The kelch repeat protein,Tea1, is a potential substrate target of the p21-activated kinase,Shk1, in the fission yeast,Schizosaccharomyces pombe

The p21-activated kinase (PAK) homolog, Shk1, is a critical component of a multifunctional Ras/Cdc42/PAK complex required for viability, polarized growth and cell shape, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. Substrate targets of the Shk1 kinase have not previously been described. Here we show that the S. pombe cell polarity factor, Tea1, is directly phosphorylated by Shk1 in vitro. We demonstrate further that Tea1 is phosphorylated in S. pombe cells and that its level of phosphorylation is significantly reduced in cells defective in Shk1 function. Consistent with a role for Tea1 as a potential downstream effector of Shk1, we show that a tea1 null mutation rescues the Shk1 hyperactivity-induced lethal phenotype caused by loss of function of the essential Shk1 inhibitor, Skb15. All phenotypes associated with Skb15 loss, including defects in actin cytoskeletal organization, chromosome segregation, and cytokinesis, are suppressed by tea1 Delta, suggesting that Tea1 is a potential mediator of multiple Shk1 functions. S. pombe cells carrying a weak hypomorphic allele of shk1 together with a tea1 Delta mutation exhibit a cytokinesis defective phenotype that is significantly more severe than that observed in the respective single mutants, providing evidence that Shk1 and Tea1 cooperate to regulate cytokinesis. In addition, we show that S. pombe cells carrying the orb2-34 allele of shk1 exhibit a pattern of monopolar growth similar to that observed in tea1 Delta cells, suggesting that Shk1 and Tea1 may regulate one or more common processes involved in the regulation of polarized cell growth. Taken together, our results strongly implicate Tea1 as a potential substrate-effector of the Shk1 kinase.     

In vitro replication of spontaneous fractures of the proximal human femur

Spontaneous fractures (i.e. caused by sudden loading and muscle contraction, not by trauma) represent a significant percentage of proximal femur fractures. They are particularly relevant as may occur in elderly (osteoporotic) subjects, but also in relation to epiphyseal prostheses. Despite its clinical and legal relevance, this type of fracture has seldom been investigated. Studies concerning spontaneous fractures are based on a variety of loading scenarios. There is no evidence, nor consensus on the most relevant loading scenario. The aim of this work was to develop and validate an experimental method to replicate spontaneous fractures in vitro based on clinically relevant loading. Primary goals were: (i) repeatability and reproducibility, (ii) clinical relevance. A validated numerical model was used to identify the most critical loading scenario that can lead to head-neck fractures, and to determine if it is necessary to include muscle forces when the head-neck region is under investigation. The numerical model indicated that the most relevant loading scenario is when the resultant joint force is applied to the head at 8 degrees from the diaphysis. Furthermore, it was found that it is not essential to include the muscles when investigating head-neck fractures. The experimental setup was consequently designed. The procedure included high-speed filming of the fracture event. Clinically relevant fracture modes were obtained on 10 cadaveric femurs. Failure load should be reported as a fraction of donor's body-weight to reduce variability. The proposed method can be used to investigate the reason and mechanism of failure of natural and operated proximal femurs.     

Insect pest control agents: Novel chiral butanoate esters (juvenogens)

During the investigation of ester derivatives (juvenogens, biochemically activated insect hormonogenic compounds) of biologically active alcohols with potential application in insect pest control, a need for availability of all existing stereoisomers of ethyl N-{2-[4-(2-butanoyloxycyclohexyl)methyl]phenoxy}ethyl carbamate occurred. They were synthesized from their chiral precursors, the corresponding stereoisomers of 2-(4-methoxybenzyl)cyclohexyl butanoate, by removing their protecting group (methyl), and by subsequent condensation of the aromatic hydroxyl moiety with ethyl N-(2-bromoethyl) carbamate. The requested enantiomers of 2-(4-methoxybenzyl)cyclohexyl butanoate were obtained by a Candida antarctica lipase-mediated transesterification and chiral resolution of the respective racemic cis- and trans-isomers of 2-(4-methoxybenzyl)cyclohexanol either directly or after a subsequent chemical esterification of the chiral precursor. In this synthesis, two convenient butanoic acid activating esters, vinyl butanoate and 2,2,2-trifluoroethyl butanoate, were employed, and the chiral precursors in the synthesis of the target molecules were obtained in 41-48% yields (i.e., 82-96% conversion), and with enantiomeric purity ee=96-98%, respectively. The enantiomeric purity of the products was determined by chiral HPLC analysis, and their absolute configuration was assigned on the basis of analyzing the (1)H and (19)F NMR spectra of their diastereoisomeric Mosher acid (3,3,3-trifluoromethyl-2-methoxy-2-phenylpropanoic acid) esters.     

Basic fibroblast growth factor stimulates hepatocyte growth factor/scatter factor secretion by human mesenchymal cells

Basic fibroblast growth factor (bFGF) together with other pleiotropic factors plays an important role in many complex physiological processes such as embryonic development, angiogenesis, and wound repair. Among these factors, hepatocyte growth factor/scatter factor (HGF/SF) which is secreted by cells of mesodermal origin exerts its mito- and motogenic activities on cells of epithelial and endothelial origin. Knowledge of the regulatory mechanisms of HGF/SF may contribute to the understanding of its role in physio-pathological processes. We observed that the secretion of HGF/SF by MRC-5 cells and by other fibroblast-derived cell cultures in conditioned media was enhanced by exposure to bFGF. HGF/SF was measured by the scatter assay, a bioassay for cell motility, and was further characterized by Western blot analysis with anti-HGF/SF antibodies. Exposure of MRC-5 cultures to 10 ng/ml of bFGF resulted already 6 h posttreatment in a threefold higher amount of scatter factor secreted into the medium as compared to untreated cultures. HGF/SF secretion was sustained after bFGF treatment for the following 72 h when increased amounts of HGF/SF were detected both in conditioned media as well as associated to the extracellular matrix. The secretion of HGF/SF in cell supernatants increased dose dependently upon treatment with bFGF starting from basal levels of 6 U/ml and reaching 27 U/ml at 30 ng/ml bFGF, plateauing thereafter. Upregulation of HGF/SF by IL-1, already described by others, was confirmed in this study. Based on our findings an articulated interaction can be speculated for bFGF, HGF/SF, and IL-1, e.g., in tissue regeneration during inflammatory processes or in wound healing. © 1996 Wiley-Liss, Inc.     

An Interaction Network of RNA-Binding Proteins Involved in Drosophila Oogenesis

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Highlights

• •Label-free and dimethyl labeling MS analysis of 6 RBPs from Drosophila ovaries.
• •Functionally related RBPs show overlapping proteomes.
• •Selective co-purification of splicing factors and translational regulators.
• •Validation of 26 novel interactions by co-immunoprecipitation.

     

Optically Active α‐Phenylethylamine as Efficient Organocatalyst in the Solvent‐free Reactions Between 2,3‐Butanedione and Conjugated Nitroolefins

(R)‐(+) and (S)‐(?)‐1‐phenylethylamine have been shown to promote highly diastereoselective and complementary enantioselective formal [3 + 2]carbocyclization reactions between 2,3‐butanedione and conjugated nitroalkenes with formation of enantiomerically rich 2‐hydroxy‐3‐nitrocyclopentanone derivatives. The reactions were carried out both in solvent and under solvent‐free conditions. The absolute configurations of the products were assigned by X‐ray and circular dichroism spectra analyses. Chirality 24:1005–1012, 2012. © 2012 Wiley Periodicals, Inc.     

Neurocognitive dysfunction in systemic lupus erythematosus: association with antiphospholipid antibodies, disease activity and chronic damage

Introduction

Systemic lupus erythematosus (SLE) is characterized by frequent neuropsychiatric involvement, which includes cognitive impairment (CI). We aimed at assessing CI in a cohort of Italian SLE patients by using a wide range of neurocognitive tests specifically designed to evaluate the fronto-subcortical dysfunction. Furthermore, we aimed at testing whether CI in SLE is associated with serum autoantibodies, disease activity and chronic damage.

Methods

Fifty-eight consecutive patients were enrolled. Study protocol included data collection, evaluation of serum levels of ANA, anti-dsDNA, anti-cardiolipin, anti-β₂-glycoprotein I, anti-P ribosomal, anti-endothelial cell, and anti-Nedd5 antibodies. SLEDAI-2000 and SLICC were used to assess disease activity and chronic damage. Patients were administered a test battery specifically designed to detect fronto-subcortical dysfunction across five domains: memory, attention, abstract reasoning, executive function and visuospatial function. For each patient, the raw scores from each test were compared with published norms, then transformed into Z scores (deviation from normal mean), and finally summed in the Global Cognitive Dysfunction score (GCDs).

Results

Nineteen percent of patients had mild GCDs impairment (GCDs 2–3), 7% moderate (GCDs 4–5) and 5% severe (GCDs≥6). The visuospatial domain was the most compromised (MDZs = −0.89±1.23). Anti-cardiolipin IgM levels were associated with visuospatial domain impairment (r = 0.331, P = 0.005). SLEDAI correlated with GCDs, and attentional and executive domains; SLICC correlated with GCDs, and with visuospatial and attentional domains impairment.

Conclusions

Anti-phospholipids, disease activity, and chronic damage are associated with cognitive dysfunction in SLE. The use of a wide spectrum of tests allowed for a better selection of the relevant factors involved in SLE cognitive dysfunction, and standardized neuropsychological testing methods should be used for routine assessment of SLE patients.     

Cardiac differentiation promotes mitochondria development and ameliorates oxidative capacity in H9c2 cardiomyoblasts

H9c2 undergoing cardiac differentiation induced by all-trans-retinoic acid were investigated for mitochondria structural features together with the implied functional changes, as a model for the study of mitochondrial development in cardiogenic progenitor cells. As the expression of cardiac markers became detectable, mitochondrial mass increased and mitochondrial morphology and ultrastructure changed. Reticular network organization developed and more bulky mitochondria with greater numbers of closely packed cristae and more electron-dense matrix were detected. Increased expression of PGC-1α proved the occurrence of mitochondrial biogenesis. Improvements in mitochondrial energetic competence were also documented, linked to better assembly between F(0) and F(1) sectors of the F(0)F(1)ATPsynthase enzyme complex.     

To what extent can linear finite element models of human femora predict failure under stance and fall loading configurations?

Proximal femur strength estimates from computed tomography (CT)-based finite element (FE) models are finding clinical application. Published models reached a high in-vitro accuracy, yet many of them rely on nonlinear methodologies or internal best-fitting of parameters. The aim of the present study is to verify to what extent a linear FE modelling procedure, fully based on independently determined parameters, can predict the failure characteristics of the proximal femur in stance and sideways fall loading configurations.