Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.     

Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.     

Is macrophage migration inhibitory factor a therapeutic target in systemic lupus erythematosus?

Systemic lupus erythematosus (SLE) is the prototype of a cluster of diseases that are characterized by a loss of self tolerance and chronic inflammation in organs including skin, kidney, brain and joints. Researchers have long debated the varying contributions of the components of the immune system to the pathogenesis of SLE, but the emigration of leucocytes from the microcirculation, and the subsequent tissue inflammation mediated by these inflammatory cells, are key features of chronic inflammation seen in SLE. Macrophage migration inhibitory factor (MIF) is a broad-spectrum pro-inflammatory cytokine. We hypothesize that MIF is an important inflammatory mediator in the perpetuation of immune activation in SLE, via its effects on activation of T and B cells, and endothelial and effector cells. As MIF exerts anti-apoptotic effects, it may also play a role in promoting abnormal survival of autoreactive lymphocytes, thus perpetuating autoimmune reactivity. In addition, MIF has a unique relationship with glucocorticoids, in that MIF can override the effects of glucocorticoids and may be important in steroid resistance. By virtue of its pluripotent functions, we propose that MIF may be a critical mediator of inflammation and damage in SLE, and that targeting of MIF may offer therapeutic benefits in this disease.     

Is Preening Behaviour Sexually Selected? An Experimental Approach

Elaborate or colourful feathers are important traits in female mate choice in birds but little attention has been given to potential costs of maintaining these traits in good condition with preening behaviour. Recent studies indicate that the time and energy required to maintain ornamental plumage in good condition reinforces the honesty of plumage trait. It has been proposed that some behaviours, whose primary function is not to transfer information, can also evolve as signalling components. Here we investigate whether the preening behaviour intensity has a signalling component: we hypothesized that if only high quality males can invest a lot of time in preening, this behaviour may be used by females as a quality signal (attractive preening hypothesis). We tested this hypothesis by using female budgerigars in mate‐choice tests in captivity. We tried to experimentally manipulate the preening behaviour of two groups of budgerigar males (treatment and control group). The proportion of time in which treated males preened in front of females was statistically higher than for control males, however, females spent similar amounts of time with treated males and control males. Moreover, males did not show significant quantitative changes in preening (for both groups) when females were present, suggesting that male budgerigars did not use this behaviour to convey information. These results are inconsistent with the ‘attractive preening’ hypothesis which predicts that preening behaviour itself provides information on condition and is used in female choice.     

Dual Utility of a Melanin‐Based Ornament in Bearded Tits

A general mechanism to account for the maintenance of sexually selected traits is the armament–ornament model, which predicts that females exploit signals used in male–male aggressive contests. Melanin‐based ornaments are good candidates to be dual utility traits because they are usually involved in male–male competition and function as signals of male dominance or fighting ability. Despite this, very few studies have investigated the dual utility of melanin‐based plumage traits and results remain generally ambiguous. In this study, we investigated the dual function of a melanin‐based trait (the black beard) in the male bearded tit, Panurus biarmicus. In a controlled aviary experiment, beard length was the best predictor of dominance in male–male competition. In a choice experiment, females showed a proximity preference for males whose beard length was experimentally elongated. Our results indicate that beard length plays an important role in both male–male competition and female choice in bearded tits, as proposed by the dual utility model.     

SGO1C is a non-functional isoform of Shugoshin and can disrupt sister chromatid cohesion by interacting with PP2A–B56

Shugoshin (SGO1) plays a pivotal role in sister chromatid cohesion during mitosis by protecting the centromeric cohesin from mitotic kinases and WAPL. Mammalian cells contain at least 6 alternatively spliced isoforms of SGO1. The relationship between the canonical SGO1A with shorter isoforms including SGO1C remains obscure. Here we show that SGO1C was unable to replace the loss of SGO1A. Instead, expression of SGO1C alone induced aberrant mitosis similar to depletion of SGO1A, promoting premature sister chromatid separation, activation of the spindle-assembly checkpoint, and mitotic arrest. In disagreement with previously published data, we found that SGO1C localized to kinetochores. However, the ability to induce aberrant mitosis did not correlate with its kinetochore localization. SGO1C mutants that abolished binding to kinetochores still triggered premature sister chromatid separation. We provide evidence that SGO1C-mediated mitotic arrest involved the sequestering of PP2A–B56 pool. Accordingly, SGO1C mutants that abolished binding to PP2A localized to kinetochores but did not induce aberrant mitosis. These studies imply that the expression of SGO1C should be tightly regulated to prevent dominant-negative effects on SGO1A and genome instability.     

Positively-Charged Semi-Tunnel Is a Structural and Surface Characteristic of Polyphosphate-Binding Proteins: An In-Silico Study

Phosphate is essential for all major life processes, especially energy metabolism and signal transduction. A linear phosphate polymer, polyphosphate (polyP), linked by high-energy phosphoanhydride bonds, can interact with various proteins, playing important roles as an energy source and regulatory factor. However, polyP-binding structures are largely unknown. Here we proposed a putative polyP binding site, a positively-charged semi-tunnel (PCST), identified by surface electrostatics analyses in polyP kinases (PPKs) and many other polyP-related proteins. We found that the PCSTs in varied proteins were folded in different secondary structure compositions. Molecular docking calculations revealed a significant value for binding affinity to polyP in PCST-containing proteins. Utilizing the PCST identified in the β subunit of PPK3, we predicted the potential polyP-binding domain of PPK3. The discovery of this feature facilitates future searches for polyP-binding proteins and discovery of the mechanisms for polyP-binding activities. This should greatly enhance the understanding of the many physiological functions of protein-bound polyP and the involvement of polyP and polyP-binding proteins in various human diseases.