A colorimetric-based amplification system for proteinases including MMP2 and ADAM8

We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.     

Sensitivity of a two-dimensional biomorphoelastic model for post-burn contraction

We consider a two-dimensional biomorphoelastic model describing post-burn scar contraction. This model describes skin displacement and the development of the effective Eulerian strain in the tissue. Besides these mechanical components, signaling molecules, fibroblasts, myofibroblasts, and collagen also play a significant role in the model. We perform a sensitivity analysis for the independent parameters of the model and focus on the effects on features of the relative surface area and the total strain energy density. We conclude that the most sensitive parameters are the Poisson’s ratio, the equilibrium collagen concentration, the contraction inhibitor constant, and the myofibroblast apoptosis rate. Next to these insights, we perform a sensitivity analysis where the proliferation rates of fibroblasts and myofibroblasts are not the same. The impact of this model adaptation is significant.

     

External calcium concentration and fertilization in the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz) (Echinodermata)

The role of external calcium ions in the fertilization of the sea urchin, Strongylocentrotusfranciscansus (A. Agassiz), was studied. When eggs were inseminated with jelly-treated sperm in artificial sea waters containing various concentrations of calcium, the percentage of fertilization decreased with decreasing concentrations of calcium. A small amount of external calcium ions was also found to be essential for fertilization by spermatozoa with reacted acrosomes. The binding capacity of jelly-treated spermatozoa to eggs was, however, not adversely affected by a calcium deficiency. These results suggest that calcium ions are required not only for the initiation of the acrosome reaction, but also for successful levels of fertilization following sperm-binding to eggs even after the acrosome reaction has occurred.     

Effect of Testosterone on Neuronal Morphology and Neuritic Growth of Fetal Lamb Hypothalamus-Preoptic Area and Cerebral Cortex in Primary Culture

Testosterone plays an essential role in sexual differentiation of the male sheep brain. The ovine sexually dimorphic nucleus (oSDN), is 2 to 3 times larger in males than in females, and this sex difference is under the control of testosterone. The effect of testosterone on oSDN volume may result from enhanced expansion of soma areas and/or dendritic fields. To test this hypothesis, cells derived from the hypothalamus-preoptic area (HPOA) and cerebral cortex (CTX) of lamb fetuses were grown in primary culture to examine the direct morphological effects of testosterone on these cellular components. We found that within two days of plating, neurons derived from both the HPOA and CTX extend neuritic processes and express androgen receptors and aromatase immunoreactivity. Both treated and control neurites continue to grow and branch with increasing time in culture. Treatment with testosterone (10 nM) for 3 days significantly (P < 0.05) increased both total neurite outgrowth (35%) and soma size (8%) in the HPOA and outgrowth (21%) and number of branch points (33%) in the CTX. These findings indicate that testosterone-induced somal enlargement and neurite outgrowth in fetal lamb neurons may contribute to the development of a fully masculine sheep brain.     

Lesion bypass of N2-ethylguanine by human DNA polymerase iota

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase iota was measured and structures of the DNA polymerase iota-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase iota opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase iota incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase iota opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase iota extends from the N2-ethyl-Gua:Cyt 3' terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase iota catalyzed reaction is well suited for N(2)-ethyl-Gua bypass. The structure of DNA polymerase iota with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase iota complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase iota polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase iota efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.