No abstract

A biocompatible heterogeneous hydrogel of poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) showing an open porous structure, viscoelastic properties similar to the neural tissue and a large surface area available for cell interaction, was evaluated for its ability to promote tissue repair and axonal regeneration in the transected rat spinal cord. After implantation, the polymer hydrogel could correctly bridge the tissue defect, from a permissive interface with the host tissue to favour cell ingrowth, angiogenesis and axonal growth occurred within the microstructure of the network. Within 3 months the polymer implant was invaded by host derived tissue, glial cells, blood vessels and axons penetrated the hydrogel implant. Such polymer hydrogel matrices which show neuroinductive and neuroconductive properties have the potential to repair tissue defects in the central nervous system by promoting the formation of a tissue matrix and axonal growth by replacing the lost of tissue.

The conformations of the phytotoxic cyclic tetrapeptide tentoxin [cyclo-(L-MeAla1-L-Leu2-MePhe[(Z) delta]3-Gly4)] have been studied in aqueous solution by two-dimensional proton nmr at various temperatures. Contrary to what is observed in chloroform, tentoxin exhibits multiple exchanging conformations in water. Aggregation phenomena were also observed. Four conformations with different proportions (51, 37, 8, and 4%) were observed at -5 degrees C. Models were constructed from nmr parameters and restrained molecular dynamics simulations. All the models exhibit cis-trans-cis-trans conformation of the amide bond sequence. The conversion from one form to another is accomplished by a conformational peptide flip consisting of a 180 degree rotation of a nonmethylated peptide bond.

A new tentoxin analogue, in which the L-methyl alanine residue is substituted by L-methylserine, has been prepared following the synthetic pathway recently described for the synthesis of tentoxin [Cavelier, F., & Verducci, J. (1995) Tetrahedron Lett. 36, 4425-4428]. Using two-dimensional homonuclear proton nuclear magnetic resonance and structural analysis, we observed that MeSer1-tentoxin, like tentoxin, adopts several conformations in aqueous solution and presents self-aggregative properties. This analogue was found to be conformationally similar to the natural toxin. It showed the same efficiency as tentoxin in inhibition of ATPase activity of the isolated chloroplast F1 proton ATPase (CF1) as well as in inhibition of the ATP synthase activity of the membrane-bound enzyme (CF0CF1) in thylakoids and proteoliposomes. At concentrations above 10 microM, MeSer1-tentoxin did not reactivate CF1 to a high extent, contrary to tentoxin. It appeared, however, to bind in the same way, since the reactivating effect of tentoxin was inhibited by MeSer1-tentoxin. These results show that it is possible, using tentoxin analogues, to separate inhibitory and activating effects on the chloroplast ATPase, despite the limited chemical difference between the two toxins.

No abstract

A new procedure for synthesis of 14C-labeled tentoxin recovery of the ATPase activity [10]. Interestingly, a tightly [14C-MePhe[(Z)A]3-tentoxin], with a high specific activity, is coupled proton transport is preserved in the partially reactidescribed. Binding experiments with CFt or CFt-E isolated from vated CF0-F1 by high concentrations of tentoxin [10], and the spinach chloroplast have been carried out using equilibrium interaction between the nucleotide binding sites, which plays a dialysis technique. The results show the presence of two classes of important role in the functioning of this complex, is severely binding sites. The association constants of the two major binding altered [11,12]. sites were derived from non-linear fitting of the binding curves.The precise binding site for tentoxin on CF1 has not been At 4°C, the first binding site has a value of K,I = 8.2 × 105 M -I yet identified. There is some evidence that tentoxin binds to in CF1 and 8.7 × 105 M -1 in CF1-E, while the second binding site the [3-subunit [3,11] although amino acids involved in the has lower affinity with K,2 = 1.5 x 104 M -1 in CF1 and 2.3 x 103 binding are still a matter of controversy [5,13]. A binding M -t in CFI-a.site located on the a-subunit or at an interface between the Key words." H+-ATPase; Chloroplast ATP synthase; subunits c~ and [3 is also a possibility [7,14]. In addition, the 14C-Labelled tentoxin; Cyclic tetrapeptide; Equilibrium binding of the 7-subunit to the ¢xl3-complex is necessary for dialysis; Spinacia oleracea L. sized by new methods, we present here an investigation of H+-ATPase. This enzyme couples the downhill proton flow tentoxin binding on spinach CF1 and CFI-~, which reveals generated by the photosynthetic redox chain with ATP synat least two types of binding sites with different affinities. thesis. Numerous studies have shown that tentoxin tightly binds to the soluble F1 moiety of the enzyme [2,4], but very little is 2. Materials and methods known about the mode of action of the toxin. The extrinsic part of chloroplastic ATP synthase, CF1, is a multi-subunit 14C-Labelled tentoxinenzyme (stoichiometry c~3133T6a) with latent ATPase activity.BocMeAla-Leu-AZPhe-GlyOMe (1) was obtained in homogeneous Removal of the E-subunit and/or chemical reduction of the phase using the synthesis scheme recently described [16] skipping the lone disulfide bond of the enzyme located on the 7-subunit methylation step of the dehydrophenylalanyl residue (AZPhe). HPLC activates ATPase activity [5]. For sensitive plants, tentoxin Rt= 23.2 min on C18 Protein&Peptide VYDAC analytical column (MeOH/H20 60/40 v/v, 0.6 ml rain-l). Using [14C]methyl iodide binding inhibits the CF1-ATPase activity at low concentration freshly synthesized [17] from [14C]methanol produced from 14CO2 (10 -8 M) [2,4,6,7] and stimulates it at a concentration of 10 -5 (specific activity 55 mCi/mmol) [18,19], selective radioactive methyla-M and higher [6][7][8][9]. The activation phenomenon also exists in tion was then performed according to [...

An improved method for the synthesis of dithioesters of amino acids and peptides has been developed. The syntheses have been carried out from the nitriles. The addition of thiol to the nitrile derivative in the Pinner step of dithioester synthesis was activated with hydrogen fluoride. A few examples of dithioester synthesis using liquid HF are described. Some novel dithioesters, which are model compounds for resonance Raman spectroscopic studies of dithioacylpapain intermediates, are described.Key words: dithioesters, amino acids, Pinner reaction, HF, isotopes.Resume : Une mCthode amCliorCe de synthkse de dithioesters d'acides amines et de peptides a Ct C dCveloppCe. Les synthkses ont Ct C accomplies ii partir des nitriles correspondants. L'addition du thiol au dCrivC nitrile lors de la synthitse du dithioester, pendant la reaction de Pinner, a Ct C activCe par le fluomre d'hydrogkne. Nous dCcrivons plusieurs exemples de synthitse de dithioesters employant du HF liquide, ainsi que plusieurs nouveaux dithioesters,composC-modtles employCs dans les Ctudes des formes passagitres de la dithioacylpapaine par la spectromCtrie de resonance de Raman.

The synthesis of a new photochemical probe for labelling tentoxin 1 binding sites in the soluble part of chloroplast F0F1 ATPsynthase is described. [Carbonyl‐14C]‐4‐benzoylbenzoyl MeSer1‐tentoxin 6 was synthesised by coupling MeSer1‐tentoxin 2 with [carbonyl‐14C]‐4‐benzoylbenzoic acid 3, itself obtained by coupling [1‐14C]‐benzoyl chloride with the dilithio derivative of 4‐bromobenzoic acid (9 ). Photolysis of the probe in methanolic solution occurs readily upon irradiation with UV light at 366 nm. Copyright © 2000 John Wiley & Sons, Ltd.