The role of endogenous bioregulators in formation of cardiogenic reflex effects on circulation

The possible role of endogenous endothelium-derived bioactive substances in organization of cardiogenic depressor reflexes under cardiac receptor stimulation (by veratrine and bradykinin) was investigated in acute experiments on anesthetized rats. The results have shown that endothelium-derived bioactive substances take part in forming of the cardiogenic depressor reflex humoral components of nervous response or nervous modulators. These data contribute to understanding of the role of endogenous endothelium-derived bioactive substances (prostacyclin) and different NOS isoforms in mechanisms of depressor reflex development and species differences in their involvement in reflex vasomotor reactions.     

Effect of Inhibition of NO Synthases on Cardiogenic Depressor Reflexes in Different Animal Species

We studied the role of the nitric oxide (NO) system in the realization of cardiogenic depressor reflexes evoked by stimulation of cardiac receptors by veratrine (reproduction of the Bezold–Jarish reflex). Acute experiments were performed on anesthetized dogs and rats: we tested the effects of inhibition of dissimilar isoforms of NO synthase (NOS) and paid special attention to possible species-related differences in realization of the reflex responses. We found that systemic inhibition of NOS by L-nitro-N-arginine (L-NNA, 30 mg/kg, i.v.) significantly decreased the depressor reflex reaction in dogs. Vasomotor dilatatory reactions of the peripheral vessels underwent considerable modifications and in some cases were converted into vasoconstrictory responses. Selective inhibition of neuronal NOS (nNOS) by 7-nitroindazole (7-NI, 25 mg/kg, i.p.) exerted no effect on the development of cardiogenic depressor reflexes in dogs. At the same time, systemic inhibition of NOS in the course of reproduction of cardiogenic depressor reflexes in rats resulted in intensification of depressor responses, while inhibition of nNOS decreased these reactions. Thus, we first demonstrated the role of NO in the realization of cardiogenic depressor reflexes under in vivo conditions and described species-related peculiarities of the involvement of the NO system in the development of these reflexes. We also demonstrated the dependence of formation of cardiogenic depressor reflexes on the predominant involvement of one NOS type or another.     

Isolation and Genetic Study of Erwinia Mutants Devoid of Common Components of the Phosphoenolpyruvate-Dependent Phosphotransferase System

Mutants of bacteria belonging the genus Erwinia(Erwinia chrysanthemi andErwinia carotovora) with pleiotropic disturbances in the utilization of many substrates were obtained through chemical and transposon mutagenesis. Genetic studies revealed that these mutants had defective ptsI or ptsH genes responsible for the synthesis of common components of the phosphoenolpyruvate-dependent phosphotransferase system, enzyme I and the HPr protein, respectively. The ptsI ⁺ allele in both Erwinia species was cloned in vivo. Mapping of obtained mutations indicated that theptsIand ptsH genes ofE. chrysanthemi do not constitute a linkage group. The ptsI gene is located at 100 min of the chromosomal map, whereas theptsH gene is located at 175 min. Sequencing of a portion of theE. chrysanthemi ptsI gene showed that a product of the cloned DNA region had up to 68% homology with the N terminus of Escherichia coli enzyme I.     

Tissue basophils in thermal damage to the skin in an experiment (morphometric analysis and statistical modelling)

By means of quantitative morphological analysis tissue basophils (TB) (mast cells) in the derm and in the subcutaneous loose connective tissue have been studied at various degree of thermal lesion of the skin. Average diameter, amount of the cells per 0.1 mm2 of the slice area and distance between them serve as criteria for estimation of the TB functional activity. Certain differences have been revealed in the TB state at a weak and severe thermal lesion of the skin. At a threshold thermal action, metabolic processes in the TB become active. Burns of the skin of the II and IIIa degree result in an increased discharge of the granular component from cytoplasm and often in death of the cells. By means of multiple linear regression equations certain dependence of the degree of the thermal skin lesion on the state of the TB population in various dermal layers and in the subcutaneous loose connective tissue is stated.     

Functional genomics by NMR spectroscopy. Phenylacetate catabolism in Escherichia coli.

Aerobic metabolism of phenylalanine in most bacteria proceeds via oxidation to phenylacetate. Surprisingly, the further metabolism of phenylacetate has not been elucidated, even in well studied bacteria such as Escherichia coli. The only committed step is the conversion of phenylacetate into phenylacetyl-CoA. The paa operon of E. coli encodes 14 polypeptides involved in the catabolism of phenylacetate. We have found that E. coli K12 mutants with a deletion of the paaF, paaG, paaH, paaJ or paaZ gene are unable to grow with phenylacetate as carbon source. Incubation of a paaG mutant with [U-13C8]phenylacetate yielded ring-1,2-dihydroxy-1,2-dihydrophenylacetyl lactone as shown by NMR spectroscopy. Incubation of the paaF and paaH mutants with phenylacetate yielded delta3-dehydroadipate and 3-hydroxyadipate, respectively. The origin of the carbon atoms of these C6 compounds from the aromatic ring was shown using [ring-13C6]phenylacetate. The paaG and paaZ mutants also converted phenylacetate into ortho-hydroxyphenylacetate, which was previously identified as a dead end product of phenylacetate catabolism. These data, in conjunction with protein sequence data, suggest a novel catabolic pathway via CoA thioesters. According to this, phenylacetyl-CoA is attacked by a ring-oxygenase/reductase (PaaABCDE proteins), generating a hydroxylated and reduced derivative of phenylacetyl-CoA, which is not re-oxidized to a dihydroxylated aromatic intermediate, as in other known aromatic pathways. Rather, it is proposed that this nonaromatic intermediate CoA ester is further metabolized in a complex reaction sequence comprising enoyl-CoA isomerization/hydration, nonoxygenolytic ring opening, and dehydrogenation catalyzed by the PaaG and PaaZ proteins. The subsequent beta-oxidation-type degradation of the resulting CoA dicarboxylate via beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA appears to be catalyzed by the PaaJ, PaaF and PaaH proteins.     

Role of NO in coronary and systemic vasodilation following cardiogenic reflexes

In acute experiments on anesthetized dogs under closed-chest conditions, we used the technique of double lumen catheterization of coronary vessels and peripheral vessel bed. We studied the role of endothelium-dependent relaxing factor/nitric oxide (EDRF/NO) in the development of parasympathetic coronary vasodilation after excitation of cardiac receptors. Under conditions of pharmacological stimulation of cardiac receptors of the left ventricle and short-lasting episodes of local myocardial ischemia, we also examined the effects of inhibition of NO synthesis on the development of cardiogenic depressor reflexes (hypotension and peripheral vasodilation). It was found that the reflex coronary dilatation following excitation of the cardiac (left ventricular) receptors significantly decreased after systemic NO synthase inhibition. Thus, NO production is one of the effector mechanisms of the development of coronary vessel dilatation; this conclusion is confirmed by changes in the dilatation level after blockade of this process with L-NNA (nitro-ω-L-arginine). We pioneered in demonstrating that after the blockade of NO synthesis peripheral vessel vasodilation decreases or disappeas altogether when cardiogenic reflexes are realized following pharmacological excitation of cardiac receptors with veratrine or catecholamine injections, and vasoconstrictor responses evoked by myocardial ischemia are significantly intensified. It is suggested that the influences of NO-dependent mechanisms exert a dual effect on sympathic control-mediated peripheral vasodilation during cardiogenic reflexes. Such mechanisms reduce central sympathetic tone and/or concurrently provide peripheral inhibition of neural sympathetic influences; in the latter case, NO-dependent cardiogenic reflexes play a crucial role in compensatory reactions after an injury to the heart.     

Structure of the complex of the colicin E2 R-domain and its BtuB receptor. The outer membrane colicin translocon

The crystal structure of the complex of the BtuB receptor and the 135-residue coiled-coil receptor-binding R-domain of colicin E3 (E3R135) suggested a novel mechanism for import of colicin proteins across the outer membrane. It was proposed that one function of the R-domain, which extends along the outer membrane surface, is to recruit an additional outer membrane protein(s) to form a translocon for passage colicin activity domain. A 3.5-A crystal structure of the complex of E2R135 and BtuB (E2R135-BtuB) was obtained, which revealed E2R135 bound to BtuB in an oblique orientation identical to that previously found for E3R135. The only significant difference between the two structures was that the bound coiled-coil R-domain of colicin E2, compared with that of colicin E3, was extended by two and five residues at the N and C termini, respectively. There was no detectable displacement of the BtuB plug domain in either structure, implying that colicin is not imported through the outer membrane by BtuB alone. It was concluded that the oblique orientation of the R-domain of the nuclease E colicins has a function in the recruitment of another member(s) of an outer membrane translocon. Screening of porin knock-out mutants showed that either OmpF or OmpC can function in such a translocon. Arg(452) at the R/C-domain interface in colicin E2 was found have an essential role at a putative site of protease cleavage, which would liberate the C-terminal activity domain for passage through the outer membrane translocon.