Biomedical subjects
Publications and source records attributed to M I Titov.
Acute experiments on cats showed that intravenous injection of thyrotropin-releasing hormone prevented blood pressure drop and produced a pronounced antiarrhythmic effect.
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.
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.
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.
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%.
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.
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The protective action of enzymatically stable analogue of Leu-enkephalin (D-ala-2-leu5-arg6), injected intraperitoneally, in the course of stress- and epinephrine induced myocardial damage was demonstrated in animal (129 white rats) experiments. Two effect of enkephalin were sufficient for the cardiac protection: enkephalin-stimulated prostacyclin biosynthesis and simultaneous inhibition of thromboxane production.
It is shown in experiments on mice that injection of Hydra Peptide Morphogen in doses of 30 micrograms/kg normalizes the content of cyclic nucleotides in the liver, disturbed as a result of injury. Hydra Peptide Morphogen did not change the content of cyclic nucleotides in the injured muscle.
The wound-healing agent Dalargin, a hexapeptide and varios of its cleavage products were tested in a wound-healing model on the basis of granulation in rat. Three days after wounding (p.op.) an increase in granulation was observed at Dalargin concentrations of 10 micrograms/ml and 50 micrograms/ml. Seven days p.op. an increase was observed already at a Dalargin concentration of 5 micrograms/ml. The cleavage products of Dalargin had no stimulating effect on granulation. A histologic differentiation of the different cell parts in the granulation tissue led to an increased total number of cells. The concentration of 5 micrograms/ml induced both an increase in the number of endothelial cells and fibroblasts, the concentration of 50 micrograms/ml induced just an increase of the number of endothelial cells. The possible bases of the mode of operation are being discussed. The stimulating effect on wound healing as described could be confirmed by the results obtained in our animal experiments.
If was shown that the addition of fibronectin antibodies exerted the inhibition of platelet aggregation. The tripeptide RGD inhibited the platelet aggregation induced by the same agents (ADP, epinephrine, thrombin, collagen) both in blood plasma and in suspension of washed platelets.
The effect of synthetic analog of leucine enkephalin hexapeptide dalargin on regeneration of peripheral nerves involved an activation of the sensory and motor fibers regeneration after transection and microsurgical restoration of the rat sciatic nerve. Endogenous opioid peptides seem to take part in the regulation of structural homeostasis of the nervous system.
Hydrolysis of Bz-Gly-Ser-Phe-Arg, C-terminal fragment of atriopeptin 2, by human cardiac angiotensin-converting enzyme has been studied. The KM for the reaction was 10(-4) M. The effect of concentration of NaCl on activity of cardiac angiotensin-converting enzyme has been determined, which allowed to regard Bz-Gly-Ser-Phe-Arg as bradykinin-like substrates. It was demonstrated that cardiac, but not pulmonary isozyme of angiotensin-converting enzyme specifically hydrolyses atriopeptin 2.
Monoclonal antibodies (MABs) to a new drug Dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg) enhancing ulcer healing have been produced. Dalargin is a synthetic analog of Leu-enkephalin. With about 40 compounds tested in competition radioimmunoassay it has been shown that specificity of the MABs is directed against the N-terminal tetrapeptide of the molecule. The MABs are sensitive to amino acid substitutions in any of the positions of the fragment and have no cross-reactivity with endogenous opioids. Their further application in pharmacokinetic studies in humans and for characterization of opioid receptors is discussed.
A series of atrial natriuretic peptides, viz., alpha-r-ANF, APII, APIII, and des-Ser5, Ser6-APII, have been obtained by condensation of earlier synthesized fragments promoted with complex F. Acetylaminomethyl protecting group were removed from cystein residues with simultaneous cyclization of the peptide. Biological activity of the atrial natriuretic peptides was studied.
At intraperitoneal injection and local application of opioid peptide dalargin induces fibroblast proliferation (3-fold increase in the mitotic index) and growth of capillaries, accelerates the maturation of granulation tissue and of scar, epitheliazation of the defect, and considerably reduces the period of healing of skin wound in rats. The stimulating action of dalargin is associated with its effect on the microcirculation system and activation of the macrophage-fibroblast interaction. Possessing the triggering mechanism, the drug induces a cascade of inflammatory-reparative reactions, which reduce the duration of all healing stages.
The possibility of transversion of low-reacting mice into high-reacting ones evaluated by the application of the natural immunostimulators. L-tyrosine and thymic polypeptides (taktivin) were compared by their ability for the phenotypical correction of the immune response of the oppositely reacting mice. It was established that under the application of L-tyrosine to oppositely reacting mice the phenotypical correction of the immune response is possible. The dramatic increase in AFC and the AFC-SI secreting IgM is observed in the mice, low-reacting to SRBC in comparison with the high-reacting lines.
The effect of synthetic opioid peptide analogue dalargin on the natural cytotoxicity of lymphocytes from the peripheral human blood has been studied in respect to target cells K-562. Natural cytotoxicity was determined by assessing the cytotoxicity of individual effector cells in agarose. It has been shown that preincubation of lymphocytes with 10(-14)--10(-8) M of dalargin enhances the binding of NK-cells with targets and the formation of conjugates with killed targets and increases the percentage of active NK-cells. In some cases bimodal kinetics of a stimulating dalargin effect on the natural cytotoxicity has been observed, with two peaks of the peptide activity being in the range of 10(14)--10(-12) and 10(-10)--10(-6) M. Naloxone at a concentration of 10(-6) M acted as an agonist of opioid receptors, enhancing the formation of effector-target conjugates and NK-cell lysis abilities. A stimulating effect of dalargin on the natural cytotoxicity was more marked than that of recombinant human alpha-interferon. The possible use of dalargin in pathological conditions characterized by disturbed NK-cell function is discussed.