Testing transgenic regulatory elements through live mouse imaging

To overcome positional and methylation effects on transgene expression, we developed a universal cloning cassette for in vivo assessment of regulatory elements using the luciferase reporter gene and the CCCD camera. Monitoring luciferase expression pattern in live mice enables screening of large numbers of transgenic founders quickly and inexpensively. We demonstrate that in the engineered transgenic mice, the chicken beta-globin 5'HS4 insulator did not always provide the desirable expression pattern, and the Island Element, responsible for the demethylation of the surrounding DNA region, was not beneficial. Both tested liver-specific and developmentally regulated promoters exhibited the expected expression pattern in most transgenic founders.     

A new UV-B absorbing mycosporine with photo protective activity from the lichenized ascomycete Collema cristatum.

A novel photo protective mycosporine was isolated from the lichenized ascomycete Collema cristatum. Biological activity was measured in terms of protection against UV-B induced membrane destruction and pyrimidine dimer formation in cultured human keratinocytes, and prevention of UV-B induced erythema. It was found that the pure isolated compound prevented UV-B induced cell destruction in a dose-dependent manner, that the compound partially prevented pyrimidine dimer formation and completely prevented UV-B induced erythema when applied to the skin prior to irradiation.     

Activin regulation of the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1

FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.     

Prostaglandins regulate melanoma-induced cytokine production in macrophages

Tumor-secreted products can affect macrophage cytokine expression and in that way alter the immune response. Prostaglandins (PGs) are found in the tumor microenvironment and have been associated with local and regional immunosuppression. We investigated whether tumor-secreted factors could induce PG synthesis in macrophages and whether these PGs could alter macrophage production of immunoregulatory cytokines. In both murine and human models, melanoma conditioned medium (MCM) induced macrophage production of PGE(2), IL-6, and TNF-alpha. PGE(2) production increased over 24 h and was accompanied by an increase in cyclooxygenase-2 (COX-2) expression, while COX-1 expression remained unchanged. In the presence of 10 microM NS398, a selective COX-2 inhibitor, MCM-stimulated PGE(2) synthesis was almost completely suppressed, while production of IL-6 and TNF-alpha proteins and mRNA also was partially abrogated. In the murine model, 200 microM NS398 resulted in more significant inhibition of cytokine protein and mRNA production. Although MCM induced NFkappaB and NF-IL-6 activation, neither dose of NS398 altered this effect. We conclude that melanoma-secreted products stimulate COX-2 expression and PGE(2) synthesis in macrophages and that inhibition of COX-2-derived PG synthesis results in partial abrogation of macrophage cytokine production.     

Structural basis of DNA recognition by p53 tetramers

The tumor-suppressor protein p53 is among the most effective of the cell's natural defenses against cancer. In response to cellular stress, p53 binds as a tetramer to diverse DNA targets containing two decameric half-sites, thereby activating the expression of genes involved in cell-cycle arrest or apoptosis. Here we present high-resolution crystal structures of sequence-specific complexes between the core domain of human p53 and different DNA half-sites. In all structures, four p53 molecules self-assemble on two DNA half-sites to form a tetramer that is a dimer of dimers, stabilized by protein-protein and base-stacking interactions. The protein-DNA interface varies as a function of the specific base sequence in correlation with the measured binding affinities of the complexes. The new data establish a structural framework for understanding the mechanisms of specificity, affinity, and cooperativity of DNA binding by p53 and suggest a model for its regulation by regions outside the sequence-specific DNA binding domain.     

The Relation between α-Helical Conformation and Amyloidogenicity

Amyloid fibrils are stable aggregates of misfolded proteins and polypeptides that are insoluble and resistant to protease activity. Abnormal formation of amyloid fibrils in vivo may lead to neurodegenerative disorders and other systemic amyloidosis, such as Alzheimer’s, Parkinson’s, and atherosclerosis. Because of their clinical importance, amyloids are under intense scientific research. It is believed that short polypeptide segments within proteins are responsible for the transformation of correctly folded proteins into parts of larger amyloid fibrils and that this transition is modulated by environmental factors, such as pH, salt concentration, interaction with the cell membrane, and interaction with metal ions. Most studies on amyloids focus on the amyloidogenic sequences. The focus of this study is on the structure of the amyloidogenic α-helical segments because the α-helical secondary structure has been recognized to be a key player in different stages of the amyloidogenesis process. We have previously shown that the α-helical conformation may be expressed by two parameters (θ and ρ) that form orthogonal coordinates based on the Ramachandran dihedrals (φ and ψ) and provide an illuminating interpretation of the α-helical conformation. By performing statistical analysis on α-helical conformations found in the Protein Data Bank, an apparent relation between α-helical conformation, as expressed by θ and ρ, and amyloidogenicity is revealed. Remarkably, random amino acid sequences, whose helical structures were obtained from the most probable dihedral angles, revealed the same dependency of amyloidogenicity, suggesting the importance of α-helical structure as opposed to sequence.     

Mean-field model of immobilized enzymes embedded in a grafted polymer layer

Two-dimensional mean-field lattice theory is used to model immobilization and stabilization of an enzyme on a hydrophobic surface using grafted polymers. Although the enzyme affords biofunctionality, the grafted polymers stabilize the enzyme and impart biocompatibility. The protein is modeled as a compact hydrophobic-polar polymer, designed to have a specific bulk conformation reproducing the catalytic cleft of natural enzymes. Three scenarios are modeled that have medical or industrial importance: 1), It is shown that short hydrophilic grafted polymers, such as polyethylene glycol, which are often used to provide biocompatibility, can also serve to protect a surface-immobilized enzyme from adsorption and denaturation on a hydrophobic surface. 2), Screening of the enzyme from the surface and nonspecific interactions with biomaterial in bulk solution requires a grafted layer composed of short hydrophilic polymers and long triblock copolymers. 3), Hydrophilic polymers grafted on a hydrophobic surface in contact with an organic solvent form a dense hydrophilic nanoenvironment near the surface that effectively shields and stabilizes the enzyme against both surface and solvent.     

Inhibition of lipid synthesis by beta beta'-tetramethyl-substituted, C14-C22, alpha, omega-dicarboxylic acids in the rat in vivo

beta beta'-Methyl-substituted alpha, omega-dicarboxylic acids (MEDICA) of C14-C18 chain length were found to inhibit liver lipid synthesis in the rat in vivo. Maximum inhibition was observed with MEDICA 16 amounting to a 50% decrease in fatty acid and cholesterol biosynthesis in the presence of 0.07 and 0.015% (w/w) of the drug in the diet, respectively. Inhibition of lipid biosynthesis by MEDICA 16 involved a reduction in cytosolic acetyl-CoA content, while the carbon flux from glucose to glycogen, protein, and carbon dioxide remained unaffected. Inhibition of lipogenesis by MEDICA 16 resulted in a 50% decrease in liver and carcass (but not brain) neutral lipid ester content at 0.25% (w/w) of the drug in the diet, as well as in a dose-dependent hypotriglyceridemic effect, with an up to 3-fold reduction in serum triacylglycerols. Inhibition of cholesterogenesis by MEDICA 16 resulted in a hypocholesterolemic effect, with 60 and 45% reductions in (very low density + low density lipoprotein) cholesterol and high density lipoprotein cholesterol, respectively.