A novel splice acceptor mutation in the DSPP gene causing dentinogenesis imperfecta type II

The dentin sialophosphoprotein (DSPP) gene (4q21.3) encodes two major noncollagenous dentin matrix proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Defects in the human gene encoding DSPP cause inherited dentin defects, and these defects can be associated with bilateral progressive high-frequency sensorineural hearing loss. Clinically, five different patterns of inherited dentin defects are distinguished and are classified as dentinogenesis imperfecta (DGI) types I, II, and III, and dentin dysplasia types I and II. The genetic basis for this clinical heterogeneity is unknown. Among the 11 members recruited from the studied kindred, five were affected with autosomal dominant DGI type II. The mutation (g.1188CG, IVS2-3CG) lay in the third from the last nucleotide of intron 2 and changed its sequence from CAG to GAG. The mutation was correlated with the affection status and was absent in 104 unaffected individuals (208 alleles) with the same ethnic and geological background. The proband was in the primary dentition stage and presented with multiple pulp exposures. The occlusal surface of his dental enamel was generally abraded, and the dentin was heavily worn and uniformly shaded brown. The dental pulp chambers appeared originally to be within normal limits without any sign of obliteration, but over time (by age 4), the pulp chambers became partially or completely obliterated. The oldest affected member (age 59) showed mild hearing loss at high-frequency (8 kHz). Permanent dentition was severely affected in the adults, who had advanced dental attrition, premature loss of teeth, and extensive dental reconstruction.     

Differential effects of gadolinium chloride on Kupffer cells in vivo and in vitro

Gadolinium chloride (GdCl) is commonly used to study the role of Kupffer cells in liver disease in vivo. The in vitro effects of GdCl on cultured Kupffer cells are poorly characterised. The aim of this study was to characterise rat Kupffer cell TNFalpha production, phagocytic function, and ED1 and ED2 antigen expression following the administration of GdCl. For in vivo experiments, rats received 10mg/kg GdCl IV or sterile saline. Lipopolysaccharide 3mg/kg IP (LPS) was administered 4h prior to sacrifice on Days 1-3, 5 or 8 following GdCl injection. Hepatic ED1 and ED2 positive macrophage numbers and TNFalpha mRNA levels were determined. For in vitro experiments, Kupffer cells were cultured in the presence of 0-270 microM GdCl for 24h following which viability, TNFalpha protein production in response to LPS (10 ng/ml), phagocytosis, and ED1 and ED2 staining were evaluated. In vivo, the proportion of ED1 positive cells which were ED2 positive was reduced from 87 to 3% and hepatic TNFalpha mRNA levels following LPS declined by 60% over Days 1-5 after injection of GdCl (P<0.01). In vitro, phagocytosis declined with increasing concentrations of GdCl. GdCl (0-27 microM) did not effect cultured Kupffer cell viability, TNFalpha production, ED1 or ED2 staining. We conclude that GdCl significantly reduces ED2 expression by Kupffer cells in vivo. In vitro, GdCl has a dose dependent effect on phagocytosis but only effects viability and TNFalpha production at high concentrations. ED2 expression of cultured Kupffer cells is not affected by GdCl.     

Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.     

Simple aromatic compounds containing propenone moiety show considerable dual COX/5-LOX inhibitory activities

For the development of safer anti-inflammatory agents, simple aromatic compounds containing propenone moiety were prepared and evaluated for their dual COX/5-LOX inhibitory activities. Among the 17 prepared compounds, most of the compounds exhibited considerable COX/5-LOX inhibitory activities. Especially compound C(15) showed the most significant dual COX/5-LOX inhibitory activity.     

Flow cytometric application of helper adenovirus (HAd) containing GFP gene flanked by two parallel loxP sites to evaluation of 293 cre-complementing cell line and monitoring of HAd in Gutless Ad production

Gutless adenoviruses (GAds), namely, all gene-deleted adenoviruses, were developed to minimize their immune responses and toxic effects for a successful gene delivery tool in gene therapy. The Cre/loxP system has been widely used for GAd production. To produce GAd with a low amount of helper adenovirus (HAd) as byproduct, it is indispensable to use 293Cre cells expressing a high level of Cre for GAd production. In this study, we constructed the HAd containing enhanced green fluorescent protein gene flanked by two parallel loxP sites (HAd/GFP). The use of HAd/GFP with flow cytometry allows one to select 293Cre cells expressing a high level of Cre without using conventional Western blot analysis. Unlike conventional HAd titration methods such as plaque assay and end-point dilution assay, it also allows one to monitor rapidly the HAd as byproduct in earlier stages of GAd amplification. Taken together, the use of HAd/GFP with flow cytometry facilitates bioprocess development for efficient GAd production.     

Chlamydia pneumoniae--an infectious risk factor for atherosclerosis?

Cardiovascular disease, of which atherosclerosis is an important component, is the leading cause of death in the western world. Although there are well-defined risk factors for atherosclerosis, these factors do not account for all incidences of the disease. Because atherosclerotic processes are typified by chronic inflammatory responses, which are similar to those that are elicited by chronic infection, the role of infection in promoting or accelerating atherosclerosis has received renewed attention. This review focuses on the accumulating evidence that chronic infection with Chlamydia pneumoniae, a ubiquitous human respiratory pathogen, might contribute to atherosclerotic lesion progression.     

Determination of kinetic parameters related to the piasmid instability: for the recombinant fermentation under repressed condition

The plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12DeltaH1Deltatrp/pPLc23trpA1 as a "host-vector" model system. The important kinetic parameters studied were the plasmid loss rate (theta) describing the rate at which the plasrnid-harboring cells lose plas-mids and the plasmid-free cells are generated per unit time and the difference in growth rates (Delta) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid-free cells to plasmid-harboring cells (Omega) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters theta and Delta were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.