive GRPRs.

Preparation of high capacity aminomethyl-polystyrene resin

Abstract Aminomethyl copoly-(styrene-1%-divinylbenzene) resins of high purity and in a variety of substitution levels have been synthesized. The substitution level was up to 96% of the available benzene rings. This was achieved by the use of FeCl3 as a catalyst. The use of FeCl3 for the introduction of other acyl type groups has produced high purity resins of controlled loading.

Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha

Since conserved mammalian polypeptides are believed to exhibit enhanced immunogenicity in avian species, hens were immunized against the poorly immunogenic, highly conserved mammalian polypeptide prothymosin alpha (ProTalpha), i.e. against either non-conjugated ProTalpha (isolated from bovine thymus) or ProTalpha conjugated to keyhole limpet hemocyanin (ProTalpha/KLH). The antibodies Y were isolated from the egg yolk and evaluated through suitable dot-blot and ELISA systems in parallel with antibodies G isolated from the antiserum of rabbits immunized against the same immunogens. As revealed, antibodies Y and G of low titer and/or affinity were obtained against non-conjugated ProTalpha, while antibodies Y against ProTalpha/KLH had a better apparent titer, could better discriminate between ProTalpha and the closely related bioactive peptide thymosin alpha 1, and were obtained at much larger quantities than the corresponding antibodies G.

(R, S) 2-fluoro (chloro)-4′-carboxy-triphenyl methanol. Novel acid labile trityl type handles for solid phase peptide synthesis

Novel trityl type handles have been prepared and applied to solid phase synthesis using 9 - fluorenylmethyloxycarbonyl (Fmoc) for Na - amino protection and acid labile protecting groups for side chain protection. The cleavage of the resultant ester can be carried out by using appropriate mild acidic cocktails 0.1 % (v/v) trifluoroacetic acid, and TFE : CH2Cl2 : CH3COOH 1 : 8 : 1 (v/v).

Synthesis and characterization of novel natural product-Gd(III) MRI contrast agent conjugates

Several novel gadolinium chelates conjugated with paclitaxel, colchicine and thyroxine have been prepared as MRI contrast agents targeted to tubulin and thyroxine-binding globulin, respectively.

Commercially available chemicals as immunizing haptens for the development of a polyclonal antibody recognizing carbendazim and other benzimidazole-type fungicides

Carbendazim is a fungicide widely used for controlling fungi affecting fruits, vegetables, field crops etc. Determination of carbendazim in water, soil and various crops is frequently required to assure compliance with national/European regulations. A polyclonal antibody recognizing carbendazim was developed by using commercially available 2-(2-aminoethyl) benzimidazole, 2-benzimidazole propionic acid and 2-mercaptobenzimidazole as immunizing haptens; each of the above derivatives was directly conjugated to the carrier protein keyhole limpet hemocyanin and a mixture of the conjugates was administered to New Zealand white rabbits. Immunochemical functionality of the antisera and the corresponding isolated antibody (whole IgG fraction) was evaluated through titer and displacement curves in an in-house developed ELISA, which employed a 2-mercaptobenzimidazole - functionalized lysine-dendrimer as the immobilized hapten. As shown with ELISA-displacement curves, the above antibody could recognize carbendazim as well as other benzimidazole-type fungicides, i.e. benomyl and thiabendazole, and also intact benzimidazole, while it did not cross-react with the structurally different pesticides carbaryl and imazalil. Considering the rather simple approach which has led to its development and its highly promising immunochemical profile, the new antibody may be exploited in immunoanalytical systems for detecting benzimidazole-type pesticides e.g. in samples of environmental interest. The above antibody is being currently tested as a biorecognition element in the novel FOODSCAN cell biosensor platform for pesticide residue detection based on the Bioelectric Recognition Assay technology.

Solid-phase synthesis of thymosin β10 using a p-cyanotrityl resin. Chemical characterization and immunochemical control of the synthetic peptide

Thymosin β10(43 amino acids) was synthesized following the Fmoc-solid-phase strategy, using a new p-cyanotrityl resin. This resin shows enhanced acid stability compared with the usual trityl ester resins, thus providing selective peptide synthesis with high yield and purity. Crude Tβ10 was purified with gel filtration and semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The purified synthetic peptide was shown to co-elute with recombinant Tβ10 in an analytical RP-HPLC system. The amino acid analysis data were in agreement with those reported for the literature primary structure of natural Tβ10. The electrospray ionization mass spectrometry (ESIMS) data showed almost identical average mass (Mr, exp)-values for the synthetic or the recombinant product, which were in agreement with the molecular mass calculated on the basis of the proposed primary structure for the peptide. Furthermore, the synthetic Tβ10 exhibited similar immunochemical characteristics compared with the recombinant material in an in vitro ELISA test using rabbit polyclonal anti-Tβ10 antiserum.

Visualization of the membrane engineering concept: evidence for the specific orientation of electroinserted antibodies and selective binding of target analytes.

Membrane engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target‐specific receptor‐like molecules on the cell surface. Previous studies have elucidated the biochemical aspects of the interaction between various analytes (including viruses) and their homologous membrane‐engineered cells. In the present study, purified anti‐biotin antibodies from a rabbit antiserum along with in‐house prepared biotinylated bovine serum albumin (BSA) were used as a model antibody‐antigen pair of molecules for facilitating membrane engineering experiments. It was proven, with the aid of fluorescence microscopy, that (i) membrane‐engineered cells incorporated the specific antibodies in the correct orientation and that (ii) the inserted antibodies are selectively interacting with the homologous target molecules. This is the first time the actual working concept of membrane engineering has been visualized, thus providing a final proof of the concept behind this innovative process. In addition, the fluorescence microscopy measurements were highly correlated with bioelectric measurements done with the aid of a bioelectric recognition assay. Copyright

Solid‐phase Synthesis of a Peptide Derivative of Thymosin alpha1 and Initial Studies on its 99mTc‐Radiolabelling

A derivative (1) of the immunopotentiating 28‐peptide thymosin alpha1 has been especially designed, so that it can be 99mTc‐radiolabelled, and synthesized following the Fmoc solid‐phase peptide synthesis approach. Derivative 1 contains the N‐terminal fragment Tα1[1‐14] as a bioactive segment, at the C‐terminus of which a 99mTc‐chelating moiety consisting of Nα,Nα‐dimethylglycine, serine and cysteine is linked through the Nɛ‐amino group of a ‘bifunctional’ lysine residue; the latter is indirectly anchored on the solid‐phase peptide synthesis resin through 6‐aminocaproic acid (dmGSCK{Nɛ‐Tα1[1‐14]}Aca). Synthetic derivative 1 was obtained at high overall yield (approximately 35%) and purity (>95%) and shown to be efficiently radiolabelled with 99mTc, thus resulting in the first, to our knowledge, so far reported 99mTc‐radiolabelled derivative of thymosin alpha1, which may be eventually used as a specific molecular tool for the in vitro/in vivo study of the mode of action of the parent bioactive peptide.