Overexpression in Escherichia coli and purification of human fibroblast growth factor (FGF-2)

Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1–155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.     

Formation of the population composition of a stationary cell culture and the participation of nonproliferating cells with a doubled DNA content in regulating its density

The conditions of the cultivation of chick embryo diploid cells were alternated (prolonged maintenance with or without medium replacement, with or without consequent cell replating in fresh medium). In different times of culture growth, the cell DNA content was assessed by cytophotometry; the percentage of non-labeled mitoses after incubating the cells with 3H-thymidine and colcemide, as well as the cell density were determined. The phenomenon, detected earlier, of the accumulation of cells containing 4c DNA during the transition of the culture from logarithmic into the stationary phase of growth, was confirmed. These cells were shown to differ in their ability to survive in conditions of stationary culture and by proliferative potential. The fraction of cells reversibly arrested in G2-period was described, by which fraction the change of the cell population size is occurring after the decrease of its proliferation rate. The transitional stage is distinguished at the beginning of the stationary phase of culture growth. During this stage the stabilization of structural and numerical composition of the population is taking place.     

Luminescent properties of NADPH: adrenodoxin reductase

The fluorescent and phosphorescent properties of NADPH-adrenodoxin reductase were investigated. It was shown that the fluorescence of protein tryptophanyls was quenched completely by acrylamide and partially by ionic quenchers (I- and Cs+). A removal of the prosthetic group from the protein causes insignificant changes in fluorescent properties of the enzyme. The denaturation of the enzyme by urea was accompanied by growth of quenching parameters. Indeed, some differences were observed in the quenching of flavin fluorescence by ionic quenchers (I- and Cs+). NADPH appeared to be an efficient quencher of NADPH-adrenodoxin reductase tryptophan fluorescence. Using F?rster's equations for non-radiative energy transfer, the distance between NADPH-binding site and tryptophanyls was evaluated to 35-40 A.     

Protein SH-group concentration and RNA synthesis during the process of induction of proliferation in the stationary phase of cell culture growth]

The dynamics of intracellular protein SH-group (PSH) content was studied cytochemically in the course of stimulation of cell proliferation in stationary cultures of an established Chinese hamster cell line and of human diploid embryo fibroblasts. The results were compared with the pattern of RNA synthesis during the prereplicative period. In Chinese hamster cells immediately after medium changing in stationary cultures there is an augmentation of PSH content in parallel withe the increase in RNA synthesis rate. Later on, the rate of RNA synthesis and PSH content are seen decreasing followed by a new increase in the rate of RNA synthesis correlated with the second rise in PSH content. In stationary cultures of human diploid fibroblasts, there is also an increase in the rate of RNA synthesis and in the content of SH after medium changing, but the second wave of RNA synthesis and the second rise in PSH content are not pronounced. The variation in PSH content reflects the shift in the cell metabolism during the prereplicative period and is not attributed to changes in cell protein content.     

Stimulation of multiplication of chick embryo cultured cells after an increase in density of the cellular population

The secondary culture of chick embryo cells in 2-3 days after seeding was super-inoculated with homologous cells. Suspended cells adhere and spread on the cell layer whereby the culture density increases quickly. After adhesion of exogenous cells to the layer the stimulation of cell proliferation takes place. This activation is not connected with methodical manipulations or with the influence of conditioned medium factors. The results suggest that the increase in cell number itself does not arrest cell multiplication. It is proposed that the known phenomenon of blocking cell proliferation in dense cultures cannot be attributed only to effects of high cell density.     

Spatiotemporal patterns of macrophage migration inhibitory factor (Mif) expression in the mouse placenta

Background  

Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy.     

Occurrence of a thermoacidophilic cell-bound exo-pectinase inAlicyclobacillus acidocaldarius

Alicyclobacillus acidocaldarius was able to degrade pectin under thermoacidophilic conditions of high temperature and acidity. Both extracellular and cell-bound pectolytic activities were found (28 and 72% of total activity, respectively). WhenA. acidocaldarius was subjected to lysozyme or sonication, more than 50% of the activity was found to be bound with the cell debris. The cell-bound enzyme presented principally exopectolytic activity. SDS-PAGE and zymogram showed that the estimated molar mass of the crude enzyme was 52 kDa. pH optimum was between 1.5 and 2.0 and the enzyme was thermostable at 70°C for 1 h at pH 2.0.     

Comparative characteristics of cyclodextrin glycosyltransferases from various groups of microorganisms]

Cyclodextrin glycosyltransferases (CGT-ase, 1.4-alpha-glucanotransferase, cyclizing, EC 2.4.1.19) produced by some thermophilic, alkalophilic and mesophilic bacterial strains, were isolated and characterized. It was shown that thermophilic and mesophilic CGT-ases represent a mixture of alpha-, beta- and gamma-cyclodextrins (CD), alpha-cyclodextrin being the predominant component. Alkalophilic enzymes produce only beta-CD and are able to produce CD not only from starch but also from maltose, melibiose, maltotriose, amylose and amylopectin. The optimal conditions for the catalytic activity of the enzymes were determined. It was found that calcium, magnesium and zinc ions have a beneficial effect on the specific activity of these enzymes. The amino acid composition of the enzymes was studied.