Phosphorylation of p27Kip1 regulates assembly and activation of cyclin D1-Cdk4

p27 mediates Cdk2 inhibition and is also found in cyclin D1-Cdk4 complexes. The present data support a role for p27 in the assembly of D-type cyclin-Cdk complexes and indicate that both cyclin D1-Cdk4-p27 assembly and kinase activation are regulated by p27 phosphorylation. Prior work showed that p27 can be phosphorylated by protein kinase B/Akt (PKB/Akt) at T157 and T198. Here we show that PKB activation and the appearance of p27pT157 and p27pT198 precede p27-cyclin D1-Cdk4 assembly in early G₁. PI3K/PKB inhibition rapidly reduced p27pT157 and p27pT198 and dissociated cellular p27-cyclin D1-Cdk4. Mutant p27 allele products lacking phosphorylation at T157 and T198 bound poorly to cellular cyclin D1 and Cdk4. Cellular p27pT157 and p27pT198 coprecipitated with Cdk4 but were not detected in Cdk2 complexes. The addition of p27 to recombinant cyclin D1 and Cdk4 led to cyclin D1-Cdk4-p27 complex formation in vitro. p27 phosphorylation by PKB increased p27-cyclin D1-Cdk4 assembly in vitro but yielded inactive Cdk4. In contrast, Src pretreatment of p27 did not affect p27-cyclin D1-Cdk4 complex formation. However, Src treatment led to tyrosine phosphorylation of p27 and catalytic activation of assembled cyclin D1-Cdk4-p27 complexes. Thus, while PKB-dependent p27 phosphorylation appears to increase cyclin D1-Cdk4-p27 assembly or stabilize these complexes in vitro, cyclin D1-Cdk4-p27 activation requires the tyrosine phosphorylation of p27. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process.     

Using FAME Analysis to Compare, Differentiate, and Identify Multiple Nematode Species

We have adapted the Sherlock^® Microbial Identification system for identification of plant parasitic nematodes based on their fatty acid profiles. Fatty acid profiles of 12 separate plant parasitic nematode species have been determined using this system. Additionally, separate profiles have been developed for Rotylenchulus reniformis and Meloidogyne incognita based on their host plant, four species and three races within the Meloidogyne genus, and three life stages of Heterodera glycines. Statistically, 85% of these profiles can be delimited from one another; the specific comparisons between the cyst and vermiform stages of H. glycines, M. hapla and M. arenaria, and M. arenaria and M. javanica cannot be segregated using canonical analysis. By incorporating each of these fatty acid profiles into the Sherlock^® Analysis Software, 20 library entries were created. While there was some similarity among profiles, all entries correctly identified the proper organism to genus, species, race, life stage, and host at greater than 86% accuracy. The remaining 14% were correctly identified to genus, although species and race may not be correct due to the underlying variables of host or life stage. These results are promising and indicate that this library could be used for diagnostics labs to increase response time.     

Noninvasive Imaging of In Vivo MuRF1 Expression during Muscle Atrophy

Numerous human diseases can lead to atrophy of skeletal muscle, and loss of this tissue has been correlated with increased mortality and morbidity rates. Clinically addressing muscle atrophy remains an unmet medical need, and the development of preclinical tools to assist drug discovery and basic research in this effort is important for advancing this goal. In this report, we describe the development of a bioluminescent gene reporter rat, based on the zinc finger nuclease-targeted insertion of a bicistronic luciferase reporter into the 3′ untranslated region of a muscle specific E3 ubiquitin ligase gene, MuRF1 (Trim63). In longitudinal studies, we noninvasively assess atrophy-related expression of this reporter in three distinct models of muscle loss (sciatic denervation, hindlimb unloading and dexamethasone-treatment) and show that these animals are capable of generating refined detail on in vivo MuRF1 expression with high temporal and anatomical resolution.     

Conducting an acute intense interval exercise session during the ramadan fasting month: what is the optimal time of the day?

This study examines the effects of Ramadan fasting on performance during an intense exercise session performed at three different times of the day, i.e., 08:00, 18:00, and 21:00?h. The purpose was to determine the optimal time of the day to perform an acute high-intensity interval exercise during the Ramadan fasting month. After familiarization, nine trained athletes performed six 30-s Wingate anaerobic test (WAnT) cycle bouts followed by a time-to-exhaustion (T(exh)) cycle on six separate randomized and counterbalanced occasions. The three time-of-day nonfasting (control, CON) exercise sessions were performed before the Ramadan month, and the three corresponding time-of-day Ramadan fasting (RAM) exercise sessions were performed during the Ramadan month. Note that the 21:00?h session during Ramadan month was conducted in the nonfasted state after the breaking of the day's fast. Total work (TW) completed during the six WAnT bouts was significantly lower during RAM compared to CON for the 08:00 and 18:00?h (p?相似文献   

HPV genotypes in high grade cervical lesions and invasive cervical carcinoma as detected by two commercial DNA assays, North Carolina, 2001-2006

Background

HPV typing using formalin fixed paraffin embedded (FFPE) cervical tissue is used to evaluate HPV vaccine impact, but DNA yield and quality in FFPE specimens can negatively affect test results. This study aimed to evaluate 2 commercial assays for HPV detection and typing using FFPE cervical specimens.

Methods

Four large North Carolina pathology laboratories provided FFPE specimens from 299 women ages18 and older diagnosed with cervical disease from 2001 to 2006. For each woman, one diagnostic block was selected and unstained serial sections were prepared for DNA typing. Extracts from samples with residual lesion were used to detect and type HPV using parallel and serial testing algorithms with the Linear Array and LiPA HPV genotyping assays.

Findings

LA and LiPA concordance was 0.61 for detecting any high-risk (HR) and 0.20 for detecting any low-risk (LR) types, with significant differences in marginal proportions for HPV16, 51, 52, and any HR types. Discordant results were most often LiPA-positive, LA-negative. The parallel algorithm yielded the highest prevalence of any HPV type (95.7%). HR type prevalence was similar using parallel (93.1%) and serial (92.1%) approaches. HPV16, 33, and 52 prevalence was slightly lower using the serial algorithm, but the median number of HR types per woman (1) did not differ by algorithm. Using the serial algorithm, HPV DNA was detected in >85% of invasive and >95% of pre-invasive lesions. The most common type was HPV16, followed by 52, 18, 31, 33, and 35; HPV16/18 was detected in 56.5% of specimens. Multiple HPV types were more common in lower grade lesions.

Conclusions

We developed an efficient algorithm for testing and reporting results of two commercial assays for HPV detection and typing in FFPE specimens, and describe HPV type distribution in pre-invasive and invasive cervical lesions in a state-based sample prior to HPV vaccine introduction.     

A quantitative assessment of the antimicrobial activity of garlic (Allium sativum)

An aqueous extract of freeze-dried garlic (Allium sativum), when incorporated into growth media, inhibited many representative bacteria, yeasts, fungi and a virus. All microorganisms tested were susceptible to garlic. Quantitative assessment of the minimum inhibitory concentrations for bacteria and yeasts showed values ranging from 0.8 to 40.0 mg garlic ml^-1. Fungal radial colony growth was inhibited by at least 25% at concentrations as low as 2.0 mg garlic ml^-1. The 50% endpoint neutralization titre for rotavirus was 2.4 to 2.8 g ml^-1. Lactic acid bacteria were the least sensitive microorganisms to the inhibitory effects of garlic. In mixed culture studies of Lactobacillus acidophilus and Escherichia coli, garlic prevented the establishment of E. coli, although the final outcome of competition was not affected.L.P. Rees, S.F. Minney and N.T. Plummer are with Interprise Ltd, Baglan Bay Industrial Park, Port Talbot SA 12 7DJ, UK. J.H. Slater and D.A. Skyrme are with the School of Pure and Applied Biology, University of Wales, Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK.     

Differential Silver Enhanced Double Labeling in Immunoelectron Microscopy

A two-sided double labeling method using protein A gold was used to demonstrate the presence of two hormones within the same anterior pituitary cell granule. A single probe size was used for both section faces but one side of the grid was silver enhanced. The use of a single probe size reduced the cost of the study and eliminated the variations in labeling efficiency that result from the use of different probe sizes.     

Improving the analysis of low heritability complex traits for enhanced genetic gain in potato

Key message

Best linear unbiased prediction (BLUP), which uses pedigree to estimate breeding values, can result in increased genetic gains for low heritability traits in autotetraploid potato.

Abstract

Conventional potato breeding strategies, based on outcrossing followed by phenotypic recurrent selection over a number of generations, can result in slow but steady improvements of traits with moderate to high heritability. However, faster gains, particularly for low heritability traits, could be made by selection on estimated breeding values (EBVs) calculated using more complete pedigree information in best linear unbiased prediction (BLUP) analysis. One complication in applying BLUP predictions of breeding value to potato breeding programs is the autotetraploid inheritance pattern of this species. Here we have used a large pedigree, dating back to 1908, to estimate heritability for nine key traits for potato breeding, modelling autotetraploid inheritance. We estimate the proportion of double reduction in potatoes from our data, and across traits, to be in the order of 10 %. Estimates of heritability ranged from 0.21 for breeder’s visual preference, 0.58 for tuber yield, to 0.83 for plant maturity. Using the accuracies of the EBVs determined by cross generational validation, we model the genetic gain that could be achieved by selection of genotypes for breeding on BLUP EBVs and demonstrate that gains can be greater than in conventional schemes.     

Model-assisted evaluation of crop load effects on stem diameter variations and fruit growth in peach

Key message

The paper identifies and quantifies how crop load influences plant physiological variables that determine stem diameter variations to better understand the effect of crop load on drought stress indicators.

Abstract

Stem diameter (D ^stem) variations have extensively been applied in optimisation strategies for plant-based irrigation scheduling in fruit trees. Two D ^stem derived water status indicators, maximum daily shrinkage (MDS) and daily growth rate (DGR), are however influenced by other factors such as crop load, making it difficult to unambiguously use these indicators in practical irrigation applications. Furthermore, crop load influences the growth of individual fruits, because of competition for assimilates. This paper aims to explain the effect of crop load on DGR, MDS and individual fruit growth in peach using a water and carbon transport model that includes simulation of stem diameter variations. This modelling approach enabled to relate differences in crop load to differences in xylem and phloem water potential components. As such, crop load effects on DGR were attributed to effects on the stem phloem turgor pressure. The effect of crop load on MDS could be explained by the plant water status, the phloem carbon concentration and the elasticity of the tissue. The influence on fruit growth could predominantly be explained by the effect on the early fruit growth stages.