Synthesis and antiviral activity of PB1 component of the influenza A RNA polymerase peptide fragments.

This study is devoted to the antiviral activity of peptide fragments from the PB1 protein - a component of the influenza A RNA polymerase. The antiviral activity of the peptides synthesized was studied in MDCK cell cultures against the pandemic influenza strain A/California/07/2009 (H1N1) pdm09. We found that peptide fragments 6-13, 6-14, 26-30, 395-400, and 531-540 of the PB1 protein were capable of suppressing viral replication in cell culture. Terminal modifications i.e. N-acetylation and C-amidation increased the antiviral properties of the peptides significantly. Peptide PB1 (6-14) with both termini modified showed maximum antiviral activity, its inhibitory activity manifesting itself during the early stages of viral replication. It was also shown that the fluorescent-labeled analog of this peptide was able to penetrate into the cell. The broad range of virus-inhibiting activity of PB1 (6-14) peptide was confirmed using a panel of influenza A viruses of H1, H3 and H5 subtypes including those resistant to oseltamivir, the leading drug in anti-influenza therapy. Thus, short peptide fragments of the PB1 protein could serve as leads for future development of influenza prevention and/or treatment agents.

[Synthesis and investigation of effects of conopressin S analogues on ion and water excretion by rat kidney].

The investigation deals with effect of analogues of conopressin S--a peptide of the vasopressin family with amino acid replacement in the 2nd and 4th positions (characteristic of invertebrate peptides)--on transport of water and ions in the rat kidney. At administration to female Wistar rats, 1-deamino-conopressin S produced a weak action on water transport and had no effect on urinary K+ and Na+ excretion. Its analogue, 1-deamino-Tyr2-conopressin S, caused antidiuretic and kaliuretic action without affecting the Na+ excretion. Estimation of significance of the variant of optic isomer of arginine in the 4th and 8th position of the molecular for the antidiuretic and kaliuretic action of the peptide showed that 1-deamino-Tyr2,D-Arg4-conopressin S and 1-deamino-Tyr2,D-Arg4,8-conopressin S did not affect the urinary K+ excretion and renal water reabsorption, whereas action of 1-deamino-Tyr2,D-Arg8-conopressin S did not differ from action of 1-deamino-Tyr2-conopressin S. Thus, it has been established that the selective kaliuretic action of analogues of conopressin S on rat kidney depends on the presence of tyrosine in the 2nd and of L-arginine, but not of D-arginine, in the 4th position of the molecule.

[Influence of new neurohypophyseal hormone analogs on sodium and water reabsorption in rat kidney].

New analogs of some neurohypophyseal hormones (oxypressin, hydrin, glumitocin, vasotocin) have been synthesized. Experiments with injection of these peptides to rats showed that substitution of C-terminal glycinamide on beta-ethanolamine (glycinol) or ethylamine in 1-deamino-arginine vasotocin resulted in loss of natriuretic but not antidiuretic activity. Analogs of oxypressin and hydrin exhibited neither natriuretic activity nor ability to affect water reabsorption. Glumitocin analog induced renal sodium ion excretion and did not influence potassium ion excretion.

[Selective effect of vasotocin analogues with amino acid substitutions in 4, 7, 8 positions on the water and cations excretion by the rat kidney].

Analogues ofarginine vasotocin with replacement of amino acids in 4, 7 and 8 positions of the hormone were synthesized. After water loading the injection of 1 x 10(-12) mol per 100 g BW 1-deamino-8-homoarginine vasotocin, 1-deamino-4-threonine-8-arginine vasotocin and 1-deamino-4-threonine-8-D-arginine vasotocin increased solute-free water reabsorption, but did not affect cations excretion (Na, K, Ca, Mg). Presence of glycine in 9th position and proline in 7th position ofa vasotocin molecule is essential for hormone interaction with a V2-receptor. In rats, injection of 5 x 10(-11) mol 1-deamino-8-homoarginine vasotocin or 1-deamino-4-threonine-8-arginine vasotocin dramatically increased urinary sodium and potassium excretion and enforced osmotically free water reabsorption but very little affected urinary magnesium and calcium excretion. The injection in the same dose of other synthesized vasotocin analogues did not affect the cations excretion. The issue of physiological mechanisms of kidney's selective response related to water, one- and bivalent cations excretion after injection of vasotocin analogues is discussed. Inhibition of cations transport is associated with activation of any V-receptor (but not V2-receptor). This effect takes place only in presence of L-arginine instead of D-arginine in the analogue.

Effect of TRH on the development of ischemic cardiac arrhythmias in cats.

Acute experiments on cats showed that intravenous injection of thyrotropin-releasing hormone prevented blood pressure drop and produced a pronounced antiarrhythmic effect.

[Mitochondrial H+-ATPase: identification of subunits of the F0 subcomplex contacting with membrane lipids]. / H+-ATP-asa mitokhondrii: identifikatsiia sub''edinits subkompleksa F0, kontaktiruiushchikh s lipidami membrany.

The subunits of the F0 membrane sector of bovine heart mitochondrial H(+)-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H(+)-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)-[12-14C]dodecanoyl]-sn- glycero-3-phosphocholine, 1-acyl-2-[11-([125I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn- glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero- 3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F0 membrane sector contact the lipids. The cross-linked products were identified by SDS-PAGE and MALDI mass spectrometry.

[A structure-activity study of a catalytic antiidiotypic antibody to the human erythrocyte acetylcholinesterase]. / Strukturno-funktsional'noe issledovanie kataliticheskogo antiidiotipicheskogo antitela k atsetilkholinésteraze éritrotsitov cheloveka.

The catalytic monoclonal antibody 9A8 (MA 9A8), antiidiotypic to the antibody AE-2 (MA AE2) produced to the active site of acetyl cholinesterase from human erythrocytes, was subjected to a structure-function study. The specific binding of MA 9A8 to MA AE2 (K 2.26 x 10(9) M-1) was shown by the method of surface plasmon resonance, and the functional activity of MA 9A8 was demonstrated. Unlike acetyl cholinesterase, this antibody specifically reacted with the irreversible phosphonate inhibitors of esterases. A peptide map of MA 9A8 was analyzed by MALDI mass spectrometry. The Ser99 residue of its heavy chain was shown to be within the active site of the catalytic antibody. A computer modeling of the MA 9A8 active site suggested the existence of a catalytic dyad formed by Ser99 and His35. A comparison of the tertiary structures of the MA 9A8 and the 17E8 monoclonal antibody, which also exhibited an esterase activity and was produced to the stable analogue of the reaction transition state, indicated a practically complete coincidence of the structures of their presumed active sites.

Enzyme mimicry by the antiidiotypic antibody approach.

The concept of "internal image" of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transition-state analog revealed structural similarity although both antibodies were elicited by two different approaches.

[MALD-MS in the quantitative analysis of peptides and proteins]. / Ispol'zovanie MALDI-MS dlia kolichestvennogo analiza peptidov i belkov.

A modified method of isotope dilution was applied to the quantitative determination of peptides and proteins by MALDI MS at subpicomolar level. The essence of the method consists in the quantitative analysis of the enzymic hydrolysis products rather than the starting compounds. This allows the measurements to be performed at a higher resolution and makes the method independent of the molecular mass of oligopeptides and proteins examined. Fragments obtained by hydrolysis of the same oligopeptide or protein in a known concentration by the same enzyme and labeled with the stable 18O isotope are used as internal standards. The label is introduced by carrying out the hydrolysis in H(2)18O, and the oligopeptide concentration is calculated from the isotope distribution between the labeled and unlabeled hydrolysis products in the mass spectrum. This method was tested in the determination of concentrations of the angiotensinogen (1-14) fragment (oligopeptide), extracellular RNAase from Bacillus amyloliquefaciens (protein) and its protein inhibitor, barstar M. Usefulness of this method in kinetic studies was also demonstrated.

Quantitation of peptides and proteins by matrix-assisted laser desorption/ionization mass spectrometry using (18)O-labeled internal standards.

A method for quantitating proteins and peptides in the low picomole and sub-picomole range has been developed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with internal (18)O-labeled standards. A simple procedure is proposed to produce such internal standards for the tested sample by enzymatic hydrolysis of the same sample (with known concentration) in (18)O-water. A mathematical algorithm was developed which uses the isotopic patterns of the substance, the internal standard, and the substance/internal standard mixture for accurate quantitation of the substance. A great advantages of the proposed method is the absence of molecular weight limitation for the protein quantitation and the possibility of quantitation without previous fractionation of proteins and peptides. Using this strategy, the peptide angiotensinogen and two proteins, RNase and its protein inhibitor, were quantified by MALDI-time-of-flight (TOF) mass spectrometry.

[MALDI-mass spectrometry for identification of new proteins in snake venoms]. / MALDI-mass-spektrometriia dlia identifikatsii novykh belkov v iade zmei.

By MALDI MS, we searched cobra venoms for new low-content polypeptides. A number of new proteins with molecular masses 7-25 kDa, characteristic of the known snake protein toxins, were identified, with the content of one of them less than 0.02%.

Peroxidase and laccase as catalysts for removal of the phenylhydrazide protecting group under mild conditions.

A new method is proposed for the removal of the phenylhydrazide protecting group by the action of peroxidase or laccase, the enzymes attributed to the class of oxidoreductases. The deblocking procedure is performed under mild oxidative conditions, i.e., aqueous solution and neutral or close to neutral pH. Such mild oxidizing agents as 1 mM H(2)O(2) and air oxygen are used for unmasking. The method is available for the deblocking of both alpha- and gamma-carboxyl groups. The enzyme-catalyzed removal of the phenylhydrazide protecting group causes no oxidative modification nor destruction of methionine or tryptophan side chains.

Copper (II) complexes as a laccase model in the catalytic removal of the phenylhydrazide protecting group under mild oxidative conditions.

Copper (II) complexes, with nitrogen-containing ligands (pyridine and imidazole), simulate perfectly the catalytic activity of laccase in the oxidative removal of the phenylhydrazide protecting group. Deblocking occurs under mild conditions, preventing the oxidation of labile amino acids. The effect of certain factors on the rate of deblocking and the degree of conversion were studied, and optimal conditions providing 100% yield of the deblocked products were chosen.

[Experimental approaches to treatment of prostatic adenoma with the luliberin analog surfagon]. / Eksperimental'nye podkhody k lecheniiu adenomy predstatel'noi zhelezy analogom liuliberina surfagonom.

Therapeutic efficacy of surfagon, an analog of LH-RH which was injected subcutaneously at a daily dose of 100 micrograms/kg for 30 days, was investigated in ACI male rats with induced prostatic adenoma. This drug was shown to be effective proceeding from a morphological study and measurement of the prostate. Measurement of blood testosterone levels by radioimmunoassay showed a decrease in this level in rats, treated with surfagon, i. e. a therapeutic effect of the drug in prostatic adenoma was accompanied by a decrease in the level of androgens.

[Effects of atrial natriuretic factor on proliferative response and natural cytotoxicity of human lymphocytes]. / Vliianie atrial'nogo natriiureticheskogo faktora na proliferatvnyi otvet i estestvennuiu tsitotoksichnost' limfotsitov cheloveka.

Effect of synthetic analogues of atrial natriuretic factor (ANF) on proliferative response and natural cytotoxic lymphocytes of human subjects was investigated in vitro. ANF-III and ANF-IV increased blast transformation lymphocytes induced by a Con-A suboptimal dose. The increase of cells activity was comparable with the effect of interleukin-2, added at a dose of 50-100 unit/ml.

[Accumulation of ATP by plasma membrane preparations enriched by particles from hypophyseal cells under the effect of thyrotropin-releasing hormone]. / Nakoplenie ATP preparatami obogashchennykh plazmaticheskimi membranami chastits iz kletok gipofiza pod vliianiem tireotropin-rilizing-gormona.

Thyrotropin-releasing hormone (TRH, thyroliberin) is endogenous tripeptide, which is synthesized in hypothalamus and is responsible for multihormonal regulation of synthesis and secretion of hormones in the anterior lobe of the hypophysis. TRH is required as an obligatory factor stimulating the GH cell lines growing in cell culture. As indicated in previous studies activation of target cells (in response to effects of insulin, growth hormone, polypeptide factors of growing, mitogens, chemostimulators) followed by fast alterations in phosphorylation of membrane components which is realized via generation of "signal" ATP in plasmatic membranes. This type of ATP was synthesized within 1 min in the preparations of particles enriched with plasmatic membranes isolated from bovine hypophyses and washed in 0.25 M sucrose, if the particles were incubated in the mixture containing TRH 30-40 mg/ml, Tris-HCl buffer, rH 7.5, ADP-Na salt Mg2+, inorganic phosphate, NaF, under conditions of NADH oxidation in presence of cytochrome c and oxygen. Accumulation of TRH-stimulated ATP in plasmatic membranes was detected, after addition of the kinases inhibitor 5'-fluorosulfonyl benzyladenosine into the incubation mixture, in II large scale experiments. The rate of TRH-stimulated synthesis of ATP was dissimilar in various preparations of hypophyses and was equal to 0.21-11.6 nmol/min/mg of protein at 30 degrees Plasmatic membrane signal ATP appears to serve as a secondary "membrane messenger" for TRH during transfer of a signal and its acceleration from the TRH receptors along the cell surface to membrane effectors--kinases.