Stimulating effect of cord blood fraction below 5 kDa and actovegin on cell growth of permanent cell lines

The influence of a calf cord blood fraction below 5 kDa and Actovegin on the adhesive and proliferative properties of cells of the BHK-21 clone 13/04 and PK-15-IECVM permanent cell lines was investigated. It was shown that the fraction and Actovegin did not affect cell adhesion, promoted cell spreading, improved cell morphology, and stimulated cellular mitotic activity. In cultures cultivated in media with various serum contents, the efficiency of the cord blood fraction below 5 kDa was higher than that of Actovegin.     

The effects of actovegin on cell proliferation of permanent lines

The influence of Actovegin on proliferation activity and mitotic regimen of cells of permanent lines PK-15-IEKVM and BHK-21 clone 13/04 was investigated. Addition of Actovegin into growth media containing bovine serums of different components and concentrations stimulates cell proliferation. Conclusion has been made that Actovegin can be used in cell culture biotechnology.     

Metalloproteinase activity is the sole factor responsible for the growth-promoting effect of conditioned medium in Trichoplusia ni insect cell cultures

Conditioned medium (CM) taken from a serum-free culture of Trichoplusia ni (BTI-Tn-5B1-4, High Five) cells on days 2 and 3, shortened the lagphase and increased the maximum cell density when added to T. ni cultures with low-inoculum cell density. Gel filtration fractions of CM, eluting at around 45kDa, stimulated cell proliferation even better than CM. A protein in the gel filtration fraction was identified by N-terminal amino acid sequencing as a proteinase, related to a snake venom metalloproteinase. Casein zymography showed, multiple metalloproteinase bands between 48 and 25kDa, as well as precursor forms above 48kDa. Metalloproteinase bands below the main band at 48kDa were autocatalytic degradation products. Metalloproteinase activity was the sole factor responsible for the growth stimulating effect of CM as shown by using the specific metalloproteinase inhibitor dl-thiorphan. Metalloproteinases have recently been shown to release growth factors from sequestering extracellular proteins. We propose that the metalloproteinase is involved in autocrine regulation of T. ni proliferation in serum-free media. In addition, a gel filtration fraction of CM, eluting at about 10kDa, inhibited cell growth. Apart from a lysozyme precursor protein and a cyclophilin-like protein, a kazal-type proteinase inhibitor could be identified in this fraction.     

Lithoautotrophic growth of the freshwater colorless sulfur bacterium Beggiatoa "leptomitiformis" D-402

The freshwater colorless sulfur bacterium Beggiatoa "leptomitiformis" D-402 was shown to be capable of lithoautotrophic growth in a batch culture under microaerobic conditions at O2 concentrations in the medium of no higher than 0.5 mg/l. The cell yield was maximum at a dissolved oxygen concentration of 0.15 mg/l. A high activity level of key enzymes of the Calvin cycle and of enzymes involved in dissimilatory oxidation of thiosulfate was recorded in the cells. The high rate of CO2 assimilation (112-139 nmol/(min mg protein)) and the cell yield (12 mg dry cells/mmol thiosulfate oxidized), 91-92% of which was accounted for by CO2 carbon, were close to those typical of autotrophic bacteria. Thiosulfate was oxidized almost completely to sulfate, and the fraction of elemental sulfur in the final products did not exceed 0.2-1.7% of the thiosulfate sulfur. The cell membrane fraction contained cytochromes (b + o) and two cytochromes c with M(r) of 23 and 26 kDa; the soluble fraction contained cytochrome c with M(r) of 12 kDa.     

Influence of cattle cord blood fraction below 5 kD on biochemical parameters of blood in experimental chronic stomach ulcer in rats

Influence of cattle cord blood fraction (below 5 kD) on lipid peroxidation product content and alkaline phosphatase activity-in peripheral blood was studied on the experimental subchronic stomach ulcer model in rats. It has been shown that the fraction administrations normalize thiobarbituric-active product content and alkaline phosphatase activity in blood, which testifies to decreasing inflammatory reaction in the mucous membrane of the stomach. The fraction administrations accelerate the processes of regeneration of the mucous membrane of the stomach up to complete healing of ulcer defects. Cord blood fraction below 5 kD from cattle possesses antiulcer activity which is analogous to the actovegin activity. It has been shown by gel-penetrating chromatography that the pattern of cord blood fraction low molecular substances is different from the actovegin pattern both qualitatively and quantitatively.     

Ectodomain shedding of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, by TIMP-2- and TIMP-3-sensitive proteolysis

Neuroglycan C (NGC) is a transmembrane-type of chondroitin sulfate proteoglycan with an epidermal growth factor (EGF)-like module that is exclusively expressed in the CNS. Because ectodomain shedding is a common processing step for many transmembrane proteins, we examined whether NGC was subjected to proteolytic cleavage. Western blotting demonstrated the occurrence of a soluble form of NGC with a 75 kDa core glycoprotein in the soluble fraction of the young rat cerebrum. In contrast, full-length NGC with a 120 kDa core glycoprotein and its cytoplasmic fragment with a molecular size of 35 kDa could be detected in the membrane fraction. The soluble form of NGC was also detectable in culture media of fetal rat neurons, and the full-length form existed in cell layers. The amount of the soluble form in culture media was decreased by adding a physiological protease inhibitor such as a tissue inhibitor of metalloproteinase (TIMP)-2 or TIMP-3, but not by adding TIMP-1. Both EGF-like and neurite outgrowth-promoting activity of the NGC ectodomain may be regulated by this proteolytic processing.     

Influence of different culture solutions on osteoblastic differentiation in cord blood and bone marrow derived progenitor cells.

Mesenchymal progenitor cells derived from cord blood (unrestringated somatic stem cells, USSC) and bone marrow (mesenchymal stem cells, MSC) are able to differentiate under defined culture conditions into at least bone, cartilage, adipose and muscle cells in vitro. The culture media and other in vitro conditions influence the osteogenic differentiation potency of both cell types. To increase and expand the number of osteoblasts in vitro an optimization of culture conditions is required. The aim of this study was to evaluate different culture media toward their osteogenic promoting capacity on human USSCs and MSCs in vitro. Immunohistochemical stainings against osteonectin (ON), osteopontin (OP) served as markers for an osteoblastic differentiation. Cellular morphology was analysed by light microscopy technique. We found significant differences between bone marrow and cord blood derived stem cells towards an osteoblastic differentiation. Considering the number of osteoblasts MesenCult seems to have advantages in bone marrow progenitor cells, whereas low glucose DMEM and HAMS-F12 promoted an osteoblastic differentiation in cord blood derived cells more than other tested media.     

"Stationary phase aging" of cell culture: an attempt of evaluation of growth medium "age" effect

Cell proliferation rate and 3H-thymidine labeling index of "young" (i. e. harvested in 3 days after subcultivation) cultured Chinese hamster cells (B11 dii-FAF28 line) have been determined in growth medium conditioned by the same cells for various periods of time during their growth and subsequent "stationary phase aging" (medium of different "age"). Cells were serially cultured in Eagle's medium with 10 % bovine serum. The experiment was conducted as follows. The "young" cells were seeded in Carrel's flasks (4500 cells/cm2) with fresh growth medium and placed at 37 degreesC. At definite time intervals, media from 3 randomly selected flasks were filtrated and stored in small glass flasks at 4 degreesC. The cells from all 3 flasks were collected by trypsin treatment and counted with hemocytometer. During the period of 26 day cultivation we collected a set of media of different "age" corresponding to certain points of the growth and "stationary phase aging" curve of the culture. Then, the "young" cells in fresh medium were seeded into tissue culture plates with cover slips placed into wells of the plates (26,600 cells/cm2) and grown at 37degreesC, 5 % CO2 for 2 h. At this point, the medium was replaced with media of different "age". 22 h later (i. e. on the first day after seeding) cell density was evaluated microscopically in all the wells. On the next day (i. e. in 2 days after seeding) 3H-thymidine was added to every well to final concentration 1.85 x 10(4) Bq/ml. After next 24 h (i. e. in 3 days after seeding) cell density was counted again, and the medium was removed. The cover slips were rinsed with Hank's solution and air-dried. Autoradiography was performed in standard manner by photoemulsion exposing for 5 days and subsequent developing in amidol developer. The relative number of nuclei with 10 and more "grains" was revealed microscopically. Based on the obtained results, two basic parameters were evaluated for every "age" medium: 1) cell proliferation activity index calculated as log2 (N3/N1), where N1 - cell density on the first day after seeding, and N3 - the same parameter on the third day after seeding; 2) cell labeling index calculated as percentage of cells with nuclei labeled by 3H-thymidine during incubation from 2nd to 3rd day of cultivation. These two indexes for cell growth in different "age" media appeared to be highly correlating (R = 0.85). Besides, it was found that the observed "age-related" diminishing of ability of the growth media of different "age" to stimulate proliferation of "young" cells cannot completely explain the "stationary phase aging" phenomenon (in particular, even for the "oldest" medium cell labeling index was 65 %). We conclude that the phenomenon is based on exactly intrinsic changes of cells, most likely on molecular level, though environmental effects cannot be entirely excluded. The authors are grateful to the Russian Basic Research Foundation for support (grants 03-04-49030 and 00-04-48049).     

Trypsin-like enzyme activity of the extracellular membrane vesicles of Bacteroides gingivalis W50

Trypsin-like enzyme activity in spent culture media from 3-d-old batch cultures of Bacteroides gingivalis W50 was measured by using the hydrolysis of N alpha-benzoyl-L-arginine-p-nitroanilide. The cell-free culture medium was fractionated by differential centrifugation at 10,000 g and 75,000 g, yielding two particulate fractions and a soluble supernatant fraction. About 80% of the total recoverable activity was associated with the particulate fractions, the remainder being in the supernatant. Electron microscopy of ruthenium-red/osmium stained ultrathin sections of the pellet fractions showed them to be composed of vesicular particles (extracellular vesicles), between 50 and 250 nm in diameter. Enzyme activity in all three fractions was enhanced by dithiothreitol. Gel-permeation chromatography of the soluble fraction yielded one peak of activity which contained 64 kDa and 58 kDa polypeptides. Enzyme activity from the vesicular fractions could be solubilized by sonication, giving a similar chromatographic profile to the supernatant fraction. The main peak of activity was composed of 64 kDa and 58 kDa polypeptides. In addition, there was a higher molecular mass enzyme activity peak composed of the 64 kDa and 58 kDa components along with 111 kDa, 93 kDa and 70 kDa polypeptides. We conclude that the trypsin-like enzyme of B. gingivalis is released as a soluble protein and is also associated with extracellular vesicles, in which it may exist as a soluble component and also as a protein complex.     

Antiapoptotic activity of 30 kDa lipoproteins family from fat body tissue of silkworm,Bombyx mori

The family of 30 kDa lipoproteins (LP1–5) is abundant in silkworm pupa fat body (FB) and hemolymph. One of its members, the 29 kDa protein decreased in concentration from peripheral (PP) FB tissue but was sustained in perivisceral (PV) FB tissue at the time of apoptosis. This study investigated the correlation of the 30 kDa proteins with FB apoptosis. Two protein fractions were purified, a 29 and a 30/31 kDa protein fraction, and they were used to test for activity against actinomycin D‐induced apoptosis in the FB tissues. Concentrations as little as 50 μg/mL of the 29 kDa protein fraction efficiently inhibited apoptosis. Less antiapoptotic activity was detected for the higher MW fraction; DNA fragmentation was observed in FB tissue treated with 50 μg/mL of the 30/31 kDa fraction. The viability of the cells in the 29 kDa protein‐supplemented culture was 40% higher than in the 31 kDa protein‐supplemented culture. However, the 30 kDa lipoproteins were not able to prevent scheduled FB degeneration during silkworm metamorphosis. Thus, it is hypothesized that the antiapoptotic 29 kDa protein needs to be proteolytically degraded by a regulatory mechanism to allow programmed cell death of FB tissue.     

Influence of the culture medium composition on the excreted/secreted proteases from <Emphasis Type="Italic">Streptomyces violaceoruber</Emphasis>

Streptomyces violaceoruber produces two different classes of mycelium, the substrate and the aerial mycelium. Since proteases have been associated with morphological turnover processes in other Streptomyces species, the presence of excretory/secretory proteolytic activities was investigated here in S. violaceoruber culture supernatants. Various polypeptide bands, with apparent molecular masses ranging from 40 to 180 kDa, were detected in soy trypticase broth (STB) culture media supernatants following 72 h of growth, using Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Zymograms showed the presence of five proteolytic enzymes (Spvio1–5), which migrated as bands of 167.7, 130.7, 110.7, 48.3 and 40.9 kDa, respectively. The characterization of these proteases by specific inhibitors showed that Spvio1–4 belong to the serine protease group and Spvio5 corresponds to a cysteine protease. Additionally, Spvio2 and 5 were inhibited by a mixture of EDTA and EGTA, indicating that both require divalent cations. The protease pattern obtained in STB enriched with glucose was identical to that obtained in STB. However, Spvio3 and 4 were absent when nitrogen was added to the culture medium. Cell death was fluorescently detected following 72 h of S. violaceoruber growth in STB and in STB that was enriched with glucose. On the contrary, no cell death was detected in nitrogen-enriched STB media. Additionally, the formation of the aerial mycelium was impaired in solid cultures of STB media enriched with nitrogen. These results demonstrate that the composition of the media influences the morphological turnover of the colony and the pattern of excreted/secreted proteases from S. violaceoruber, and suggest that Spvio3 and 4 are involved in the aerial mycelium formation.     

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.     

In vitro culture of Keratinocytes from human umbilical cord blood mesenchymal stem cells: the Saigonese culture

There have been many attempts to acquire and culture human keratinocytes for clinical purposes including from keratotome slices in media with fetal calf serum (FCS) or pituitary extract (PE), from skin specimens in media with feeder layers, from suction blister epidermal roofs’ in serum-free culture and from human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs) in media with skin feeder layers. Conversely this study was designed to investigate whether keratinocytes could be obtained directly from hUCB MSCs in vitro. It is widely established that mesenchymal stem cells from human umbilical cord blood have multipotent capacity and the ability to differentiate into disparate cell lineages hUCB MSCs were directly induced to differentiate into keratinocytes by using a specific medium composed of primary culture medium (PCM) and serum free medium (SFM) in a ratio 1:9 for a period of 7 days and tested by immunostain p63 and K1-K10. Cells thus cultured were positive in both tests, confirming the possibility to directly obtain keratinocytes from MSCs hUCB in vitro.     

Convergence of Cells from the Progenitor Fraction of Adult Olfactory Bulb Tissue to Remyelinating Glia in Demyelinating Spinal Cord Lesions

Background

Progenitor cells isolated from adult brain tissue are important tools for experimental studies of remyelination. Cells harvested from neurogenic regions in the adult brain such as the subependymal zone have demonstrated remyelination potential. Multipotent cells from the progenitor fraction have been isolated from the adult olfactory bulb (OB) but their potential to remyelinate has not been studied.

Methodology/Principal Findings

We used the buoyant density gradient centrifugation method to isolate the progenitor fraction and harvest self-renewing multipotent neural cells grown in monolayers from the adult green-fluorescent protein (GFP) transgenic rat OB. OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient. The progenitor fraction was isolated and these cells were plated in growth media with serum for 24 hrs. Cells were then propagated in N2 supplemented serum-free media containing b-FGF. Cells at passage 4 (P4) were introduced into a demyelinated spinal cord lesion. The GFP⁺ cells survived and integrated into the lesion, and extensive remyelination was observed in plastic sections. Immunohistochemistry revealed GFP⁺ cells in the spinal cord to be glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and neurofilament negative. The GFP⁺ cells were found among primarily P0⁺ myelin profiles, although some myelin basic protein (MBP) profiles were present. Immuno-electron microscopy for GFP revealed GFP⁺ cell bodies adjacent to and surrounding peripheral-type myelin rings.

Conclusions/Significance

We report that neural cells from the progenitor fraction of the adult rat OB grown in monolayers can be expanded for several passages in culture and that upon transplantation into a demyelinated spinal cord lesion provide extensive remyelination without ectopic neuronal differentiation.     

Fibroblasts contribute to the deposition of laminin 5 in the extracellular matrix

Laminin 5 (alpha3beta3gamma2) is specifically present in the basal lamina underneath epithelia with secretory or protective functions, where it is essential for anchoring basal epithelial cells to the underlying extracellular matrix. Laminin 5 is produced by epithelial cells as a 480-kDa precursor that is converted into forms of 440 and 400 kDa. To analyse the processing of laminin 5, we have used monolayer and co-cultures of epithelial cells and fibroblasts. The processing of the 180-kDa laminin alpha3 chain to 165 kDa in the cell culture medium, and to both 165 and 145 kDa polypeptides in the cell layer, are not modified by the presence of fibroblasts. In contrast, cleavage of the laminin gamma2 chain, occurring in the cell culture medium and in the cell layer, is enhanced by the presence of fibroblasts. Further analysis by immunofluorescence staining and laser-scanning microscopy reveals that deposited laminin 5 is present in a fibroblast-associated filamentous meshwork. Only laminin 5 containing a fully processed gamma2 chain is present in this fibroblast-associated fraction. These studies show that, although laminin 5 is a product of epithelial cells, fibroblasts contribute to its integration into the extracellular matrix architecture.     

The role of fibrin clot degradation products in the regulation of vital functions of PC-12 cells

The products of the fibrin clot hydrolysis performed by PC-12 cells modulated dose-dependently the rate of cell proliferation and favored their survival when seeded in suboptimal density. Co-incubation of PC-12 cells with fibrin degradation products enhanced cell adhesion to tissue culture plastic, as well as the number of nicotinic acetylcholine receptor alpha3 and alpha5 subunits expressed. It was demonstrated that, in fact, such a heterogeneous and comprehensive influence was a sum of effects exerted by different fibrin fragments. Low molecular weight fraction (below 30 kDa), but not a purified alphaC-domain, stimulated PC-12 cell proliferation, diminished their adhesion to plastic, increased nicotinic receptor expression and caused processes outgrowth. On the contrary, high molecular weight products, in particular D, DD and E fragments, enhanced PC-12 adhesion to plastic and, as a result, slowed cell division. Both high and low molecular weight fragments favored the survival of PC-12 cells. These results showed that fibrin degradation products support the vital functions of neuron-like cells, favor their contacts with extracellular surrounding and act as neurotrophic factors.     

The character of growth of mycobacterium tuberculosis in vitro in dependence on peripheral human blood's components

Blood plasma, cell mass, white blood cells (WBC) and erythrocytes were investigated for their relation to cord formation of M. tuberculosis, cultivated in blood according to Pryce's method. It was found that cord formation was related to blood cell and followed the zone of sedimentation of WBC. In lysed blood cord formation could be revealed on the whole glass surface, but disappeared if the lysed blood was filtered through Seitz filter - evidences which were accepted as indications that cord formation was dependent on WBC. However, destroying WBC in lysed blood by freezing and thawing or eliminating them by centrifugation did not disturb cord formation and addition of WBC to simple media did not result in cord formation. It was concluded that cord formation was called forth by lysed erythrocytes and this was confirmed through adding erythrocytes stromata to simple media, where cord formation appeared. The practical value of the technique applied and the eventual role of erythrocyte stromata's lipids in cord formation are discussed.     

Influence of Actovegin on proliferation of transplanted cell lines

The influence of the drug Actovegin on the proliferative activity and mitotic regime of cells of transplanted RK-15-1EKVM and VNK-21 clone 13/04 lines is studied. The stimulating effect of the drug on cell proliferation when added to a growth medium containing cattle blood serum of differing compositions and at different concentrations is demonstrated. It is concluded that Actovegin shows promise for use in bioengineering of cell cultures.