Use of moving aeration membrane bioreactor for the efficient production of tissue type plasminogen activator in serum free medium

A moving aeration-membrane (MAM) bioreactor was employed for the production of 2 μg/mL of tissue type Plasminogen Activator (tPA) in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating conditions, shear stress was as low as 0.65 dynes/cm² at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2 μg tPA/mL while maintaining a high cell density of 1.0×10⁷ viable cells/mL.     

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding–impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.     

The protein-tyrosine kinase Syk interacts with the C-terminal region of tensin2

Syk is a 72-kDa protein-tyrosine kinase that regulates signaling through multiple cell surface receptors including those for antigens, immunoglobulins and proteins of the extracellular matrix. As part of its function, Syk binds a variety of downstream effectors through interactions that are often mediated by motifs that recognize phosphotyrosines. In a search for novel Syk-interacting proteins by yeast two-hybrid analysis, we identified tensin2 as a Syk-binding protein. Syk interacts with a fragment of tensin2 located near the C-terminus that contains SH2 and PTB domains. In epithelial cells, tensin2 localizes both to focal adhesions and to large cytoplasmic puncta. It is within these punctuate structures that Syk and tensin2 are co-localized. The clustering of Syk within these structures leads to its phosphorylation on tyrosine.     

Risk Assessment of the Abandoned Jukjeon Metal Mine in South Korea Following the Korean Guidelines

Toxic metal contamination in the vicinity of Korean abandoned metal mines has been reported. A risk assessment for these metals was performed for the inhabitants in the area of the abandoned Jukjeon metal mine. Soil, groundwater, and crop samples were collected around the mine. After pretreatment of these samples, metal concentrations were measured and then a risk assessment was performed using the Korean soil-contamination risk assessment guidelines. Phytoaccumulation of metals in crops was observed in soybeans (As and Zn), red peppers (Zn), sweet potatoes (As and Zn), and cabbage (Cu), which had higher metal concentrations than soils in the area. The metal intake rate was highest for inhalation of soil. Cancer risk was highest from ingestion of As-contaminated crops. The sum of carcinogenic risks was 6.29 × 10^–3. The non-carcinogenic risk was highest for ingestion of As-contaminated crops (8.17). Most of the risks were attributable to As, Pb, and Hg contamination, therefore these three metals must be considered as the principal metals toxic to human health in the sampled area. In particular, the inhalation of metal-contaminated soil should be considered for risk assessment along with ingestion of water and crops in abandoned mine areas.     

Thioredoxin 1 as a serum marker for breast cancer and its use in combination with CEA or CA15-3 for improving the sensitivity of breast cancer diagnoses

Background

Influenza B and C are single-stranded RNA viruses that cause yearly epidemics and infections. Knowledge of RNA secondary structure generated by influenza B and C will be helpful in further understanding the role of RNA structure in the progression of influenza infection.

Findings

All available protein-coding sequences for influenza B and C were analyzed for regions with high potential for functional RNA secondary structure. On the basis of conserved RNA secondary structure with predicted high thermodynamic stability, putative structures were identified that contain splice sites in segment 8 of influenza B and segments 6 and 7 of influenza C. The sequence in segment 6 also contains three unused AUG start codon sites that are sequestered within a hairpin structure.

Conclusions

When added to previous studies on influenza A, the results suggest that influenza splicing may share common structural strategies for regulation of splicing. In particular, influenza 3′ splice sites are predicted to form secondary structures that can switch conformation to regulate splicing. Thus, these RNA structures present attractive targets for therapeutics aimed at targeting one or the other conformation.     

Telomere length and risk of melanoma,squamous cell carcinoma,and basal cell carcinoma

Background: Telomeres help maintain chromosomal structure and may influence tumorigenesis. We examined the association between telomere length and skin cancer in a clinic-based case-control study of 198 melanoma cases, 136 squamous cell carcinoma (SCC) cases, 185 basal cell carcinoma (BCC) cases, and 372 healthy controls. Methods: Cases were histologically confirmed patients treated at the Moffitt Cancer Center and University of South Florida Dermatology Clinic in Tampa, FL. Controls self-reported no history of cancer and underwent a skin cancer screening exam at study enrollment to rule out the presence of skin cancer. Quantitative real time PCR was used to measure telomere length in peripheral blood samples. Results: Melanoma patients had longer telomeres than controls (odds ratio (OR) = 3.75; 95% confidence interval (CI): 2.02–6.94 for highest versus lowest tertile) (P for trend = <0.0001). In contrast, longer telomere length was significantly inversely associated with SCC (OR = 0.01; 95% CI: 0.00–0.05 for highest versus lowest tertile) (P for trend = <0.0001) and BCC (OR = 0.10; 95% CI: 0.06–0.19 for highest versus lowest tertile) (P for trend = <0.0001). Conclusion: Telomere length may be involved in the development of skin cancer, although the effect on cancer risk differs for melanoma and non-melanoma carcinomas. Our findings suggest that long telomere length is positively associated with melanoma while inversely associated with SCC and BCC.     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

Synthesis and biological evaluation of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors

A series of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles 14a-ae, 16a, 16b, and 21a-c has been prepared and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-(4-methoxyphenyl)-3-(6-methylpyridin-2-yl)-1H-pyrazole-1-carbothioamide (14n) inhibited ALK5 phosphorylation with IC(50) value of 0.57 nM and showed 94% inhibition at 100 nM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.     

Homogeneity of recombination rate within a conserved region on BTA3 that contains QTL

The D3S20-D3S34-D3S3 region on BTA3 contains quantitative trait loci (QTL) controlling milk production traits. This region also displays extensive conservation of synteny among several species including cattle, humans, mice and sheep. In this study, we evaluated the adjacent intervals D3S20-D3S34 and D3S34-D3S3 for differences in recombination rate (theta) among bulls in order to assess the suitability of population-based estimates of theta for marker assisted selection and to explore the relationship between variation in theta and chromosome breakpoints associated with mammalian evolution. Using sperm typing, thetaD3S20-D3S34 and theta D3S34-D3S3 were estimated for six triply heterozygous bulls. Recombination frequency ranged from 6.2 to 12.5% and from 9.7 to 19.2% for the D3S20-D3S34 and D3S34-D3S3 intervals, respectively. However, significant variation in theta was not detected between bulls for either interval (D3S20-D3S34 chi(2)5 d.f.=2.59, P < 0.90; D3S34-D3S3 chi(2)5 d.f.=3.72, P < 0.75). The observed differences in theta were most readily attributed to differences in allele-specific amplification efficiencies among bulls. Our results suggest that the positions of QTL in this region can be reliably determined from population data and therefore accurate marker-assisted selection can be performed for desirable alleles without concern for variation in theta. Furthermore, when considered with results of earlier studies, these findings support a correlation between the existence of evolutionary breakpoints or chromosome rearrangements and variation in theta.