Human embryonic kidney cells (HEK-293 cells): characterization and dose-response relationship for modulated release of nerve growth factor for nerve regeneration

The development of engineered constructs to bridge nerve gaps may hold the key to improved functional outcomes in the repair of injured peripheral nerves. These constructs must be rendered bioactive by providing the growth factors required for successful peripheral nerve regeneration. Previous studies demonstrated that harvested human and rat dermal fibroblasts could be genetically engineered to release nerve growth factor (NGF) both in vitro and in vivo. The use of fibroblasts, however, has the potential to cause scarring, and the expression of NGF from those cells was transient. To overcome these potential difficulties, human embryonic kidney cells were modified for use with the ecdysone-inducible mammalian expression system. These cells (hNGF-EcR-293) have been engineered and regulated to secrete human NGF in response to the ecdysone analogue ponasterone A. HEK-293 cells were transfected with human NGF cDNA with the ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, Calif.). Stable clones were then selected. Ponasterone A, an analogue of ecdysone, was used as the inducing agent. The secretion of NGF into the medium was analyzed with two different methods. After 24 hours of exposure to the inducing agent, cell medium was transferred to PC-12 cells seeded in 12-well plates, for determination of whether the secreted NGF was bioactive. Medium from untreated or ponasterone A-treated hNGF-EcR-293 cells was deemed bioactive on the basis of its ability to induce PC-12 cell differentiation. The concentrations of secreted NGF were also quantified with an enzyme-linked immunosorbent assay, in triplicate. NGF production was measured in successive samples of the same medium during a 9-day period, with maximal release of 9.05 +/- 2.6 ng/ml at day 9. Maximal NGF production was 8.46 +/- 2.1 pg/10(3) cells at day 9. These levels were statistically significantly different from levels in noninduced samples (p 相似文献   

Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (alpha, beta, and gamma), and also their two-domain combinations, viz. alphabeta and betagamma in Escherichia coli. After purification, alpha and beta showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs alphabeta and betagamma showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK.HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK.HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels.     

Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact

Sodium channels isolated from mammalian brain are composed of alpha, beta1, and beta2 subunits. The auxiliary beta subunits do not form the ion conducting pore, yet play important roles in channel modulation and plasma membrane expression. beta1 and beta2 are transmembrane proteins with one extracellular V-set immunoglobulin (Ig) protein domain. It has been shown recently that beta1 and beta2 interact with the extracellular matrix proteins tenascin-C and tenascin-R. In the present study we show that rat brain beta1 and beta2, but not alphaIIA, subunits interact in a trans-homophilic fashion, resulting in recruitment of the cytoskeletal protein ankyrin to sites of cell-cell contact in transfected Drosophila S2 cells. Whereas alphaIIA subunits expressed alone do not cause cellular aggregation, beta subunits co-expressed with alphaIIA retain the ability to adhere and recruit ankyrin. Truncated beta subunits lacking cytoplasmic domains interact homophilically to produce cell aggregation but do not recruit ankyrin. Thus, the cytoplasmic domains of beta1 and beta2 are required for cytoskeletal interactions. It is hypothesized that sodium channel beta subunits serve as a critical communication link between the extracellular and intracellular environments of the neuron and may play a role in sodium channel placement at nodes of Ranvier.     

Recombinant BCG approach for development of vaccines: cloning and expression of immunodominant antigens of M. tuberculosis

In spite of major advances in our understanding of the biology and immunology of tuberculosis, the incidence of the disease has not reduced in most parts of the world. In an attempt to improve the protective efficacy of Mycobacterium bovis bacille Calmette-Guérin (BCG), we have developed a generic vector system, pSD5, for expression of genes at varying levels in mycobacteria. In this study, we have cloned and overexpressed three immunodominant secretory antigens of M. tuberculosis, 85A, 85B and 85C, belonging to the antigen 85 complex. All the genes were cloned under the control of a battery of mycobacterial promoters of varying strength. The expression was analysed in the fast-growing strain M. smegmatis and the slow-growing vaccine strain M. bovis BCG. The recombinant BCG constructs were able to express the antigens at high levels and the majority of the expressed antigens was secreted into the medium. These results show that by using this strategy the recombinant BCG approach can be successfully used for the development of candidate vaccines against infections associated with mycobacteria as well as other pathogens.     

The dynamics of natural selection and vicariance in the Dominican anole: patterns of within-island molecular and morphological divergence

The larger islands of the Lesser Antilles are ecologically and geologically complex and are inhabited by single, but morphologically variable, Anolis species. Although earlier work has indicated that a large part of the morphological variation in Anolis oculatus from Dominica can be attributed to selection, a history of recurrent volcanic activity over the last few million years suggests that vicariance may have also played a significant role. We report a study of variation in the cytochrome b gene of mitochondrial DNA across the island to address this issue. We uncovered a very high degree of polymorphism, with an overall gene diversity of 0.97 and a nucleotide diversity of 0.04. Sequences, on average, differ by 3.82% and the maximum pairwise divergence (corrected for multiple hits) is 9.29%. Most haplotypes are restricted to single localities (a pattern not changed by increasing the sample size). Phylogenetic analysis revealed the presence of two distinct lineages on the island with strong phylogeographic structure. One of these is geographically restricted to a relatively small part of the central Caribbean coast. Sublineages were also discernible within the other more widely distributed lineage, but resolution within and support for these sublineages was poor. The phylogeographic pattern is not congruent with generalized body shape and scalation, but is significantly correlated with color pattern. Even when correcting for this lineage effect with partial Matrix correspondence tests, the relationship between color pattern and vegetation is reaffirmed, suggesting that although both vicariance and selection have played a role in the morphological differentiation of this species, selection for current environmental conditions has been more important. We discuss the causes of the phylogeographic structure in light of the volcanic history of the island and highlight the exceptional instance of congruence between all morphological character systems and lineage boundaries, which occurs at the transition between the northern and southern Caribbean ecotypes.     

Stimulation of the chemiluminescence of human polymorphonuclear leukocytes in various stages of the intraerythrocyte development of Plasmodium falciparum

The synchronized cultures of Plasmodium falciparum were used to stimulate in vitro the chemiluminescence of human polymorphonuclear leukocytes in the presence of immune serum. The schizonts were concentrated by Percoll gradient centrifugation method (density 1.085 and osmolarity 285 mOsmol), and placed in culture, treated 6 hours later by sorbitol. Under incubation at constant temperature and pressure, the rate of synchronization reached 85% for schizonts during 5 replicative cycles. Every asexual stages of Plasmodium falciparum were used separately to stimulate polymorphonuclear leukocytes: merozoites were the most effective, followed by schizonts, trophozoites, and lastly supernatants of cultures containing degradation products of parasites.     

Purification and characterization of Streptomyces griseus catechol O-methyltransferase

A soluble (100,000 x g supernatant) methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to catechols was present in cell extracts of Streptomyces griseus. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7. 5 and 35 degrees C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the K(m) for 6,7-dihydroxycoumarin was 500 +/- 21.5 microM, and that for S-adenosyl-L-methionine was 600 +/- 32.5 microM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of S-adenosyl-L-methionine, with a K(i) of 224 +/- 20.6 microM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg(2+), p-chloromercuribenzoic acid, and N-ethylmaleimide.     

Toxic effect of dimethoate on the crosslinking of gingival and uteral collagen in female albino rats fed with normal and high protein diets

The effect of dimethoate on gingival and uteral collagen crosslinking was studied in normal and high protein diets fed female albino rats. The gingival and uteral samples were collected from all the groups of rats and percent reversibility of neutral salt soluble collagen gel and the solubility of insoluble collagen in KCNS or urea were determined. The analysis of gingival and uteral samples showed alpha 1 and alpha 2 sub units of neutral salt soluble collagen appreciably increased beta chains and aldehyde content significantly decreased in dimethoate treated animals compared to controls. The results indicate that due to the effect of dimethoate, the crosslinking and maturation of collagen are impaired.