p21Cip1 and p27Kip1 induce distinct cell cycle effects and differentiation programs in myeloid leukemia cells

The cyclin-dependent kinase (Cdk) inhibitors p21(Cip1) and p27(Kip1) have been proposed to exert redundant functions in cell cycle progression and differentiation programs, although nonoverlapping functions have also been described. To gain further insights into the relevant mechanisms and to detect possible functional differences between both proteins, we conditionally expressed p21(Cip1) and p27(Kip1) in K562, a multipotent human leukemia cell line. Temporal ectopic expression of either p21(Cip1) or p27(Kip1) arrested proliferation, inhibited Cdk2 and Cdk4 activities, and suppressed retinoblastoma phosphorylation. However, whereas p21(Cip1) arrested cells in both G(1) and G(2) cell cycle phases, p27(Kip1) blocked the G(1)/S-phase transition. Furthermore, although both p21(Cip1) and p27(Kip1) associated with Cdk6, only p27(Kip1) significantly inhibited its activity. Most importantly, each protein promoted differentiation along a distinct pathway; p21(Cip1) triggered megakaryocytic maturation, whereas p27(Kip1) resulted in the expression of erythroid markers. Consistently, p21(Cip1) and p27(Kip1) were rapid and transiently up-regulated when K562 cells are differentiated into megakaryocytic and erythroid lineages, respectively. These findings demonstrate distinct functions of p21(Cip1) and p27(Kip1) in cell cycle regulation and differentiation and indicate that these two highly related proteins possess unique biological activities and are not functionally interchangeable.     

Characterization of an <Emphasis Type="Italic">Eco</Emphasis>RI family of satellite DNA from two species

We have cloned and sequenced a 321bp band of repetitive DNA from Eptesicus fuscus and E. serotinus observed after gel electrophoresis of EcoRI digested genomic DNA in both species. Southern blot analysis of genomic DNA (from both species) digested with the same enzyme showed the existence of a ladder pattern indicating that the repetitive DNA is arrayed in tandem. The repetitive sequences have a monomer unit of 321bp which is composed of two subunits of 160bp, suggested by the existence of a 160bp band in the ladder of E. fuscus and by the presence of some direct repeats found in the analysis of the consensus sequence. Analysis of the methylation status demonstrated that cytosines in CCGG sequences in this satellite DNA are methylated in E. fuscus but not in the E. serotinus. Alignment of the sequenced clones showed that several nucleotide positions are diagnostic species-specific and consequently the phylogenetic analysis grouped the monomer units from both species in two clearly separated groups.     

Cadmium exposure modifies lactotrophs activity associated to genomic and morphological changes in rat pituitary anterior lobe

Cadmium (Cd) is widely used in industrial applications and is an important contaminant of agricultural products. As an endocrine disruptor, Cd modifies the hormone release of pituitary anterior lobe (PAL). This work was undertaken to evaluate a possible association between phospholipase D (PLD) and prolactin mRNA expressions and the activity of lactotrophs and folliculostellate cells (FSC) in PAL of Cd exposed adult male Wistar rats (Cd, 0.133 mM per liter for 2 months). The PALs were submitted to immunohistochemical and morphometric analysis to determine the percentage of lactotrophs (PRL-ir) and FSC (S-100-ir). Cultured PAL cells were stained with Hoechst 33258 to determine the presence of alterations in nuclear morphology consistent with apoptosis. The expressions of PLD and prolactin mRNA were assessed by RT-PCR. Cd treated rats showed a decrease of PLD mRNA levels that can be associated to both high number of apoptotic cells and increase of S-100 protein expression in FSC. Cd decreased prolactin mRNA expression, number of lactotrophs and percentage of PRL-ir suggesting a low availability of prolactin to be secreted from PAL. Cd modifies the lactotrophs activity of pituitary gland through biochemical, genomic and morphological changes and contributes directly or indirectly to the levels of serum prolactin.     

Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.     

A biomechanics-based method for the quantification of muscle selectivity in a musculoskeletal system

In this paper, we have developed a novel and simple method to quantify the ability to selectively activate our muscles in an effective pattern to achieve a particular task. In the context of this study, we define an effective pattern as that in which muscles whose mechanical contribution to the task is greatest, are mostly active, while the antagonist muscles are mostly silent. This new method uses biomechanical parameters to project the multi-channel EMGs into a three-dimensional artificial torque space, where the EMGs are represented as muscle activation vectors. Using the muscle activation vectors we defined a simple scalar, the muscle selection index, to quantify muscle selectivity. We demonstrate that by using this index we are able to quantify the muscle selectivity during the generation of isometric shoulder or elbow torques in brain-injured and able-bodied subjects. This method can be used during both static and dynamic motor tasks in a multi-articular musculoskeletal system.     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

Fluorescent silica particles for monitoring oxygen levels in three-dimensional heterogeneous cellular structures

Bacterial biofilms are a major obstacle challenging the development of more effective therapies to treat implant infections. Oxygen availability to bacterial cells has been implicated in biofilm formation and planktonic cell detachment; however, there are insufficient tools available to measure oxygen concentrations within complex three‐dimensional structures with ～1 µm resolution. Such measurements may complement measures of biofilm structure and cell activity to provide a more comprehensive understanding of biofilm biology. Thus, we developed oxygen‐sensing microparticles specifically designed to characterize oxygen transport through the volume of bacterial biofilms. The Stöber method was used to synthesize monodisperse silica microparticles of approximately the same size as a bacterium (～1 µm). Two fluorophores, oxygen‐sensitive Ru(Ph₂phen₃)Cl₂, and the reference fluorophore Nile blue chloride were immobilized on the surface of the particles. We demonstrate application of the microparticles toward measuring the oxygen concentration profiles within a live Staphylococcus aureus biofilm. Biotechnol. Bioeng. 2012; 109: 2663–2670. © 2012 Wiley Periodicals, Inc.     

S-layer proteins of Lactobacillus acidophilus inhibits JUNV infection

We investigated the regulation of Hsp27 phosphorylation by protein kinase C δ (PKCδ) during etoposide-induced apoptosis. The phosphorylation of Hsp27 at Ser78 was temporally correlated with the proteolytic activation of PKCδ during apoptosis. Hsp27 phosphorylation was dependent on the activity of PKCδ since treatment with rottlerin, a chemical inhibitor of PKCδ, or overexpression of a PKCδ dominant negative mutant abolished the phosphorylation. In addition, recombinant PKCδ phosphorylated Hsp27 at Ser78 in vitro. Moreover, caspase-3 was specifically activated following Hsp27 phosphorylation at Ser78. Pull-down assays using a phosphomimetic Hsp27 mutant revealed that binding between Hsp27 and cytochrome c was abolished by the phosphorylation. These results suggest that Hsp27 dissociates from cytochrome c following PKCδ-mediated phosphorylation at Ser78, which allows formation of the apoptosome and stimulates apoptotic progression.     

Accelerating the domestication of trees using genomic selection: accuracy of prediction models across ages and environments

? Genomic selection is increasingly considered vital to accelerate genetic improvement. However, it is unknown how accurate genomic selection prediction models remain when used across environments and ages. This knowledge is critical for breeders to apply this strategy in genetic improvement. ? Here, we evaluated the utility of genomic selection in a Pinus taeda population of c. 800 individuals clonally replicated and grown on four sites, and genotyped for 4825 single-nucleotide polymorphism (SNP) markers. Prediction models were estimated for diameter and height at multiple ages using genomic random regression best linear unbiased predictor (BLUP). ? Accuracies of prediction models ranged from 0.65 to 0.75 for diameter, and 0.63 to 0.74 for height. The selection efficiency per unit time was estimated as 53-112% higher using genomic selection compared with phenotypic selection, assuming a reduction of 50% in the breeding cycle. Accuracies remained high across environments as long as they were used within the same breeding zone. However, models generated at early ages did not perform well to predict phenotypes at age 6 yr. ? These results demonstrate the feasibility and remarkable gain that can be achieved by incorporating genomic selection in breeding programs, as long as models are used at the relevant selection age and within the breeding zone in which they were estimated.