[On the use of type I tropocollagen for local injection therapy of spine, upper and lower extremity disorders].

A meeting of Interdisciplinary Expert Panel with leading specialists in the field of orthopedics/traumatology, surgery, rheumatology, and neurology was held in Moscow on February 10, 2023. The purpose of the meeting was to discuss the current status of local injection therapy (LIT) in Russia and the rationale behind the use of collagen-based products for various musculoskeletal disorders. The experts considered the following issues: (1) General contraindications to the use of medical products based on tropocollagen as well as an algorithm for actions in case of adverse events; (2) Guidelines regarding LIT in general and LIT using tropocollagen in particular, including in combination with other LIT products; (3) Particular indications and approaches to the treatment of patients with abnormal changes in appendicular joints and spine with damage to both intra-articular structures and periarticular soft tissue.

[Analysis of diversity and identification of the genovariants of plague agent strains from Mongolian foci].

The genetic diversity of Yersinia pestis strains from the Mongolian natural plague foci has been investigated. A total of 32 strains isolated from western, eastern, and central aimaks, as well as from the territory of the Gobi region, have been studied. Twenty-four strains belong to the main Y. pestis subspecies, while eight belong to other subspecies. There is only one strain of biovar medievalis (genovariant 2.MED1) among the strains of the main subspecies, while the rest of the subspecies belong to the biovar antiqua. Biovar antiqua strains are split into three groups. Strains from the eastern part of the country were classified as genovariant 2.ANT3, and those from the western and central regions were classified as genovariant 3.ANT2, which was endemic for Mongolia. One strain from the Bayan-Ulegeiskii aimak had the rare genovariant 4.ANT. None of the strains of the biovar antiqua belonged to its ancient 0.ANT branch, which is inconsistent with the commonly accepted idea that ancient marmot's plague agent race originates from Mongolia. Six out of eight strains of the minor subspecies belonged to the ulegeica subspecies, which are endemic to Mongolia, one strain belonged to the microtus group, and the last belonged to a previously uncharacterized variant of the minor subspecies.

[Standard algorithm of molecular typing of Yersinia pestis strains].

AIM: Development of the standard algorithm of molecular typing of Yersinia pestis that ensures establishing of subspecies, biovar and focus membership of the studied isolate. Determination of the characteristic strain genotypes of plague infectious agent of main and nonmain subspecies from various natural foci of plague of the Russian Federation and the near abroad. MATERIALS AND METHODS: Genotyping of 192 natural Y. pestis strains of main and nonmain subspecies was performed by using PCR methods, multilocus sequencing and multilocus analysis of variable tandem repeat number. RESULTS: A standard algorithm of molecular typing of plague infectious agent including several stages of Yersinia pestis differentiation by membership: in main and nonmain subspecies, various biovars of the main subspecies, specific subspecies; natural foci and geographic territories was developed. The algorithm is based on 3 typing methods--PCR, multilocus sequence typing and multilocus analysis of variable tandem repeat number using standard DNA targets--life support genes (terC, ilvN, inv, glpD, napA, rhaS and araC) and 7 loci of variable tandem repeats (ms01, ms04, ms06, ms07, ms46, ms62, ms70). The effectiveness of the developed algorithm is shown on the large number of natural Y. pestis strains. Characteristic sequence types of Y. pestis strains of various subspecies and biovars as well as MLVA7 genotypes of strains from natural foci of plague of the Russian Federation and the near abroad were established. CONCLUSION: The application of the developed algorithm will increase the effectiveness of epidemiologic monitoring of plague infectious agent, and analysis of epidemics and outbreaks of plague with establishing the source of origin of the strain and routes of introduction of the infection.

[Comparative genetic characteristic of vaccine strain of Yersinia pestis EV and its putative "virulent derivatives"].

AIM: To perform a comparison of genetic characteristics of vaccine strain EV and its putative "virulent derivatives", obtained after passages through highly susceptible animals, in order to identify the strains-"revertants" and establish their possible origin. MATERIALS AND METHODS: Yersinia pestis EV strains and its putative "virulent derivatives" were used in the study. Polymerase chain reaction and DNA-DNA hybridization were used for genetic analysis. RESULTS: Comparison of several genetic characteristics of vaccine strain EV and its putative "virulent derivatives" allowed to establish that virulent "revertants" are not derivatives of vaccine strain EV because they do not belong to East biovar, do not have ribotype characteristic for EV strain and contain pigmentation area, which is absent in vaccine strain. CONCLUSION: Obtained results evidence against possibility of reversion of vaccine EV strain to virulent forms in organisms of highly susceptible animals and confirm its safety for vaccination.

[Genomic polymorphism of the main subspecies of the plague agent strains].

Genomic fingerprinting analysis of plague agent strains of the main subspecies isolated in natural foci of various types in the Russian Federation and neighboring countries suggests their genetic polymorphism, while they are similar in phenotypic properties. The strains of the main subspecies, Y. pesis subsp. Pestis, fall into four genetic variants, each of them being associated with specific carrier species. The conserved genomic fingerprinting profile of each genovariant of Y. pesis subsp. Pestis strains ensures the suggested methodic approach to be promising for the intraspecies differentiation of plague agent strains (including atypical strains). Correlation of genovariants with carrier species permits their application for research into enzootic territories, where carrier change-over takes place.

[Effect of nocodazole on the activation of transcription factors STAT1 and STAT3 in A431 cells]. / Vliianie nokodazola na aktivatsiiu transkriptsionnykh faktorov STAT1 i STAT3 v kletkakh A431.

The STAT transcription factors (signal transducers and activators of transcription), STAT1 and STAT3, are involved in signal transduction from growth factors and different cytokine receptors. STAT1 and STAT3 activation mechanisms are not sufficiently investigated, but they are known to depend upon both cell type and stimulus for either of them. Recently, we have shown that nocodazole blocked EGF-induced STAT1 transport to the nucleus. Here, we have compared STAT1 and STAT3 activation in response to IFNgamma, IFNalpha and epidermal growth factor (EGF) in A431 cells. We have shown the STAT1 activation by all these agents; unlike, STAT3 was activated by EGF only. STAT1 and STAT3 activation upon EGF is blocked by both nocodazole and Src-kinase family inhibitor. STAT1 activation upon IFNgamma influence is blocked by nocodazole, but does not depend on the activity of Src-family kinases. The increased STAT3 phosphorylation results from a combined action of Src-kinase inhibitor and IFNgamma. IFNalpha-induced activation of STAT1 was not inhibited by either nocodazole or Src-kinase inhibitor. Taken together, the data obtained suggest that the activation of both STAT1 and STAT3 in A431 cells is accomplished by different mechanisms.

[Genotyping Yersinia pestis strains from various natural foci]. / Genotipirovanie shtammov Yersinia pestis iz razlichnykh prirodnykh ochagov.

Seven genetic variants of Yersinia pestis were detected by finger-printing of 85 strains of this bacterium from natural foci by means of a BX probe. Variants of Y. pestis strains correlate with certain species of carriers.

Isolation and characterization of myeloperoxidase from leukocytes of rat peritoneal fluid.

Myeloperoxidase (MPO) was isolated from rat peritoneal leukocytes with a yield of 51% and A430/A280 = 0.75 - 0.80, and its physicochemical properties were studied. The molecular weight of the MPO is about 150 kD. The MPO was assayed for amino acid content. We used substrate mixture containing phenol, 4-aminoantipyrine, and H2O2 to detect 10(-10) M of the enzyme. The MPO was localized in rat blood neutrophils using polyclonal anti-MPO antibodies and secondary fluorescein isothiocyanate-labeled antibodies. Immunofluorimetric assay (IFMA) was developed for quantitative measurement of the MPO. The MPO and leukocytes can iodinate BSA using NaI or thyroxine as the source of iodine.

[The association of a protein regulator of intracellular Rab7 membrane transport with endosomes containing an epidermal growth factor receptor with activated and inactivated tyrosine kinase]. / Assotsiatsiia belkovogo reguliatora vnutrikletochnogo membrannogo transporta Rab7 s éndosomami, soderzhashchimi retseptor épidermal'nogo faktora rosta s aktivirovannoi i neaktivirovannoi tirozinkinazoi.

By double indirect immunofluorescent microscopy, Rab7, traditionally considered as a late endosomal marker, has been demonstrated to colocalize with an internalized epidermal growth factor receptor (EGFR) possessing active and inactive tyrosine kinase (TK). The epidermal growth factor (EGF), which induces TK activity of EGFR, and monoclonal antibody Mab 108, which does not, have been exploited as ligands to stimulate endocytosis of EGFR. Colocalization between EGFR and Rab7 has been detected at both early (10 min) and delayed (60 and 120 min) endocytosis of EGFR, while it turned out to be much more obvious at the later ones. A comparison between EGFR-mediated and peroxidase fluid-phase endocytoses has revealed that Rab7 failed to be recruited on endosomal structures, containing peroxidase, even after 180 min endocytosis. Subcellular fractionation of endosomes containing 125I-EGF and 125I-Mab 108 in Percoll density gradients, in parallel with the analysis of ligand degradation, have verified the efficient transition of EGF-receptor complexes into the late endosomes and retention of Mab 108-receptor complexes within the early (sorting) endosomes. Taken together, the data cotained suggest that endogenous Rab7 is able to be recruited not only on late but also on maturating sorting EGFR-containing endosomes, thus mediating sorting along the EGFR endocytotic pathway.

[The characteristics of the transport into the nucleus of conjugates with normal and mutant nucleophilic sequences]. / Kharakteristika transporta v iadro kon''iugatov s normal'noi i mutantnoi nukleofil'nymi posledovatel'nostiami.

A study was made of the transport of conjugates of a fluorescently labeled protein and a synthetic peptide, corresponding to the nuclear localization sequence of the SV 40 large T-antigen, into the nuclei of cultured human (HeLa and A431) and murine (HER14) cells. A possibility for such conjugates to be transported into the nuclei of digitonin-permeabilized cells, without addition of exogenous cytosol, was demonstrated. A quantitative comparison of the transport levels of constructions with normal or altered (K128T) peptide sequence was performed, and a low selectivity of nuclear transport with this alteration in the digitonin-permeabilized cell system was revealed, whereas constructions with the mutant sequence, when injected into live cells, remained in the cytoplasm. ATP-dependent transport of constructions with the mutant sequence into permeabilized cell nuclei was demonstrated, with a considerable part of this transport being NEM-insensitive. A suggestion is put forward that there are several variants of the nucleophilic sequence containing protein transport in the digitonin-permeabilized cell system.

[The production and characteristics of polyclonal antibodies to the threonine-containing site of the cytoplasmic domain of the EGF receptor]. / Poluchenie i kharakteristika poliklonal'nykh antitel k treoninsoderzhashchemu uchastku tsitoplazmaticheskogo domena retseptora EFR.

A conjugate of a synthetic polypeptide to hemocyanine was used as an antigen for obtaining polyclonal antibodies to the site of cytoplasmic domain of the epidermal growth factor (EGF) receptor. The amino acid sequence of the peptide used was the same as that of the EGF receptor from residue 650 to 661. In the A431 cell solubilizate the obtained antibodies interact with the phosphorylated protein with 170 kDa molecular weight (MW), by immunoblotting recognize the protein of this MW, and as evidenced by immunofluorescence, their distribution in the cell is the same as that of monoclonal antibodies to the EGF receptor. It is concluded that the obtained antibodies may recognize the EGF receptor. Moreover, these antibodies in solubilizates of A431, CHO cells, and normal human fibroblasts by immunoblotting recognize 74 and 76 kDa proteins.

[Eicosanoic acid levels in children with atopic dermatitis]. / Soderzhanie éikozanoidov u detei s atopicheskim dermatitom.

Column chromatography followed by RIA was used to measure the blood plasma content of arachidonic acid metabolites (leukotrienes C4, B4, C4/D4/E4, prostaglandins F2 alpha, E2, 6-keto-prostaglandin F1 alpha, thromboxane B2) in 146 children aged 6 months to 14 years with atopic dermatitis. The data obtained were compared to the healthy children's parameters. The majority of the patients manifested activation of the system of arachidonic acid metabolism. The intensity of changes in the content of eicosanoids was found to depend on the clinical pattern and spreading of skin lesions.

[Cytoskeletal changes in A-431 cells under the action of epidermal growth factor]. / Izmenenie tsitoskeleta kletok A-431 pri deistvii épidermal'nogo faktora rosta.

By the use of rhodamine-phalloidin, the distribution of actin in A-431 cells during the action of epidermal growth factor (EGF) has been studied. Changes in the pattern of staining are observed in 30-60 s after addition of the EGF. Microvilli and wrinkles are created on the cell surface. Following a 5-10 min action of EGF, rhodamine-phalloidin stained intensely ruffles and cell borders. After 60 min, the ruffling of cell surface disappeared, and actin was seen concentrating on the cell borders only. Electron microscopy of the EGF-treated A-431 cells lysed by Triton X-100 also revealed some vigorous fibrillar bunches on the cell edges.

[The immunofluorescent detection of phosphorylated receptors of the epidermal growth factor during their internalization in A-431 cells]. / Immunofluorestsentnoe vyiavlenie fosforilirovannykh retseptorov épidermal'nogo faktora rosta v protsesse ikh internalizatsii v kletkakh A-431.

Phosphorylated receptors of the epidermal growth factor (EGF) were localized in the human epidermoid carcinoma cells using immunofluorescent staining with antibody to phosphotyrosine. The application of EGF at 4 degrees C was seen to induce a characteristic fluorescence of the cell margins, whereas no cell staining occurs in the absence of EGF. After a 1 hour incubation of cells at 37 degrees C, within which the internalized EGF receptor complexes are accumulated in the juxtanuclear compartment near the para-Golgi region, the staining with antiphosphotyrosine antibody reveals the receptors in this region. It is concluded that the internalized EGF-receptor complexes may remain in the phosphorylated state.

[Transport of monosaccharides in model systems: inhibition of sugar transport in frog muscle fibers, treated with glutaraldehyde]. / Transport monosakharidov v modelnykh sistemakh: ingibirovanie transporta sakharov v myshechnykh voloknakh liagushki, obrabotannykh glutaral'degidom.

The entry of D-xylose, a permeable non-metabolizing glucose analog, in the frog muscle fibers was examined. The sugar transport system activity was established in the frog muscle fibers treated by 0.3% glutaraldehyde. The basal transport as well as insulin activated D-xylose transport was seen preserved. Sugar transport inhibitors, phlorizin, phloretin and cytochalasine B reduce the rate of D-xylose transport in this "glutar model" of a muscle fiber. A long treatment by glutaraldehyde (3.5 hours at 4 degrees C, and 1 hour at 20 degrees C) leads to a 40% decline in the entry rate of D-xylose.

[Effect of amines on insulin-stimulated processes: the transport of sugars and synthesis of glycogen in frog sartorius muscles]. / Vliianie aminov na insulinstimuliruemye protsessy: transport sakharov i sintez glikogena v portniazhnykh myshtsakh liagushki.

Amines and amides were found to inhibit the process of stimulation of sugar transport in muscle tissue (N. A. Vinogradova et al., 1978). The present paper reports results of experiments on frog sartorius muscles acted upon by amines and other substances that inhibit induction of cultured cell proliferation. The stimulation of sugar transport induced by insulin, 2,4-dinitrophenol, or KCl was found to be inhibited by dansylcadaverine (1 mM), 5-methoxytryptamine (2 mM), or methylamine (100 mM). Such substances as chloroquine, bacitracin, or monensin exerted no effect. Besides, dansylcadaverine (1 mM), and 5-methoxytryptamine inhibited the stimulation of insulin induced glycogen synthesis. Dansylcadaverine was ineffective at concentrations lower than 0.5 mM. It is suggested that the inhibitory action of amines depends on their influence on the processes of membrane protein phosphorylation.

[Effect of dansylcadaverine on the epidermal growth factor-stimulated phosphorylation of uridine]. / Vliianie dansilkadaverina na stimulirovannoe épidermal'nym faktorom rosta fosforilirovanie uridina.

The increase of uridine phosphorylation during the first hour after epidermal growth factor (EGF) stimulation (1.25 nM) of Swiss 3T3 cells is completely blocked by 100 microM dansylcadaverine (DC). Lack of the effect of DC on uridine transport, uridine kinase activity in cell homogenate, intracellular ATP concentration and plasma membrane permeability for phosphorylated uridine derivatives makes it possible to propose the inhibition by DC (100 microM) of the activated state of uridine kinase. The rapidity of the inhibition of EGF effect and the lack of influence of DC (in tested concentration) upon the clustering of EGF-receptor complexes, rate of their internalization (Sorkin, 1985; Nikol'skii et al., 1987) and pH value of intracellular compartments (Sorkin et al., 1985; Teslenko et al., 1986) may suggest an association of DC inhibitory action with blocking of some steps of the receptor mediated endocytosis. Accumulation of DC in cell membranes, rather than in intracellular compartments with acidic pH, is a necessary factor for its blocking effect. Possibilities of DC action through the influence on calmodulin-dependent proteins or EGF-induced cell protein phosphorylation are discussed.

[Effect of amines on the binding, internalization and degradation of epidermal growth factor and the induction of DNA synthesis in a 3T3 cell culture]. / Vliianie aminov na sviazyvanie, internalizatsiiu, degradatsiiu épidermal'nogo faktora rosta i induktsiiu sinteza DNK v kul'ture kletok 3T3.

The role of intracellular processing of epidermal growth factor (EGF) in the induction of proliferation of quiescent Swiss 3T3 cells was studied using various inhibitors. The number of amines (dansylcadaverine, chloroquine, cystamine, 5-methoxytryptamine) dimethylurea and monensin were shown to block the mitogenic effect of EGF. The majority of these substances while used in concentrations sufficient to inhibit the proliferation do not significantly influence 125I-EGF binding and internalization. The level of EGF degradation was reduced only by chloroquine. The inhibitory effect of amines and monensin on the generation of proliferative signal was supposed to take place at the stages of EGF processing in "specialized" endosomes and in Golgi apparatus.

[Stimulating action of epidermal growth factor and insulin on uridine phosphorylation in 3T3 and 3T6 cells]. / Stimuliruiushchee deistvie épidermal'nogo faktora rosta i insulina na fosforilirovanie uridina v kletkakh 3T3 i 3T6.

The influence of epidermal growth factor (EGF) and insulin on uridine phosphorilation was investigated in cell cultures Swiss 3T3 and 3T6, arrested in the medium with serum content of 0.5%. It is shown that following 5-10 minutes the addition of EGF into the culture medium in concentrations from 0.15 to 51 nM results in the increase in the rate of uridine phosphorilation which reaches the maximum value, similar to that of the stimulating effect of 10% serum. Insulin in the 4-85 nM concentrations also enhanced the rate of uridine phosphorilation and exerted a potential influence on EGF effect only in high concentrations. The investigation of the dynamics of binding and internalization of 125I-EGF showed that the number of EGF molecules on membrane increase during 10 minutes after addition of EGF into the medium and then begin to decrease. The binding of EGF with not more than 2% of the total number of cell receptors was shown by approximal estimation to be enough for stimulation of uridine phosphorilation. A conclusion is drawn that the presense of single growth factor EGF in enough for maximum stimulation of early reaction of cells on the proliferation stimulus realized at the posttranscriptional level, while both the additional factors and higher concentrations of EGF are necessary for the maximum induction of DNA synthesis.