[Effects of novel antiviral agents on HIV-1 replication].

Two groups of the antiviral agents: 1) adamantane- and norbornen-containing compounds with in-built cholesterol to potentiate the membranotropic properties and 2) synthetic matrix protein peptides (peptides A and B) were found to have effects on HIV replication. The agents of the former group produced antiviral activity only when added in combination with the virus. Peptide A (matrix protein 43-60 amino acids) inhibited viral replication when added in both the early and late periods. Fluorescein-labeled peptide A was detectable in the cytoplasm and nucleus (although adsorption of a portion of the peptides cannot be excluded onto the cell surface). Peptide A was shown to inhibit Gag precursor p55 transport from the nuclei to the plasma membrane, the site of virus assembly. Peptide B had no antiviral activity.

[Identification and expression of haponin, a new protein from HL-60 cells].

We found a new protein haponin (an HLDF-alike protein) in promyelocyte HL-60 cells that is immunoreactive to polyclonal antibodies against HLDFbeta. Determination of the partial primary structure of the protein allowed us to reveal an immunogenic peptide of haponin and, on the basis of the amino acid sequence of this peptide, the degenerate primers were synthesized, which enabled us to clone the full-size cDNA of haponin. The stable heterologous expression of this cDNA in E. coli cells (Rosetta strain) was obtained. Preparations of natural and recombinant proteins exhibited antigenic cross-reactivity to polyclonal antibodies against this peptide.

Epitope mapping of human Fas using peptide phage display.

Sandwich EIA for measurement of soluble Fas was developed on the basis of SA-7 and SA-8 monoclonal antibodies to full-length human Fas. The threshold sensitivity of the test system is 0.3 ng/ml. Several isoforms of soluble Fas were identified. The structure of SA-7 and SA-8 antibody epitopes was determined using the peptide phage library. It was shown that SA-7 antibody epitope is determined by amino acid residues 129-134 (CKPNFF), while SA-8 antibody epitope is determined by amino acid residues 94-99 (KAHFSS) of full-length Fas. Hence, sandwich EIA on the basis of SA-7 and SA-8 monoclonal antibodies for detection of soluble Fas in human serum is to detect the following Fas isoforms: FasExo6Del, FasExo4Del, FasExo4,6Del, FasExo4.7Del, and FasExo8Del.

Hexapeptides HLDF-6 and PEDF-6 restore memory in rats after chronic intracerebroventricular treatment with beta-amyloid peptide Abeta(25-35).

Effects of homologous peptides HLDF-6 and PEDF-6 on behavior of animals with experimental Alzheimer's disease induced by chronic intracerebroventricular administration of beta-amyloid peptide Abeta(25-35) were studied in the zoosocial recognition test and Morris water maze. Peptides HLDF-6 and PEDF-6 possessed neuroprotective activity and counteracted the toxic effect of Abeta(25-35). Peptides HLDF-6 and PEDF-6 mainly improved long-term memory and working memory, respectively.

Synthesis of 10- and 9-membered dilactams derived from aspartic and glutamic acids.

9- and 10-membered bridged dipeptides derived from L-aspartic acid and L- or D-glutamic acid were synthesized using aminoacyl incorporation reaction. Key intermediates containing internal pyroglutamyl moiety were prepared via side chain to backbone cyclization of related protected dipeptide derivatives of glutamic acid.

Preparation and reactivity of aminoacyl pyroglutamates. Facile synthesis of 10-membered-ring cyclic dipeptides derived from 1,4-diaminobutyric and glutamic acids.

A number of protected proline-containing dipeptides Boc-Xaa-Pro-OBu(t) were converted via epimerization-free oxidation with RuO4 to dipeptides with an internal pyroglutamic acid residue, Boc-Xaa-Glp-OBu(t). The latter were subjected to oxidative Hoffman-type rearrangement induced by PhI[OC(O)CF3]2 to give N-(aminoacyl)-pyroglutamates. The behavior of these derivatives under basic conditions was studied, and for two such a derivatives an aminoacyl incorporation reaction was observed, producing otherwise poorly accessible 10-membered-ring dilactams derived from 1,4-diaminobutyric and glutamic acids in practicable yields.

Different mechanisms of protective and differentiative activities of homological peptides TGENHR and TQVEHR.

Previously we identified a six-membered fragment 354TQVEHR359 of the C-terminal part of the PEDF (Pigment Epithelium-Derived Factor) differentiation factor molecule that shares homology with fragment 41TGENHR46 of the HLDF (Human Leukemia Differentiation Factor) differentiation factor molecule, which is responsible for its differentiation activity. HLDF has been isolated from the culture medium of human promyelocytic leukemia cell line HL-60. Hexapeptides HLDF-6 (TGENHR) and PEDF-6 (TQVEHR) corresponding to these HLDF and PEDF molecule fragments, which were previously shown to induce cell differentiation (Kostanyan et al. (2000) Russian Journal of Bioorganic Chemistry, 26, 505-511), also have neuroprotective properties. Both peptides prevent degeneration of Purkinje cells of rat cerebellar vermis upon chemical hypoxia induced by sodium azide in vivo; this effect is also observed on a behavioral level. Peptide HLDF-6 but not PEDF-6 promotes survival of HL-60 cells upon chemical hypoxia. Peptides HLDF-6 and PEDF-6 affect different second messenger biosynthesis systems in HL-60 cells. HLDF-6 diminishes cyclic AMP level in those cells due to adenylate cyclase inhibition, while PEDF-6 inhibits phosphatidylinositol-specific phospholipase C stimulated by aluminum tetrafluoride anions.

Synthesis of 9-membered dilactams derived from 1,3-diaminopropionic and glutamic acids.

Previously unknown 9-membered bridged dipeptides derived from l or d isomers of 1,3-diaminopropionic acids and l-glutamic acid were synthesized using aminoacyl incorporation reaction. Key intermediates containing internal pyroglutamyl moiety were prepared via side chain to backbone cyclization of related protected dipeptide derivatives of glutamic acid.

[Bacteriorhodopsin retains the conformation of Val69-Gly72 fragment during functioning]. / Sokhranenie konformatsii fragmenta Val69-Gly72 bakteriorodopsina pri ego funktsionirovanii.

Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of functioning bacteriorhodopsin molecule. Using the methods of solid phase enzyme immunoassay, peptide phage display, and 1H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4, which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.

[Biological role of a neurotrophic factor fragment from pigment epithelium: structure-functional homology with a differentiation factor for the HL-60 cell line]. / Biologicheskaia rol' fragmenta neirotrfnogo faktor iz pigmentnogo épiteliia: strukturno-funktsional'naia gomologiia s faktorom differentsirovki kletok linii HL-60.

It was shown that the full-size neurotrophic factor from pigment epithelium (PEDF) induces the cell differentiation of the human promyelocyte leukemia cell line HL-60. A structural analysis of PEDF revealed in its C-terminal region a six-membered peptide fragment PEDF-(352-357) (PEDF-6) whose sequence is highly homologous to the 41-46 fragment of the active site of the human leukocyte differentiation factor HLDF (HLDF-6). The biological effect of PEDF and synthetic peptides PEDF-6 and HLDF-6 on the HL-60 cells and the early gastrula ectoderm of Xenopus laevis embryos was studied. On the basis of the structural and functional homologies of HLDF, PEDF, and their homologous peptides and the computer models of the spatial structures of the full-size PEDF and the PEDF with the C-terminal fragment split off tby the cleavage of the Leu380-Thr381 bond in the serpin loop, a hypothesis on the functional role of the serpin loop in PEDF was put forward.

[Biologically active fragment of the differentiation factor from HL-60 cell line. Identification and properties]. / Biologicheski aktivnyi fragment faktora differentsirovki kletok linii HL-60. Identifikatsiia i svoistva.

Six-membered peptide fragment TGENHR (HLDF-6) was identified in the HL-60 cell culture of human promyelocyte leukemia treated with retinoic acid when studying the differentiation factor HLDF of this cell line. HLDF-6 retains the ability of the full-size factor to induce the differentiation and arrest the proliferation of the starting HL-60 cells. It was shown that the synthetic peptide HLDF-6 has no specific receptors on the surface of the HL-60 cells but can affect the binding of interleukin IL-1 beta, a cytokine involved in proliferation, to the cell surface. It was found on a model of transplantable NSO myeloma that HLDF-6 has an antitumor activity.

[The endogenous differentiation factor of the HL-60 cells shows a nuclease activity]. / Endogennyi faktor differentsirovki kletok linii HL-60 obladaet nukleaznoi aktivnost'iu.

A structural homology between the endogenous differentiation factor of the HL-60 cell line of promyelocyte leukemia (HLDF) and several DNA/RNA-binding and DNA/RNA-hydrolyzing proteins was revealed, and expression of the hldf gene in prokaryotic systems was studied. On the basis of these experiments, the amino acid sequence of an 8-membered fragment of HLDF with potential nuclease activity was identified. The synthetic octapeptide RRWHRLKE was shown to be capable of the cleavage of RNA, linear DNA from phage lambda, and all forms of plasmid DNA. We established that treatment of the HL-60 cell culture with this peptide (10(-6) M) results in an increase in the number of apoptotic cells and suggested that HLDF is involved in processes of apoptosis.

Zn(2+)-mediated structure formation and compaction of the "natively unfolded" human prothymosin alpha.

Human recombinant prothymosin alpha (ProTalpha) is known to have coil-like conformation at neutral pH; i.e., it belongs to the class of "natively unfolded" proteins. By means of circular dichroism, SAXS, and ANS fluorescence, we have investigated the effect of several divalent cations on the structure of this protein. Results of these studies are consistent with the conclusion that ProTalpha conformation is unaffected by large excess of Ca(2+), Mg(2+), Mn(2+), Cu(2+), and Ni(2+). However, Zn(2+) induces compaction and considerable rearrangement of the protein structure. This means that ProTalpha can specifically interact with Zn(2+) (K(D) approximately 10(-3) M), and such interactions induce folding of the natively unfolded protein into a compact partially folded (premolten globule-like) conformation. It is possible that these structural changes may be important for the function of this protein.

[Conformational states of cyclic dipeptides with lactam bond between omega-functions of lysine and aspartic or glutamic acid residues]. / Konformatsionnye sostoianiya tsiklicheskikh dipeptidov s laktamnoi sviaz'iu mezhdu omega-funktsiiami ostatkov lizina i asparaginovoi ili glutaminovoi kisloty.

The conformations of H-Lys-Asp-OH and H-Glu-Lys-OH cyclic dipeptides were subjected to theoretical analysis by the method of atom-atom potentials with flexible geometry. Constants of spin-spin coupling of vicinal protons were calculated for the theoretical conformers of both dipeptides. CD and NMR spectra were measured for both peptides synthesized, and the calculated and experimental values of spin-spin coupling constants were compared. These coincided well for the dipeptide containing Asp residue, and we concluded that it exists in solution as one conformer. For the Glu-containing dipeptide, the existence of minor conformers, which increase the difference between experimental and theoretical spin-spin coupling constants, was shown to be possible.

[Total solid phase synthesis of gamma-subunit of cGMP phosphodiesterase from bovine retina and physicochemical properties of synthetic protein]. / Polnyi tverdofaznyi sintez gamma-sub''edinitsy fosfodiesterazy cGMP setchatki glaza byka i nekotorye fiziko-khimicheskie svoistva sinteticheskogo belka.

The 87-membered polypeptide with the sequence of the gamma subunit of cGMP phosphodiesterase from bovine retina rods (PDE gamma) was synthesized by the solid phase method. Two synthetic approaches, which were based on the Boc/Bzl-strategy, were used; both syntheses were carried out in a continuous-flow reactor with swellographic monitoring. In the first approach, five Arg residues were coupled in the form of Boc-Arg(Z)2-OH and the final cleavage of the peptide from the support was effected by the mixture of CF3SO2SiMe3 and thionisole in trifluoroacetic acid. There resulted a heterogeneous, ornitine-rich, and absolutely inactive peptide material which was insoluble in aqueous alkali. In the second approach, Arg(Tos) and the HF low-high cleavage procedure were used, which resulted in a homogeneous polypeptide (according to HPLC and capillary electrophoresis) that manifested correct molecular mass under ion-spray mass spectrometry and the full functional activity characteristic of the native protein. The effect of zinc salts on the PDE gamma fluorescence in solutions and on its solubility was established. This demonstrated a significant PDE gamma affinity with Zn2+ ions and appeared to be connected with the functioning of the protein in the retina cells. For the first time, the dynamics of the peptidylpolymer swelling in different solvents was studied during the synthesis of peptides with very long sequences.

[Fragments 183-198 and 125-145 of the alpha-subunits of the Torpedo californica nicotinic acetylcholinergic receptor binds alpha-bungarotoxin and neurotoxin II from Naja naja oxiana]. / Fragmenty 183-198 i 125-145 alpha-sub''edinitsy nikotinovogo atsetilkholinovogo retseptora Torpedo californica sviazyvaiut alph-bungarotoksin i neirotoksin II Naja naja oxiana.

Interaction of the mono[125I]iodinated alpha-bungarotoxin and neurotoxin II Naja naja oxiana with the synthetic peptides corresponding to the fragments of the alpha-subunit of nicotinic acetylcholine receptor from Torpedo californica was studied. It was found that both toxins bind to the fragments alpha 186-198, alpha 183-198 and alpha 125-145 adsorbed to the 96-well P.E.T.G. assay plates (COSTAR). Acm-groups on Cys residues did not prevent toxin binding by the peptides studied. Determination of the binding parameters showed that alpha-bungarotoxin interacts with fragment alpha 125-145 less effectively than neurotoxin II. The data obtained demonstrate the presence of different toxin-binding sites on alpha-subunit and confirm the model of multipoint neurotoxin-receptor interaction.

A swellographic approach to monitoring continuous-flow solid-phase peptide synthesis.

A new type of practical low pressure continuous-flow reaction system has been developed, allowing continuous "dead volume free" handling of polystyrene-based peptide-resins (PR) under conventional Boc/Bzl solid-phase peptide synthesis (SPPS). The reaction system offers a unique opportunity for direct recording of bed volume changes accompanying chemical and physical manipulations with PRs. A new monitoring principle, called "swellography," based on a straightforward interpretation of PR bed volume dynamics, is described. It seems to provide a basis for a novel, automated, non-invasive feedback control system. This novel technique does not complicate SPPS practice; and, moreover, it provides useful information about the swelling behavior of PRs in the real time of SPPS. Although the approach is not directly applicable for quantitation of deprotection and coupling reactions, it does give a good opportunity for real-time optimization of solvent and re-agent usage during the washing and neutralization steps of SPPS. A number of 8- to 16-membered peptides were synthesized manually with complete swellographic monitoring. The practical merits of the new approach are discussed in some detail.