Age-dependent changes in Fibroblast growth factor 2 (FGF-2) expression in mouse cerebellar neurons

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor that regulates many neuronal functions and survival. We have characterised FGF-2 expression immunohistochemically in the cerebellum of young (4 months) and old (22 months) mice. About half of the population of the granule cells (GC), and all Purkinje cells (PC) expressed FGF-2 in all folia of the cerebellum at both ages. FGF-2 showed differential intracellular localization: predominantly localised to the nuclei of GC and present mainly in the cytosol of PC. There was a statistically significant (P = 0.0028) reduction in the number of FGF-2-positive GC in the cerebella of old (41.3+/-0.91%) compared to young (48.5+/-1.67%) mice, whereas no statistically significant age-dependent difference occurred in the number of FGF-2 positive PC. These results indicate a possible role of FGF-2 in cerebellar ageing.     

Enhanced fibronectin-mediated cell adhesion of human osteoblast by fibroblast growth factor, FGF-2

Using specific recombinant human fibronectin peptide (hFNIII9-10) that contains the binding site for integrin, we found that the fibroblast growth factor, FGF-2, enhances fibronectin-mediated adhesion in human osteoblast-like MG63 cells. The mechanism of the synergistic adhesion was due to the activation of extracellular-regulated kinase (ERK)-type MAPK upon interaction of integrin to hFNIII9-10 and its downstream activation of signaling pathways.     

Production of poly(3-hydroxybutyrate) from whey by fed-batch culture of recombinant Escherichia coli in a pilot-scale fermenter

Production of poly(3-hydroxybutyrate) [P(3HB)] from wheyby fed-batch culture of recombinant Escherichia coli CGSC 4401 harboring a plasmid containing the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes was examined in a 30 l fermenter supplying air only. With lactose below 2 g l^–1, cells grew to 12 g dry cell l^–1 with 9% (w/w) P(3HB) content. Accumulation of P(3HB) could be triggered by increasing lactose to 20 g l^–1. By employing this strategy, 51 g dry cell l^–1 was obtained with a 70% (w/w) P(3HB) content after 26 h. The productivity was 1.35 g P(3HB) l^–1 h^–1. The same fermentation strategy was used in a 300 l fermenter, and 30 g dry cell l^–1 with 67% (w/w) P(3HB) content was obtained in 20 h.     

Immunolocalization of fibroblast growth factor-2 (FGF-2) in the developing root and supporting structures of the murine tooth

Summary Epithelio–mesenchymal interactions are active during the development of the root of the tooth and are regulated by a variety of growth factors, such as fibroblast growth factors. FGF-2, 3, 4, and 8 have all been shown to play a role in the development of the crown of the tooth, but less is known about the factors that govern root formation, particularly FGF-2. The aim of this study was thus to elucidate the spatial and temporal expression of FGF-2 in the root of the developing tooth, as this growth factor is believed to be a mediator of epithelio–mesenchymal interactions. Parasagittal sections of the maxillary and mandibular arches of post-natal mice were utilized and the roots of the molar teeth were studied. Immunocytochemistry utilizing an antibody to FGF-2 was performed on sections of teeth at various stages of development. Intense immunostaining for FGF-2 was observed in differentiating odontoblasts at the apical end of the tooth and in the furcation zone of the developing root at all the stages examined. FGF-2 localization was also observed in cementoblasts on post-natal days 16, 20 and 24. The pattern of localization of FGF-2 in the developing root suggests that this growth factor may participate in the signaling network associated with root development.     

Expression of lexA targeted ribozyme in Escherichia coli BL-21 (DE3) cells

Coding sequences for a hammerhead ribozyme designed to cleave lexA mRNA in a targeted manner was cloned under phage T7 promoter and expressed in E. coli strain BL-21 (DE3) expressing T7 RNA polymerase under the control of IPTG-inducible lac UV-5 promoter. Ribozyme expression in vivo was demonstrated by RNase protection assay. Also, total RNA extracted from these transformed cells following induction by IPTG, displays site-specific cleavage of labeled lexA RNA in an In vitro reaction. The result demonstrates the active ribozyme in extracts of cell transformed with a recombinant cassette and goes beyond the earlier demonstration of the stability of In vitro synthesized ribozyme in cell extracts. The observed rise in lexA mRNA rules out any role for protease activity or resulting fragments of lexA protein in de-repression of RNA. (Mol Cell Biochem 271: 197–203, 2005)     

Overexpression of an alkaline lipase gene from Proteus vulgaris K80 in Escherichia coli BL21/pKLE

Over-expression of Proteus vulgaris K80 lipase gene in Escherichia coli BL21 (DE3)/pKLE was achieved, with the enzyme being produced in an as active soluble form, using the T7 RNA polymerase system in a modified M9 salt or M9ZB medium. d-Lactose (55 mM) was used to induce gene expression and gave twice the lipase activity achieved with 0.4 mM IPTG. The expression of the lipase gene depended on the feeding rate of glucose being optimal at 12 g l^–1 h^–1.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

Fedbatch design for periplasmic product retention in Escherichia coli

The feed profile of glucose during fedbatch cultivation could be used to influence the retention of the periplasmic product ZZ-cutinase. An increased feed rate led to a higher production rate but also to an increased specific leakage, which reduced the periplasmic retention. Three growth rates: 0.3, 0.2 and 0.1 h(-1) where studied and resulted in 20, 9 and 6%, respectively, of the total ZZ-cutinase accumulating in the medium. It was also shown that leakage during fedbatch production of a Fab fragment was also influenced by the feed rate in a similar manner to ZZ-cutinase. If intracellular product accumulation is desired the advantage of a high productivity, resulting from a high substrate feed rate, is diminished because of a reduced product retention. Biochemical analysis revealed that the growth rate, resulting from a glucose limited feed, influenced the outer membrane protein compositions with respect to OmpF and LamB, whilst OmpA was largely unaffected. As the feed rate increased the amount of total outer membrane protein decreased. When ZZ-cutinase was produced there were further reductions in outer membrane protein accumulation, by 82, 100 and 22% for OmpF, LamB and OmpA, respectively, and the total reduction was almost 60% with a high product formation rate. We suggest that the reduced titre of the outer membrane proteins, OmpF and LamB, may have contributed to a reduced ability for the cell to retain recombinant protein secreted to the periplasm.     

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.     

Site-directed mutagenesis of bovine FGF-2 cDNA allows the production of the human-form of FGF-2 in Escherichia coli

Human fibroblast growth factor-2 can be used to induce angiogenesis in ischemias and wound healing. Site-directed mutagenesis of bovine FGF-2 cDNA was performed in order to produce the human-form of FGF-2 in E. coli. The mitogenic, angiogenic and neurotrophic activities of the recovered protein were analysed by [³H]thymidine uptake to DNA of cultured fibroblasts, rabbit ear dermal ulcers wound healing and neuronal differentiation of PC12 cells.     

Expression, purification, and bioactivity of human tumstatin from Escherichia coli

Tumstatin is a M(r) 28,000 C-terminal NC1 fragment of type alpha3 (IV) collagen that inhibits pathological angiogenesis and suppresses proliferation of endothelial cells and growth of tumors. We report here high cytoplasmic expression of recombinant human tumstatin in Escherichia coli and its purification, in vitro refolding, and inhibitory activity analysis. Human tumstatin was expressed in the bacterial cytoplasm as an insoluble N-terminal polyhistidine tagged protein, which accounted for more than 30% of total bacterial protein in BL21 (DE3) cells. After extraction and solubilization in guanidine-HCl, recombinant protein was purified to homogeneity using a simple one-step Ni(2+)-chelate affinity chromatography and then refolded by dialysis against acidic pH buffers with gradually decreasing concentrations of denaturant. The renatured recombinant tumstatin could specifically inhibit endothelial cell proliferation in a dose-dependent manner, and suppress bFGF-induced angiogenesis in chick embryo chorioallantoic membrane and tumor growth in mouse B16 melanoma xenograft models.     

Expression of human transforming growth factor β1 in E. coli (I) --Direct expression in cytoplasm

PCR technique is used to amplify the mature peptide gene of human transforming growth factor pl (hTGFβ1); the gene is verified by full-length sequence analysis. In DHSa/pBV220 expression system, hTGFβ1 attains expression in the cytoplasm ofE. coli up to 16%. The recombinant protein is proved to be the monomer of hTGFPl by N-terminal amino acids analysis and immunoblotting. After refolding of the monomer proteinin vitm in glutathione system or CHPAS/DMSO system, the dimeric protein accumulates to 30% in the refolding mixture. The recombinant protein is purified to homogeneity on silver staining, and is shown to have strong biological activity from MTT bioassay on MvlLu cells. Project supported by the National Natural Science Foundation of China (Grant No. 39580015)     

Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily that has been used for bone grafting. We were interested in exploring the functions of BMP-2 in other disease areas and focused on expressing and purifying active BMP-2 proteins. We have developed a new approach which involves using FoldIt refolding buffer to refold BMP-2 followed by a heparin affinity column to separate correctly folded dimer from monomer. A high yield of 29.4 mg BMP-2 dimer per gram cell wet weight was achieved. The purified BMP-2 dimer was shown to possess the same level of activity as BMP-2 from CHO cells as tested by the induction of alkaline phosphatase activity in C2C12 cells. This approach has potential application in refolding and purifying other homodimeric proteins.     

Study of the thermokinetic properties of copper(II) on Escherichia coli growth

By using an LKB2277 Bioactivity Monitor, stop-flow mode, the power-time curves of Escherichia coli at 37°C affected by Cu(II) were determined. Some parameters, such as growth rate constants k, inhibitory ratio I, the heat output Q_log in the log phase, and the generation times G were obtained. According to these parameters, we found that a low concentration of Cu(II) (0–20 μg/mL) had an promoting action on the growth of E. coli, but a high concentration of Cu(II) (40–100 μg/mL) had an inhibitory action. The toxicity of Cu(II) can also be expressed as the half-inhibitory concentration IC₅₀; the value is 69.7 μg/mL. The assay is quantitative, inexpensive, and versatile.     

Structural and immunological characterization of recombinant ovomucoid expressed in Escherichia coli

The expression of recombinant allergens is becoming new insights of an important diagnosis and the therapy of allergies as well as molecular approaches to immunological and structural studies of allergens. Ovomucoid is a major food allergens in the hen's egg white which causes immediate food-hypersensitivity reactions mainly in children. A gene coding for the cDNA representing an entire ovomucoid molecule has been cloned in Escherichia coli under the control of T5 promoter fused with six-Histidine tag at the amino terminal end. Upon induction, the E. coli cells, harbouring this construct, expressed the recombinant protein as a soluble fraction and the recombinant ovomucoid protein was purified to electrophoeretic homogeneity using Ni²⁺ nitrilotriacetic acid agarose affinity chromatography. Immunoblot analysis showed that human IgE and IgG binding activities of the recombinant ovomucoid was identical to that of native analogue. The antigenicity and allergenicity of recombinant ovomucoid were almost same as that of native form when tested with an ELISA using six individual patient's serum. CD spectra indicated that that the recombinant ovomucoid has more -helix and less -structure than native form. These results show that the recombinant ovomucoid constructed in this study could be used for further studies on the immunological and structural studies of ovomucoid.     

Antibacterial effect of honey on the in vitro and in vivo growth of Escherichia coli

The study investigated the antibacterial effect of honey against pathogenic Escherichia coli. Honey showed inhibitory activity against the growth of E. coli (ATCC 25922) in agar plate assay. In liquid culture (48 h, 37 °C) the growth rate of bacterial cells decreased in the presence of honey (9.6 × 10⁵ c.f.u./ml) compared with sucrose (2.87 × 10⁸ c.f.u./ml). Rats fed with honey and orally inoculated with E. coli excreted significantly (P < 0.05) less bacterial cells in faeces compared to controls. Animals acclimatized to feeding of honey prior to E. coli inoculation showed a significant decrease in excreted bacterial load compared with the group provided with honey after bacterial inoculation. Consumption of honey also enhanced the concentration of short chain fatty acids in the intestine of rats (83 mM) compared with the control group (44.5 mM). The results show that honey possessed significant antibacterial activity against E. coli under in vitro and in vivo conditions, and indicate the potential benefit of consumption of honey regularly on the microbiological constitution of animals feeding on it.     

Fed-batch cultivation ofEscherichia coli YK537 (pAET-8) for production ofphoA promoter-controlled human epidermal growth factor

Secretion of the expressed heterologous proteins can reduce the stress to the host cells and is beneficial to their recovery and purification. In this study, fed-batch cultures ofEscherichia coliYK537 (pAET-8) were conducted in a 5-L fermentor for the secretory production of human epidermal growth factor (hEGF) whose expression, was under the control of alkaline phosphatase promoter. The effects of feeding of glucose and complex nitrogen sources on hEGF production were investigated. When the fed-batch culture was conducted in a chemically defined medium, the cell density was 9.68 g/L and the secreted hEGF was 44.7 mg/L in a period of 60 h. When a complex medium was used and glucose was added in pH-stat mode, the secreted hEGF was improved to 345 mg/L. When the culture was fed with glucose at a constant specific rate of 0.25 gg⁻¹h⁻¹, hEGF reached 514 mg/L. The effects of adding a solution containing yeast extract and tryptone were further studied. Different rate of the nitrogen source feeding resulted in different levels of phosphate and acetic acid formation, thus affected hEGF expression. At the optimal feeding rate, hEGF production achieved 686 mg/L.     

Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris

Tryptophan hydroxylase (TPH) from several mammalian species has previously been cloned and expressed in bacteria. However, due to the instability of wild type TPH, most successful attempts have been limited to the truncated forms of this enzyme. We have expressed full-length human TPH in large amounts in Escherichia coli and Pichia pastoris and purified the enzyme using new purification protocols. When expressed as a fusion protein in E. coli, the maltose-binding protein-TPH (MBP-TPH) fusion protein was more soluble than native TPH and the other fusion proteins and had a 3-fold higher specific activity than the His-Patch-thioredoxin-TPH and 6xHis-TPH fusion proteins. The purified MBP-TPH had a V(max) of 296 nmol/min/mg and a K(m) for L-tryptophan of 7.5+/-0.7 microM, compared to 18+/-5 microM for the partially purified enzyme from P. pastoris. To overcome the unfavorable properties of TPH, the stabilizing effect of different agents was investigated. Both tryptophan and glycerol had a stabilizing effect, whereas dithiothreitol, (6R)-5,6,7,8,-tetrahydrobiopterin, and Fe(2+) inactivated the enzyme. Irrespective of expression conditions, both native TPH expressed in bacteria or yeast, or TPH fusion proteins expressed in bacteria exhibited a strong tendency to aggregate and precipitate during purification, indicating that this is an intrinsic property of this enzyme. This supports previous observations that the enzyme in vivo may be stabilized by additional interactions.