Biomedical subjects

J Bódi

Publications and source records attributed to J Bódi.

11 recordsLinked to original sources

PubMed2001

An alternative solid phase peptide fragment condensation protocol with improved efficiency.

The success of solid phase peptide synthesis is often limited by the aggregation of the growing peptide chains on the resin. Working from the results of a study of model coupling reactions in solution between Z-Gly-Phe-OH and H-Phe-OBzl, we have achieved higher efficiency in the repetitive solid phase fragment condensation of VGVAPG, in a 3:1 chloroform-phenol solvent system, using diisopropylcarbodiimide (DIC) as coupling agent, and a combination of 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt) and its tetrabutyl ammonium salt as additive, than in DMF with DIC and HODhbt alone.

Amino Acid Sequence↗

PubMed2001

Antagonist and agonist activities of the mouse agouti protein fragment (91-131) at the melanocortin-1 receptor.

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91-131) (AP91-131) at the melanocortin type-1 receptor (MC1-R) were assessed using B 16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91-131 was about 3-fold less potent than the natural agonist alpha-melanocyte-stimulating hormone (alpha-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-alpha-MSH (Ki 6.5 +/- 0.8 nmol/l). Alpha-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP9 l -131; the IC50 values for AP91-131 in thetwo assay systems were 91 +/- 22 nM and 95 +/- 15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91-131 with IC50 values of 9.6+/-1.8 nM and 5.0+/-2.4 nM, respectively. This indicates inverse agonist activity of AP91-131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91-131 in the adenylate cyclase and melanin assays. On the other hand, AP91-131 inhibited cell growth similar to alpha-MSH (IC50 11.0 +/- 2.1 nM; maximal inhibition 1.8-fold higher than that of alpha-MSH). Furthermore, MC1-R was down-regulated by AP91-131 with about the same potency and time-course as with alpha-MSH. These results demonstrate that AP91-131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different alpha-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.

Adenylyl Cyclases↗

PubMed2000

Combined solid-phase and solution approach for the synthesis of large peptides or proteins.

In the synthesis of large peptides or proteins, highly homogeneous segments are indispensable for a convergent strategy either on a solid-phase resin or in solution. Employing Boc/Bzl chemistry to prepare fully protected segments with a free alpha-carboxyl group from the solid support, base-labile linkers are profitable for practical peptide synthesis since they require no special equipment. For this purpose, an N-[9-(hydroxymethyl)-2-fluorenyl]succinamic acid (HMFS) linker was adopted. Consequently, there must be high compatibility between the protecting groups of the segment and the anchoring group which is cleavable by treatment with morpholine or piperidine in DMF. Instead of using the 2-bromobenzyloxycarbonyl (BrZ) group for the Tyr residue and the formyl (For) group for the Trp residue, both of which are the most susceptible protecting groups under these base-catalysed conditions, the base-resistant 3-pentyl (Pen) and cyclohexyloxycarbonyl (Hoc) groups were introduced to the respective side-chain functional groups. By applying the present strategy, the authors were able to rapidly synthesize homogeneous protected segments for use in the subsequent segment coupling in solution. In the present paper, the utility of the combined solid-phase and solution approach is demonstrated by synthesizing muscarinic toxin 1 (MTX1) which binds to the muscarinic acetylcholine receptors.

Chromatography, High Pressure Liquid↗

PubMed1999

Synthesis and cytotoxic activity of N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates.

As analogs of the widely used anti-tumor agents, N-(2-chloroethyl)-N-nitrosoureas, N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates were synthesized by nitration following the reaction of the appropriate amines or alcohols with 2-chloroethyl isocyanate. All tested compounds exert cytotoxic effect with IC50 values of 10(-4) to 10(-6) M and most of them show somewhat higher cytotoxicity in nitrogen than in air.

Air↗

PubMed1998

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence.

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris. HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (lambdamax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

Amino Acid Sequence↗

PubMed1997

In vitro cytotoxic effect of difluoromethylomithine increased nonspecifically by peptide coupling.

Difluoromethylomithine (DFMO)-peptide conjugates were synthesized as prodrugs to improve the cytotoxic efficacy of DFMO. All conjugates inhibited cell growth in different cell lines more effectively than DFMO itself. The best cytotoxic effect was achieved in all cell lines by DFMO-Glu-His-Phe-Arg-Trp-Gly-OMe, where the carrier peptide is a melanotropin hormone fragment. Although this conjugate is capable of displacing labeled melanotropin from its receptor, its cytotoxic effect on the receptor-positive human melanoma cell line has not been proven to be receptor-mediated. The differences in the cytotoxicities of the congeners seem to be influenced, at least in part, by the nature of the carrier molecule.

Antineoplastic Agents↗

PubMed1996

Effect of Ca2+ on the secondary structure of linear and cyclic collagen sequence analogs.

To investigate the role of secondary structure in the substrate specificity of human 72 kDa type IV collagenase, we synthesised linear and cyclic collagen sequence analogs. As Ca2+ plays a crucial role in the enzyme activity, the CD and FTIR spectra of the peptides were also measured in the presence of Ca2+. Most of the linear, but none of the cyclic peptides form stable 1:1 Ca2+ complexes. The cyclic hexapeptides adopt significantly different backbone conformations comprising not only beta-turns but also the less frequent gamma-turns. Consequently, in the cyclopeptides the scissile Gly-Ile(Leu) bond is embedded into a different conformational environment, but in spite of that none of them is a substrate or an inhibitor of the enzyme. The best substrate Ac-Pro-Leu-Gly-Leu-Ala-Gly-D-Lys-OH binds Ca2+, but does not form a stable 1:1 Ca2+ complex, which suggests that instead of a folded structure an extended flexible conformation is preferred by the enzyme.

Amino Acid Sequence↗

PubMed1992

Synthesis and characterization of some collagen sequence analogs.

Some analogs of natural collagen sequences (773-779) were synthesized. The peptides were hydrolyzed at the Gly-Ile bond not only by crude collagenase isolated from normal rat liver, but also by the bacterial Clostridium histolyticum collagenase. The reason for this unusual cleavage site in the latter case may lie in the unordered secondary structure of the substrates measured by CD spectroscopy.

Amino Acid Sequence↗

PubMedDate not supplied

Solution synthesis of human midkine, a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five disulphide bonds.

Human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121-residue peptide was assembled from two large fully protected intermediates, Boc-(1-59)-OH and H-(60-121)-OBzl, in CHL/TFE(3:1, v/v) using water-soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N-terminal half domains [(1-59) and (60-121)] were also synthesized by the same procedure used for the hMK synthesis. The C-half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N-half one showed much less activity in general.

Amino Acid Sequence↗

PubMedDate not supplied

The effect of N-terminal substitutions on the biological activity of MSH fragments.

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of alpha-melanotropin, [Glp5]alpha-MSH(5-10), [Glp5,D-Phe7]alpha-MSH(5-10), [Sar5,D-Phe7]alpha-MSH(5-10), [Nle4,D-Phe7]alpha-MSH(4-10), [N-carbamoyl]alpha-MSH(5-10), and formyl and acetyl derivatives of alpha-MSH(5-10), [Gly5]alpha-MSH(5-10) and [Gly5,D-Phe7]alpha-MSH(5-10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.

Amino Acid Sequence↗

PubMedDate not supplied

Synthesis of an O-palmitoylated 44-residue peptide amide (PLTX II) blocking presynaptic calcium channels in Drosophila.

PLTX II, a presynaptic calcium channel blocker in Drosophila isolated from the plectreurys spider venom, is a 44-residue peptide containing ten Cys residues and an O-palmitoylated threonine amide at the carboxy-terminus. In this study, the palmitoylated peptide was synthesized in solution by applying our maximum protection strategy using the HF method at the final deprotecting step. Before designing the synthesis, we examined the stability of the palmitoyl moiety under the conditions for the synthesis of the peptide using several model peptides. The O-palmitoyl group was confirmed to be stable during elongation of the peptide bonds, but was partially removable during the deprotection reaction in HF. The depalmitoylation reaction in HF was temperature- and time-dependent. Therefore, the decision was made to protect the Asp residues with benzyl ester, since it is more susceptible to HF than cyclohexyl ester, which is now commonly used in the Boc-based, solid-phase synthesis. Thus, the HF reaction was carried out at -10 degrees or -15 degrees C for 1 h in order to reduce the extent of the depalmitoylation reaction. The resulting palmitoylated and depalmitoylated products were separated, the remaining Acm groups were removed using Hg(OAc)2, and then the completely deprotected peptides were folded to their native forms. The final palmitoylated peptide was proven to be identical with the natural one using various HPLC systems and by bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗