Differential regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.     

AUTOGAMOUS AUXOSPORULATION IN PINNULARIA NODOSA (BACILLARIOPHYCEAE)1

Auxosporulation of the freshwater epipelic diatom Pinnularia nodosa (Ehrenb.) W. Sm. was studied in a clonal culture. Interphase cells possessed two chloroplasts with invaginated pyrenoids. The nucleus contained a single small body of heterochromatin at one end, also visible during most of meiotic prophase. During auxosporulation, induced by transfer of stationary‐phase cells to fresh medium and suppressed by high nitrogen (N), an unpaired mother cell produced a single auxospore. Although meiosis II and nuclear fusion were not observed, indirect evidence indicated that auxosporulation was autogamous (rarely reported in pennate diatoms), rather than apomictic; paedogamy was excluded. The protoplast produced after meiosis either (1) matured into a “pseudozygote,” via an asymmetrical contraction after meiosis I to form a single spherical cell at one end of the mother cell (pathway 1); or (2) constricted into two spherical cells (pathway 2). In pathway 2, the “pseudogametes” never fused and only one or none developed into a pseudozygote and then into an auxospore. Pathway 2 could be suppressed by continuous light. During metamorphosis of the spherical pseudozygote into an elongate young auxospore, a complete covering of thin siliceous incunabular strips was formed, separate from the organic wall formed around the pseudozygote when first formed and from the perizonium. Mature auxospores produced via pathway 2 had 60% of the volume as those produced via pathway 1 and had smaller chloroplasts (through loss of fragments during protoplast cleavage), but they achieved exactly the same lengths, suggesting that absolute length is monitored during expansion.     

A MOLECULAR SYSTEMATIC APPROACH TO EXPLORE DIVERSITY WITHIN THE SELLAPHORA PUPULA SPECIES COMPLEX (BACILLARIOPHYTA)1

The common and cosmopolitan freshwater benthic diatom Sellaphora pupula (Kütz.) Mereschk. is a model system for studying the nature of species in microalgae; the biological significance of morphological variation in this species complex has been widely demonstrated. The aim of this study was to establish a two‐gene phylogeny (18S rDNA and rbcL) for 23 Sellaphora taxa, including 19 S. pupula aggregate (agg.) demes or species, S. bacillum (Ehrenb.) D. G. Mann, and S. laevissima (Kütz.) D. G. Mann. A range of analyses on separate and combined data sets indicated that Sellaphora is a monophyletic group containing four major clades. Of the traditionally recognized species, S. bacillum and S. laevissima are natural groups, but S. pupula is paraphyletic or polyphyletic because S. bacillum groups with S. pupula“small lanceolate” and S. lanceolata. Thickened bars at the poles of valves within the core “pupula–bacillum” group may be a morphological synapomorphy; the fossil record suggests that this group is at least 12 million years old. Otherwise, there was no clear pattern in the distribution of different morphologies among the major clades; each clade was also heterogeneous with respect to mating system. More intensive and extensive sampling will doubtless uncover even greater diversity; the challenge lies in its interpretation. Our results demonstrate the limitations of paleoecological, ecological, and biogeographical research based on morphospecies.     

The control of size in animals: insights from selector genes

How size is controlled during animal development remains a fascinating problem despite decades of research. Here we review key concepts in size biology and develop our thesis that much can be learned by studying how different organ sizes are differentially scaled by homeotic selector genes. A common theme from initial studies using this approach is that morphogen pathways are modified in numerous ways by selector genes to effect size control. We integrate these results with other pathways known to regulate organ size in developing a comprehensive model for organ size control.     

Oxidative stress promotes ligand-independent and enhanced ligand-dependent tumor necrosis factor receptor signaling

Tumor necrosis factor (TNF) receptor 1 (TNFR1, p55) and 2 (TNFR2, p75) are characterized by several cysteine-rich modules in the extracellular domain, raising the possibility that redox-induced modifications of these cysteine residues might alter TNFR function. To test this possibility, we examined fluorescence resonance energy transfer (FRET) in 293T cells transfected with CFP- and YFP-tagged TNFRs exposed to the thiol oxidant diamide. Treatment with high concentrations of diamide (1 mm) resulted in an increase in the FRET signal that was sensitive to inhibition with the reducing agent dithiothreitol, suggesting that oxidative stress resulted in TNFR self-association. Treatment of cells with low concentrations of diamide (1 mum) that was not sufficient to provoke TNFR self-association resulted in increased TNF-induced FRET signals relative to the untreated cells, suggesting that oxidative stress enhanced ligand-dependent TNFR signaling. Similar findings were obtained when the TNFR1- and TNFR2-transfected cells were pretreated with a cell-impermeable oxidase, DsbA, that catalyzes disulfide bond formation between thiol groups on cysteine residues. The changes in TNFR self-association were functionally significant, because pretreating the HeLa cells and 293T cells resulted in increased TNF-induced NF-kappaB activation and TNF-induced expression of IkappaB and syndecan-4 mRNA levels. Although pretreatment with DsbA did not result in an increase in TNF binding to TNFRs, it resulted in increased TNF-induced activation of NF-kappaB, consistent with an allosteric modification of the TNFRs. Taken together, these results suggest that oxidative stress promotes TNFR receptor self-interaction and ligand-independent and enhanced ligand-dependent TNF signaling.     

Micro-mechanical modeling of the cement-bone interface: the effect of friction, morphology and material properties on the micromechanical response

In order to gain insight into the micro-mechanical behavior of the cement-bone interface, the effect of parametric variations of frictional, morphological and material properties on the mechanical response of the cement-bone interface were analyzed using a finite element approach. Finite element models of a cement-bone interface specimen were created from micro-computed tomography data of a physical specimen that was sectioned from an in vitro cemented total hip arthroplasty. In five models the friction coefficient was varied (mu=0.0; 0.3; 0.7; 1.0 and 3.0), while in one model an ideally bonded interface was assumed. In two models cement interface gaps and an optimal cement penetration were simulated. Finally, the effect of bone cement stiffness variations was simulated (2.0 and 2.5 GPa, relative to the default 3.0 GPa). All models were loaded for a cycle of fully reversible tension-compression. From the simulated stress-displacement curves the interface deformation, stiffness and hysteresis were calculated. The results indicate that in the current model the mechanical properties of the cement-bone interface were caused by frictional phenomena at the shape-closed interlock rather than by adhesive properties of the cement. Our findings furthermore show that in our model maximizing cement penetration improved the micromechanical response of the cement-bone interface stiffness, while interface gaps had a detrimental effect. Relative to the frictional and morphological variations, variations in the cement stiffness had only a modest effect on the micro-mechanical behavior of the cement-bone interface. The current study provides information that may help to better understand the load-transfer mechanisms taking place at the cement-bone interface.     

Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions

Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from the in vivo situation in important aspects. Here we introduce a straightforward methodology to compare cell lines to their cognate primary cells and to derive a comparative functional phenotype. We used SILAC (stable isotope labeling by amino acids in cell culture) for quantitative, mass spectrometry-based comparison of the hepatoma cell line Hepa1-6 with primary hepatocytes. The resulting quantitative proteome of 4,063 proteins had an asymmetric distribution, with many proteins down-regulated in the cell line. Bioinformatic analysis of the quantitative proteomics phenotypes revealed that Hepa1-6 cells were deficient in mitochondria, reflecting re-arrangement of metabolic pathways, drastically up-regulate cell cycle-associated functions and largely shut down drug metabolizing enzymes characteristic for the liver. This quantitative knowledge of changes provides an important basis to adapt cell lines to more closely resemble physiological conditions.     

Topological information embodied in local juxtaposition geometry provides a statistical mechanical basis for unknotting by type-2 DNA topoisomerases

Topoisomerases may unknot by recognizing specific DNA juxtapositions. The physical basis of this hypothesis is investigated by considering single-loop conformations in a coarse-grained polymer model. We determine the statistical relationship between the local geometry of a juxtaposition of two chain segments and whether the loop is knotted globally, and ascertain how the knot/unknot topology is altered by a topoisomerase-like segment passage at the juxtaposition. Segment passages at a "free" juxtaposition tend to increase knot probability. In contrast, segment passages at a "hooked" juxtaposition cause more transitions from knot to unknot than vice versa, resulting in a steady-state knot probability far lower than that at topological equilibrium. The reduction in knot population by passing chain segments through a hooked juxtaposition is more prominent for loops of smaller sizes, n, but remains significant even for larger loops: steady-state knot probability is only approximately 2%, and approximately 5% of equilibrium, respectively, for n=100 and 500 in the model. An exhaustive analysis of approximately 6000 different juxtaposition geometries indicates that the ability of a segment passage to unknot correlates strongly with the juxtaposition's "hookedness". Remarkably, and consistent with experiments on type-2 topoisomerases from different organisms, the unknotting potential of a juxtaposition geometry in our polymer model correlates almost perfectly with its corresponding decatenation potential. These quantitative findings suggest that it is possible for topoisomerases to disentangle by acting selectively on juxtapositions with "hooked" geometries.