Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage

The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC(Cdh1)) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC(Cdh1) ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage.     

Functional training improves club head speed and functional fitness in older golfers

Functional training programs have been used in a variety of rehabilitation settings with documented success. Based on that success, the concept of functional training has gained popularity in applied fitness settings to enhance sport performance. However, there has been little or no research studying the efficacy of functional training programs on the improvement of sport performance or functional fitness. Thus, it was the purpose of this study to determine the effect of a progressive functional training program on club head speed and functional fitness in older male golfers. Eighteen male golfers (age: 70.7 +/- 9.1 [SD] years) were randomly assigned to an exercise (N = 11) or control (N = 7) group. The exercise group participated in an 8-week progressive functional training program including flexibility exercises, core stability exercises, balance exercises, and resistance exercises. Pre- and postmeasurements included club head speed of a driver by radar (exercise and Control) and Fullerton Senior Fitness Test measurements (exercise only). One-way analysis of covariance was performed on club head speed measurements using pretest measurements as the covariate. Paired t-tests were performed to analyze Senior Fitness Test variables. After the intervention, maximal club head speed increased in the exercise group (127.3 +/- 13.4 to 133.6 +/- 14.2 km x hr(-1)) compared with the control group (134.5 +/- 14.6 to 133.3 +/- 11.2 km x hr(-1); p < 0.05). Additionally, improvements (p < 0.05) were detected for most Senior Fitness Test variables in the exercise group. In summary, this functional training program resulted in significant improvements in club head speed and several components of functional fitness. Future research should continue to examine the effect of functional training programs on sport performance and functional fitness in older adults.     

Gene microarray analysis using angular distribution decomposition.

Clustering techniques have been widely used in the analysis of microarray data to group genes with similar expression profiles. The similarity of expression profiles and hence the results of clustering greatly depend on how the data has been transformed. We present a method that uses the relative expression changes between pairs of conditions and an angular transformation to define the similarity of gene expression patterns. The pairwise comparisons of experimental conditions can be chosen to reflect the purpose of clustering allowing control the definition of similarity between genes. A variational Bayes mixture modeling approach is then used to find clusters within the transformed data. The purpose of microarray data analysis is often to locate groups genes showing particular patterns of expression change and within these groups to locate specific target genes that may warrant further experimental investigation. We show that the angular transformation maps data to a representation from which information, in terms of relative regulation changes, can be automatically mined. This information can be then be used to understand the "features" of expression change important to different clusters allowing potentially interesting clusters to be easily located. Finally, we show how the genes within a cluster can be visualized in terms of their expression pattern and intensity change, allowing potential target genes to be highlighted within the clusters of interest.     

Stability of biocatalysts

Despite their many favorable qualities, the marginal stability of biocatalysts in many types of reaction media often has prevented or delayed their implementation for industrial-scale synthesis of fine chemicals and pharmaceuticals. Consequently, there is great interest in understanding effects of solution conditions on protein stability, as well as in developing strategies to improve protein stability in desired reaction media. Recent methods include novel chemical modifications of protein, lyophilization in the presence of additives, and physical immobilization on novel supports. Rational and combinatorial protein engineering techniques have been used to yield unmodified proteins with exceptionally improved stability. Both have been aided by the development of computational tools and structure-guided heuristics aimed at reducing library sizes that must be generated and screened to identify improved mutants. The number of parameters used to indicate protein stability can complicate discussions and investigations, and care should be taken to identify whether thermodynamic or kinetic stability limits the observed stability of proteins. Although the useful lifetime of a biocatalyst is dictated by its kinetic stability, only 6% of protein stability studies use kinetic stability measures. Clearly, more effort is needed to study how solution conditions impact protein kinetic stability.     

New alternative phosphorylation sites on the cyclin dependent kinase 1/cyclin a complex in p53-deficient human cells treated with etoposide: possible association with etoposide-induced apoptosis

Cell cycle arrest is a major cellular response to DNA damage preceding the decision to repair or die. Many malignant cells have non-functional p53 rendering them more “aggressive” in nature. Arrest in p53-negative cells occurs at the G2M cell cycle checkpoint. Failure of DNA damaged cells to arrest at G2 results in entry into mitosis and potential death through aberrant mitosis and/or apoptosis. The pivotal kinase regulating the G2M checkpoint is Cdk1/cyclin B whose activity is controlled by phosphorylation. The p53-negative myeloid leukemia cell lines K562 and HL-60 were used to determine Cdk1 phosphorylation status during etoposide treatment. Cdk1 tyrosine 15 phosphorylation was associated with G2M arrest, but not with cell death. Cdk1 tyrosine 15 phosphorylation also led to suppression of nuclear cyclin B-associated Cdk1 kinase activity. However cell death, associated with broader tyrosine phosphorylation of Cdk1 was not attributed to tyrosine 15 alone. This broader phosphoryl isoform of Cdk1 was associated with cyclin A and not cyclin B. Alternative phosphorylations sites were predicted as tyrosines 4, 99 and 237 by computer analysis. No similar pattern was found on Cdk2. These findings suggest novel Cdk1 phosphorylation sites, which appear to be associated with p53-independent cell death following etoposide treatment.     

Growth rate and its variability in erect Antarctic bryozoans

Climate is altering rapidly in parts of the Arctic and Antarctic but we know little about how marine organisms are responding to, or might respond to such changes. Knowledge of within-taxon variability is the vital context (currently missing) to interpretation of environmental signals. We investigated growth in six species and three genera of erect Antarctic bryozoans, an ideal model taxon to investigate such response. Cellarinella margueritae, C. nodulata, C. rogickae, C. watersi, Melicerita obliqua and Stomhypselosaria watersi, extended 3.4, 5.2, 4.6, 4.1, 4.9 and 4.5 mm year⁻¹ and synthesised 24, 55, 45, 176, 34 and 46 mg CaCO³ year⁻¹, respectively. The maximum ages of these species ranged from 11 to 15 years except M. obliqua, which reached 32 years. This is the first investigation of growth rates of closely related Antarctic invertebrate species and reports the slowest growth rates of bryozoans known from anywhere to date. Our data coupled with that from literature shows that Antarctic bryozoan growth varies <<10¹ between species, 10¹ between genera, 10² between morphologies and is ∼10¹ slower than in tropical or temperate regions. However, within encrusting types the slowest growing species grow at similar rates from poles to tropics. Age was a strong confounding factor across our Antarctic study species but age-standardised data showed a possible decline in annual growth from 1992 to 2003. We identify several factors increasing this environmental signal strength, including (1) the importance of generic (though not necessarily species) identification and (2) use of dry-mass or ash-free dry-mass as the measures of growth.     

Platelet-associated NAD(P)H oxidase contributes to the thrombogenic phenotype induced by hypercholesterolemia

Elevated cholesterol levels promote proinflammatory and prothrombogenic responses in venules and impaired endothelium-dependent arteriolar dilation. Although NAD(P)H oxidase-derived superoxide has been implicated in the altered vascular responses to hypercholesterolemia, it remains unclear whether this oxidative pathway mediates the associated arteriolar dysfunction and platelet adhesion in venules. Platelet and leukocyte adhesion in cremasteric postcapillary venules and arteriolar dilation responses to acetylcholine were monitored in wild-type (WT), Cu,Zn-superoxide dismutase transgenic (SOD-TgN), and NAD(P)H oxidase-knockout (gp91(phox-/-)) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. HC elicited increased platelet and leukocyte adhesion in WT mice versus ND. Cytosolic subunits of NAD(P)H oxidase (p47phox and p67phox) were expressed in platelets. This was not altered by hypercholesterolemia; however, platelets and leukocytes from HC mice exhibited elevated generation of reactive oxygen species compared to ND mice. Hypercholesterolemia-induced leukocyte recruitment was attenuated in SOD-TgN-HC and gp91(phox-/-)-HC mice. Recruitment of platelets derived from WT-HC mice in venules of SOD-TgN-HC or gp91(phox-/-)-HC recipients was comparable to ND levels. Adhesion of SOD-TgN-HC platelets paralleled the leukocyte response and was attenuated in SOD-TgN-HC recipients, but not in WT-HC recipients. However, gp91(phox-/-)-HC platelets exhibited low levels of adhesion comparable to those of WT-ND in both hypercholesterolemic gp91(phox-/-) and WT recipients. Arteriolar dysfunction was evident in WT-HC mice, compared to WT-ND. Overexpression of SOD or, to a lesser extent, gp91(phox) deficiency restored arteriolar vasorelaxation responses toward WT-ND levels. These findings reveal a novel role for platelet-associated NAD(P)H oxidase in producing the thrombogenic phenotype in hypercholesterolemia and demonstrate that NAD(P)H oxidase-derived superoxide mediates the HC-induced arteriolar dysfunction.