Determination of the pH dependence,substrate specificity,and turnovers of alternative substrates for human ornithine aminotransferase

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.     

Genomic Stability of Lyophilized Sheep Somatic Cells before and after Nuclear Transfer

The unprecedented decline of biodiversity worldwide is urging scientists to collect and store biological material from seriously threatened animals, including large mammals. Lyophilization is being explored as a low-cost system for storage in bio-banks of cells that might be used to expand or restore endangered or extinct species through the procedure of Somatic Cell Nuclear Transfer (SCNT). Here we report that the genome is intact in about 60% of lyophylized sheep lymphocytes, whereas DNA damage occurs randomly in the remaining 40%. Remarkably, lyophilized nuclei injected into enucleated oocytes are repaired by a robust DNA repairing activity of the oocytes, and show normal developmental competence. Cloned embryos derived from lyophylized cells exhibited chromosome and cellular composition comparable to those of embryos derived from fresh donor cells. These findings support the feasibility of lyophylization as a storage procedure of mammalian cells to be used for SCNT.     

Differential Gene Expression in Thrombomodulin (TM; CD141)+ and TM? Dendritic Cell Subsets

Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin⁺ dendritic cells are tolerogenic while thrombomodulin⁻ dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived dendritic cells were treated with soluble thrombomodulin and expression of surface markers was determined. Treatment with thrombomodulin reduces the expression of maturation markers and increases the expression of TM on the DC surface. Thrombomodulin treated and control dendritic cells were sorted into thrombomodulin⁺ and thrombomodulin⁻ dendritic cells before their mRNA was analyzed by microarray. mRNAs encoding pro-inflammatory genes and dendritic cells maturation markers were reduced while expression of cell cycle genes were increased in thrombomodulin-treated and thrombomodulin⁺ dendritic cells compared to control dendritic cells and thrombomodulin⁻ dendritic cells. Thrombomodulin-treated and thrombomodulin⁺ dendritic cells had higher expression of 15-lipoxygenase suggesting increased synthesis of lipoxins. Thrombomodulin⁺ dendritic cells produced more lipoxins than thrombomodulin⁻ dendritic cells, as measured by ELISA, confirming that this pathway was upregulated. There was more phosphorylation of several cell cycle kinases in thrombomodulin⁺ dendritic cells while phosphorylation of kinases involved with pro-inflammatory cytokine signaling was reduced. Cultures of thrombomodulin⁺ dendritic cells contained more cells actively dividing than those of thrombomodulin⁻ dendritic cells. Production of IL-10 is increased in thrombomodulin⁺ dendritic cells. Antagonism of IL-10 with a neutralizing antibody inhibited the effects of thrombomodulin treatment of dendritic cells suggesting a mechanistic role for IL-10. The surface of thrombomodulin⁺ dendritic cells supported activation of protein C and procarboxypeptidase B2 in a thrombomodulin-dependent manner. Thus thrombomodulin treatment increases the number of thrombomodulin⁺ dendritic cells, which have significantly altered gene expression compared to thrombomodulin⁻ dendritic cells in key immune function pathways.     

The callovian (Middle Jurassic) crinoids from northern Lithuania

Callovian strata in northern Lithuania (Papilė area) have yielded some crinoid taxa[Chariocrinus andreae (Desor),Balanocrinus berchteni Hess —Pugin,B. subteres (Münster inGoldfuss) andIsocrinus nicoleti (Desor)], which have not been described from this area. The only form known previously wasBalanocrinus pentagonalis (Goldfuss).Palaeocomaster jaegeri n. sp. is the first free-living crinoid recorded from the Callovian of eastern Europe. It is characterized by possessing a very low, strongly flattened and narrow radial cavity. A very similar crinoid assemblage is known from the Callovian glacially-derived clays exposed in the Łuków region (eastern Poland), which were originally located near the Baltic Sea during the Middle Jurassic. It is considered that the allochthonous Callovian deposits from Poland are facies equivalents of the deposits known from Lithuania. The sole significant exception is the domination of free-moving comatulids (Comatulida) in the sediments known from the Papilė region, which may suggest that the Lithuanian sea basin was a little shallower than that located farther west in the Baltic area.      

Experimental tumbling of <Emphasis Type="Italic">Dreissena polymorpha</Emphasis>: implications for recognizing durophagous predation in the fossil record

Shell damage left by predators constitutes an important source of information on predator–prey interactions. However, recognition of the origins of shell damage can often be controversial, and needs to be assessed cautiously. More specifically, differentiation between predation- and abiotic-induced shell damage remains challenging. Here, we show the results of tumbling experiments using a bivalve species Dreissena polymorpha in order to determine rates and patterns of shell damage induced by physical forces in high-energy conditions. It is demonstrated that, in contrast to durophagous fish and crab predation, abiotic-induced fragmentation and damage are typically characterized by the presence of distinct abrasive scratches and wear scars on the surface of shell fragments. Furthermore, fragmented shells typically reveal a wide size distribution, and a different degree of sphericity and roundness resulting from abrasion. Importantly, large shell fragments commonly display smooth edges. These data suggest that durophagous predation, which typically induces fragmentation into large and angular shell fragments bearing no wear scars, can be reliably recognized both in present-day environments and in the fossil record.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. II. Binding of cytochrome c to oxidase-containing cardiolipin/phosphatidylcholine membranes.

Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, whereas the final saturation level of bound protein is independent of salt concentration and corresponds to approximately 53% of the total cco molecules present in the membrane. This suggests a 1:1 binding stoichiometry between the two proteins. The nonspecific binding component is governed by electrostatic interactions between cyt c and the membrane lipids and results in a partially ionic strength-reversible protein-membrane association. Thus, hydrophobic interactions between cyt c and the membrane, which are the predominant mode of binding in the absence of cco, are greatly suppressed. Both the amount of nonspecifically bound protein and the binding affinity can be varied over a broad range by changing the ionic strength and the extent of CL incorporation into the membrane. Under conditions approximating the physiological state in the mitochondrion (i.e., 20 mol% CL and medium ionic strength), 1-1.5 cyt c molecules are bound to the lipid phase per molecule of cco, with a dissociation constant of 0.1 microM. The possible physiological significance of these observations is discussed.     

Diversity dynamics of post‐Palaeozoic crinoids – in quest of the factors affecting crinoid macroevolution

Following the end‐Permian biotic crisis which led to the near extinction of crinoids, this echinoderm class rebounded rapidly during the Mesozoic, resulting in forms with important morphological and behavioural novelties. However, quantitative patterns of crinoid diversity during the Mesozoic remain largely unexplored. Here, we report results of analyses of the evolutionary dynamics of post‐Palaeozoic crinoid genera spanning a time interval between 250 and 70 Myr. We show that crinoids reached their Mesozoic peak of genus‐level richness during the Late Jurassic. We also document a major reorganization of different ecological crinoid groups in the Mesozoic. More specifically, the diversity of sessile forms generally increased towards the mid‐Mesozoic but decreased significantly starting in the Cretaceous, whereas the number of motile crinoid genera increased linearly during the Mesozoic. The possible role of biotic and abiotic factors in crinoid evolution is discussed.     

cMyc-p53 feedback mechanism regulates the dynamics of T lymphocytes in the immune response

Activation and proliferation of T cells are tightly regulated during the immune response. We show here that kinetics of proliferation of PHA activated T cells follows the expression of cMyc. Expression of p53 is also elevated and remains high several days after activation. To investigate the role of p53 in activated T cells, its expression was further elevated with nultin-3 treatment, a small molecule that dissociates the E3 ubiquitin protein ligase MDM2 from p53. Concomitantly, cMyc expression and proliferation decreased. At the other end of the cMyc-p53 axis, inhibition of cMyc with 10058-F4 led to down regulation of p53, likely through the lower level of cMyc induced p14ARF, which is also known to dissociate the p53-MDM2 complex. Both compounds induced cell cycle arrest and apoptosis. We conclude that the feedback regulation between cMyc and p53 is important for the T cell homeostasis. We also show that the two compounds modulating p53 and cMyc levels inhibited proliferation without abolishing the cytotoxic function, thus demonstrating the dichotomy between proliferation and cytotoxicity in activated T cells.