Normal distribution and medullary-to-cortical shift of Nestin-expressing cells in acute renal ischemia.

Nestin, a marker of multi-lineage stem and progenitor cells, is a member of intermediate filament family, which is expressed in neuroepithelial stem cells, several embryonic cell types, including mesonephric mesenchyme, endothelial cells of developing blood vessels, and in the adult kidney. We used Nestin-green fluorescent protein (GFP) transgenic mice to characterize its expression in normal and post-ischemic kidneys. Nestin-GFP-expressing cells were detected in large clusters within the papilla, along the vasa rectae, and, less prominently, in the glomeruli and juxta-glomerular arterioles. In mice subjected to 30 min bilateral renal ischemia, glomerular, endothelial, and perivascular cells showed increased Nestin expression. In the post-ischemic period, there was an increase in fluorescence intensity with no significant changes in the total number of Nestin-GFP-expressing cells. Time-lapse fluorescence microscopy performed before and after ischemia ruled out the possibility of engraftment by the circulating Nestin-expressing cells, at least within the first 3 h post-ischemia. Incubation of non-perfused kidney sections resulted in a medullary-to-cortical migration of Nestin-GFP-positive cells with the rate of expansion of their front averaging 40 microm/30 min during the first 3 h and was detectable already after 30 min of incubation. Explant matrigel cultures of the kidney and aorta exhibited sprouting angiogenesis with cells co-expressing Nestin and endothelial marker, Tie-2. In conclusion, several lines of circumstantial evidence identify a sub-population of Nestin-expressing cells with the mural cells, which are recruited in the post-ischemic period to migrate from the medulla toward the renal cortex. These migrating Nestin-positive cells may be involved in the process of post-ischemic tissue regeneration.

[Antioxidant effect on the growth of Saccharopolyspora erythraea and biosynthesis of erythromycins].

Oxidative stress in the mycelium of submerged culture of S. erythraea, an organism producing erythromycins, was observed. The stress could be eliminated by addition of antioxidants, such as butyloxyanisole, alpha-tocopherol, cytochrome C to the culture. In the presence of the antioxidants the mycelium lysis decreased, the level of the decrease being higher in the medium preferably containing carbohydrates vs. the medium mainly containing lipids (soybean oil). The antioxidants had no specific effect on the biosynthesis of erythromycins.

[Hematopoiesis on cellulose acetate membranes in the presence of lipopolysaccharides]. / Krovetvorenie na atsetat-tselliuloznykh membranakh pod vliiniem lipopolisakharida.

The formation of hemopoietic colonies on acetate cellulose membranes in the peritoneal cavity of mice was markedly enhanced after the injection of bacterial lipopolysaccharide. In addition to granulocytic-macrophagal differentiation, the foci of erythropoiesis appeared. The stimulating effect of lipopolysaccharide was not expressed in nonirradiated mice and during the formation of hemopoietic foci on acetate cellulose membranes in the subcutaneous connective tissue.

[Hemopoiesis on stromal sublayers formed by constant lines of fibroblasts]. / Krovetvorenie na stromal'nykh podsloiakh, obrazovannykh postoiannymi liniiami fibroblastov.

We studied the formation of hemopoietic colonies on an artificial sublayer in the peritoneal cavity of mice under the influence of stromal sublayers consisting of fibroblasts of six constant cell limes. All studied lines of stromal cells supported the formation of granulocytic foci and some of them supported the formation of erythroid foci as well. It was shown that hemopoiesis was preserved or, in some cases, enhanced on sublayers of fixed (metabolically inactive) cells. The treatment of fibroblasts by E. coli lipopolysaccharide did not lead, as a rule, to significant stimulation of hemopoiesis.

[A study of hemopoiesis on a sublayer of peritoneal cells in the presence of hemopoietic cytokines]. / Issledovanie gemopoc'za na podsle peritoneal'nykh kletok pod vliianiem krovetvornykh tsitokinov.

We studied the effects of erythropoietin and thrombopoietin on the clonogenic capacity and direction of differentiation of the hemopoietic cells that form colonies on acetate cellulose membrane in the peritoneal cavity of mice. An increased level of erythropoietin in the blood of recipient mice after blood letting led to the appearance of erythroid colonies upon transplantation of syngeneic hemopoietic cells but did not affect the differentiation of transplanted xenogeneic (guinea pig) hemopoietic cells. Erythropoietin transported top the stromal sublayer by a polymeric carrier also induced erythroid differentiation, while thrombopoietin transported in a similar way somewhat enhanced megakaryocytopoiesis.

Studies on clonogenic hemopoietic cells of vertebrate in space: problems and perspectives.

Hemopoietic tissues were studied in vertebrates launched aboard the Soviet (Russian) biosatellites ("Cosmos-1129, 1514, 1667, 1887 and 2044"; "Bion-10 and 11") between 1980 and 1996. In the bone marrow of rats exposed to spaceflight conditions, a statistically significant decrease in cell number was revealed in the progenitor cell compartment accounting for the compensatory response of granulocyte-macrophage (CFU-gm) and erythrocyte lineages (BFU-e and CFU-e) and in the compartment of multipotent hemopoietic stem cells (CFU-s), which is responsible for the permanent renewal of hemopoietic tissue. The number of stromal fibroblastic progenitors (CFC-f) in the bone marrow of these rats was also reduced. Apparently, changes in the hemopoietic stroma damage the hemopoietic microenvironment and, hence, may be responsible for changes observed in the hemopoietic tissue proper. Attempts were made to develop methods for analyzing morphologically indiscernible clonogenic hemopoietic cells of newts, and studies on the effects of spaceflight factors on these cells were performed. The results showed that the numbers of clonogenic cells in newts of the flight group newts were significantly lower than in control newts. The data obtained are used as the basis for formulating the problems to be studied, drawing up a program for further research on the effects of spaceflight factors on stem and other clonogenic hemopoietic cells, and developing new experimental models for analyzing stem cells, the state of the hemopoietic stroma, etc.

[Use of the copolymer ethylenevinylacetate for studying the effect of hemopoietic cytokines]. / Ispol'zovanie kopolimera étilenvinilatsetata dlia issledovaniia éffekta krovetvornykh tsitokinov.

Copolymer ethylenevinylacetate is known as a polymeric carrier, which ensures a lasting release of biologically active substances. An attempt has been made to use it for testing the effects of hemopoietic cytokinins. It was shown that erythropoietin, embedded in this polymer, induces formation of erythropoietic foci on acetate-cellulose membranes in the peritoneal cavity of mice.

[Experimental research on the cellular interactions during hemopoiesis]. / Eksperimental'noe issledovanie mezhkletochnykh vzaimodeistvii pri gemopoéze.

We present a review of experimental studies performed at the Laboratory of Histogenesis of the Institute of Developmental Biology, Russian Academy of Sciences, on the problem of cell interactions during hemopoiesis. Special attention has been given to original experimental models, such as production of hemopoietic foci on underlayers of fibroblasts encapsulating a foreign body in the peritoneal cavity of rodents (after intraperitoneal transplantation of hemopoietic cells) and repopulation of ectopic hemopoietic territories under the kidney capsule of mice by syngeneic or xenogeneic hemopoietic cells. We describe the competitive interactions of genetically different hemopoietic cells after the transplantation of their mixtures to irradiated mice (multicomponent radiation chimeras). Xenogeneic and multicomponent chimeras have also been obtained in long-term bone marrow culture. We have examined characteristics of hemopoiesis on stromal cell underlayers produced by cells of various origins in vitro and then transplanted into the peritoneal cavity of irradiated mice. We discuss the results obtained and possible mechanisms of these phenomena.

[Serine/threonine type protein kinase activity in cell-free extracts of Streptomyces lividans]. / Proteinkinaznaia aktivnost' serin/treoninovogo tipa v beskletochnykh ekstraktakh Streptomyces lividans.

Cell-free extracts of Streptomyces lividans prepared at the physiological ionic strength from the early logarithmic phase culture showed an endogenous protein kinase activity. Incubation of the salt soluble fraction of S.lividans with [gamma-32P]ATP led to incorporation of the labelled phospate into 6 to 7 polypeptide chains of 12-100 kDa. Addition of heparine, polylysine, spermine, phosphatidylserine, kanamycin, cAMP and cGMP did not change the spectrum or level of the polypeptide phosphorylation in the extract while Ca2+ ions stimulated the protein phosphorylation. It was shown that the protein kinase(s) catalyzed binding of the phosphate groups to the serine and thereonine residues in the polypeptides with M(r) 100 and 35 kDa.

[The NADP-diaphorase reaction as a neurohistologic method]. / NADF-diaforaznaia reaktsiia kak neirogistologicheskii metod.

We present evidence about the possible use of histochemical NADP diaphorase reaction to visualize elements of the nervous system.

[Determination of the nucleotide sequence of the silent aminoglycoside phosphotransferase gene in Streptomyces rimosus P3]. / Opredelenie nukleotidnoi posledovatel'nosti "molchashchego" gena aminoglikozidfosfotransferazy Streptomyces rimosus P3.

The nucleotide sequence BgIII-Pstl of the DNA fragment containing the aph gene cloned from Streptomyces rimosus P3 not producing aminoglycoside antibiotics was determined. The aph gene was shown to encode neomycin phosphotransferase differing by the substrate specificity from the enzyme encoded by the aph genes from the organisms producing aminoglycoside antibiotics. Comparison of the cloned aph VIII gene and its product with the aph genes and their products from the antibiotic-producing actinomycetes and clinical microbial strains permitted to consider it as a representative of the third group genes which are likely widely distributed in soil microorganisms not producing aminoglycoside antibiotics. The gene length was 777 nucleotide pairs at the average GC content of 67 per cent. Comparison of the nucleotide and protein sequences of the S.rimosus gene with those of the genes cloned from the aminoglycoside-producing organisms (S.fradiae and Micromonospora chalcea) revealed high homology in the 3'-end region of the genes. However, the homology percentage by DNA for the S.rimosus gene was lower than that for the genes from the other organisms on their comparison with each other: 56-67 and 82-86 per cent respectively. Comparison of the profiles of the protein hydrophilic properties revealed differences in the region of the first 110 amino acids of the gene under the investigation. The amino acid sequence contained all the highly conservative regions characteristics of aminoglycoside phosphotransferases but had several amino acid substitutes detected for the first time including those in the region responsible for the antibiotic binding.

[Determination of the nucleotide sequence and characterization of the novel aminoglycoside phosphotransferase aphVIII gene from the Streptomyces rimosus strain]. / Opredelenie nukleotidnoi posledovatel'nosti i kharakteristika novogo aminoglikozidfosfotransferaznogo gena aphVIII ia shtamma Streptomyces rimosus]

The nucleotide sequence of a BglII-PstI DNA fragment that contains the cloned aphVIII gene from the Streptomyces rimosus P3 strain, the producer of oxytetracycline, was determined. It was established that the aph gene encodes neomycin phosphotransferase that differs by substrate specificity from neomycin phosphotransferases encoded by aph genes in producers of aminoglycoside antibiotics and clinical bacterial strains. The gene was shown to be 777 bp in length with the mean GC content equal to 67%. The amino acid sequence possesses all highly conserved regions typical for aminoglycoside phosphotransferases; however, this sequence contained several amino acid substitutions that have been detected for the first time, including those in the domain responsible for the association with antibiotics.

[NO synthetase in the cells of different tissues and organs of the mouse]. / Issledovanie NO-sintetazy v kletkakh razlichnykh tkanei i organov myshi.

Cellular NO synthase was assayed in bone marrow, spleen, thymus, lymph nodes, epidermis, hypodermic connective tissue, small gut, peritoneal exudate, liver, kidney, and heart by cytochemical and immunofluorescent methods. As a rule the enzymatic activity was found in differentiated cells that finished proliferation (mature hemopoietic cells, epithelium of gut villi, etc.). The enzyme activity was undetectable in actively reproducing low-differentiated cells (basal layer of the epidermis, crypt cells of the small gut, or blast hemopoietic forms). NO is proposed to participate in regulation of tissue-specific terminal differentiation (maturation) of the cells.

Experimental analysis of cell interactions during hemopoiesis.

The principal approach was to study hemopoiesis on different stromal cell underlayers (fibroblasts or fibroblast-like cells covering a foreign body implanted into the peritoneal cavity of mice or other rodents) after intraperitoneal transplantation of syngeneic, allogeneic, and xenogeneic hemopoietic cells. The data obtained are compared with the results of experiments on repopulation of ectopic hemopoietic territories (under the mouse kidney capsule) by syngeneic and xenogeneic hemopoietic cells. Competitive cell interactions are described that occur during repopulation of the hemopoietic stroma or formation of the hemopoietic foci on cellulose acetate membranes (CAMs) in the peritoneal cavity of irradiated mice by genetically different hemopoietic cells transplanted to these animals (multicomponent radiation chimeras). The model of xenogeneic and multicomponent radiation chimeras was reproduced in long-term bone marrow cultures, where hemopoietic cells of different genotypes coexisted, without any competitive cell elimination. The second part of this review deals with hemopoiesis on stromal cell underlayers, formed by cells of different origin, different stages of development, and obtained from other sources. These underlayers were formed on CAMs in vitro and then transferred into the peritoneal cavity of irradiated mice, which subsequently received intraperitoneal injections of donor hemopoietic cells. Specific features of hemopoiesis on stromal underlayers formed by the following cell types are described: (1) fibroblasts from mouse embryos at different developmental stages; (2) fibroblasts from the skin, liver, and bone marrow of 17-day mouse fetuses and newborn mice; (3) fibroblasts from the monolayer cultures of mouse and rat bone marrow; (4) 3T3 cell line; (5) hepatocytes of 17-day mouse fetuses or sexually mature rats; (6) newborn mouse kidney cells; and (7) cells transgenic for the erythropoietin gene. The phenomena observed in these experiments and their probable mechanisms are discussed.

Blood and clonogenic hemopoietic cells of newts after the space flight.

Ribbed newts were used for studying the effect of space flight on board of the biosatellite (Cosmos-2229) on blood and clonogenic hemopoietic cells. In blood of newts of the flight group, the relative proportion of neutrophils increased, whereas that of lymphocytes and eosinophils decreased. Space flight did not result in loss of the ability of newt blood cells to incorporate H3-thymidine. Analysis of clonogenic hemopoietic cells was performed using the method of hemopoietic colony formation on cellulose acetate membranes implanted into the peritoneal cavity of irradiated newts. To analyze reconstitution of hemopoiesis after irradiation donor hemopoietic cells from flight or control newts were transplanted into irradiated newts whose hemopoietic organs were investigated. The newt can be considered an adequate model for studying hemopoiesis under the conditions of the space flight.

[Study of antimutagenic properties of bio-ginseng in mammalian cells in vitro and in vivo]. / Izuchenie antimutagennykh svoistv biozhen'sheniia na kletkakh mlekopitaiushchikh in vitro i in vivo.

The impact was studied of bio-ginseng produced from ginseng callus cells on the rate of chromosome rearrangements in Chinese hamster cells and in continuous tumor cells of mice (Ehrlich strain). Bio-ginseng reduced rate of spontaneous SCE as well as the level of mitomycin C-induced chromosome aberrations in Chinese hamster cells. It protected ascitic tumor cells (Ehrlich strain) against the mutagen action of urea nitrosomethyl.

[The characteristics of the hematopoietic cells forming colonies on cellulose acetate membranes and their distinctions from other clonogenic cells]. / Kharakteristika krovetvornykh kletok, obrazuiushchikh kolonoii na atsetat-tselliuloznykh membranakh, ikh otlichiia ot drugkikh klonogennykh kletok.

In order to characterize hemopoietic cells forming colonies on membranes of cellulose acetate (CFU-acm) implanted into the peritoneal cavity of mice, we studied the effect of factors stimulating and inhibiting granulocytopoiesis on these cells. Proliferation of CFU-acm can be controlled by humoral factors and this allows us to conclude that they are not identical to CFUs and probably belong to the compartment of early hemopoietic cells of the granulocyte series. We also present evidence for fractional composition, ultrastructure and bone marrow origin of cells belonging to the layer providing for hemopoietic microenvironment for the resulting foci of hemopoiesis; we also present evidence for the role of fibroblasts (fibroblast-like cells) in the maintenance of hemopoiesis. Experiments on transplantation of bone marrow from several rodent species to syn-, allo- and xenogenic recipients allowed us to study interactions of hemopoietic elements of colonies with cells of the underlying layer.

Decrease in the number of progenitors of erythrocytes (BFUe, CFUe), granulocytes and macrophages (GM-CFC) in bone marrow of rats after a 14-day flight onboard the Cosmos-2044 Biosatellite.

A decrease in the number of progenitors of erythrocytes (BFUe, CFUe) and of granulocytes and macrophages (GM-CFC) in bone marrow was found in rats exposed to microgravitation during a 14-day flight onboard the Cosmos-2044 biosatellite when compared to control rats maintained under conditions of gravitation on the ground. The number of progenitors of both lineages of haemopoiesis was also decreased in synchronous control rats, thus suggesting that the pool of progenitors is influenced also by the action of the nonspecific space flight factors.