Spin asymmetries for electron-thallium scattering calculated with the relativistic convergentclose-coupling method

Spin asymmetries for elastic and inelastic scattering of electrons from thallium are presented. Thalliumis a heavy target (Z 81) and the spin asymmetries can be caused by relativistic effects (spin-orbit interactions) in addition to exchange effects

De Novo Generated Human Red Blood Cells in Humanized Mice Support Plasmodium falciparum Infection

Immunodeficient mouse–human chimeras provide a powerful approach to study host specific pathogens like Plasmodium (P.) falciparum that causes human malaria. Existing mouse models of P. falciparum infection require repeated injections of human red blood cells (RBCs). In addition, clodronate lipsomes and anti-neutrophil antibodies are injected to suppress the clearance of human RBCs by the residual immune system of the immunodeficient mice. Engraftment of NOD-scid Il2rg[superscript -/-] mice with human hematopoietic stem cells leads to reconstitution of human immune cells. Although human B cell reconstitution is robust and T cell reconstitution is reasonable in the recipient mice, human RBC reconstitution is generally poor or undetectable. The poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages. Delivery of plasmid DNA encoding human erythropoietin and interleukin-3 into humanized mice by hydrodynamic tail-vein injection resulted in significantly enhanced reconstitution of erythrocytes. With this improved humanized mouse, here we show that P. falciparum infects de novo generated human RBCs, develops into schizonts and causes successive reinvasion. We also show that different parasite strains exhibit variation in their ability to infect these humanized mice. Parasites could be detected by nested PCR in the blood samples of humanized mice infected with P. falciparum K1 and HB3 strains for 3 cycles, whereas in other strains such as 3D7, DD2, 7G8, FCR3 and W2mef parasites could only be detected for 1 cycle. In vivo adaptation of K1 strain further improves the infection efficiency and parasites can be detected by microscopy for 3 cycles. The parasitemia ranges between 0.13 and 0.25% at the first cycle of infection, falls between 0.08 and 0.15% at the second cycle, and drops to barely detectable levels at the third cycle of infection. Compared to existing mouse models, our model generates human RBCs de novo and does not require the treatment of mice with immunomodulators.Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology

Pulsatile ex vivo perfusion of human saphenous vein grafts under controlled pressure conditions increases MMP-2 expression

<p>Abstract</p> <p>Background</p> <p>The use of human saphenous vein grafts (HSVGs) as a bypass conduit is a standard procedure in the treatment of coronary artery disease while their early occlusion remains a major problem.</p> <p>Methods</p> <p>We have developed an <it>ex vivo </it>perfusion system, which uses standardized and strictly controlled hemodynamic parameters for the pulsatile and non-static perfusion of HSVGs to guarantee a reliable analysis of molecular parameters under different pressure conditions. Cell viability of HSVGs (n = 12) was determined by the metabolic conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) into a purple formazan dye.</p> <p>Results</p> <p>Under physiological flow rates (10 mmHg) HSVGs remained viable for two weeks. Their exposure to arterial conditions (100 mmHg) was possible for one week without important reduction in viability. Baseline expression of matrix metalloproteinase-2 (MMP-2) after venous perfusion (2.2 ± 0.5, n = 5) was strongly up-regulated after exposure to arterial conditions for three days (19.8 ± 4.3) or five days (23.9 ± 6.1, p < 0.05). Zymographic analyses confirmed this increase on the protein level. Our results suggest that expression and activity of MMP-2 are strongly increased after exposure of HSVGs to arterial hemodynamic conditions compared to physiological conditions.</p> <p>Conclusion</p> <p>Therefore, our system might be helpful to more precisely understand the molecular mechanisms leading to an early failure of HSVGs.</p

Deletion of PKBα/Akt1 Affects Thymic Development

BACKGROUND: The thymus constitutes the primary lymphoid organ for the majority of T cells. The phosphatidyl-inositol 3 kinase (PI3K) signaling pathway is involved in lymphoid development. Defects in single components of this pathway prevent thymocytes from progressing beyond early T cell developmental stages. Protein kinase B (PKB) is the main effector of the PI3K pathway. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether PKB mediates PI3K signaling in the thymus, we characterized PKB knockout thymi. Our results reveal a significant thymic hypocellularity in PKBalpha(-/-) neonates and an accumulation of early thymocyte subsets in PKBalpha(-/-) adult mice. Using thymic grafting and fetal liver cell transfer experiments, the latter finding was specifically attributed to the lack of PKBalpha within the lymphoid component of the thymus. Microarray analyses show that the absence of PKBalpha in early thymocyte subsets modifies the expression of genes known to be involved in pre-TCR signaling, in T cell activation, and in the transduction of interferon-mediated signals. CONCLUSIONS/SIGNIFICANCE: This report highlights the specific requirements of PKBalpha for thymic development and opens up new prospects as to the mechanism downstream of PKBalpha in early thymocytes

The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli : a comparison with the traditional gene fusion technology

The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.The financial support of the EMBL Heidelberg, Germany and Fundacao para a Ciencia e Tecnologia (FCT), Portugal, is acknowledged: the fellowship SFRH/BD/46482/2008 to Sofia J. Costa and the project PTDC/CVT/103081/2008. The authors wish to acknowledge Anne-Claude Gavin for providing four of the constructs for this study (RVS167, SPO14, YPK1, and YPK2) and Emmanuel Poilpre for the experimental help (both from the EMBL Heidelberg, Germany)

Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes.

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 µm; IIB, K(d) = 8 µm; IIC, K(d) = 1.0 µm). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade

Novel tumor suppressive function of Smad4 in serum starvation-induced cell death through PAK1–PUMA pathway

DPC4 (deleted in pancreatic cancer 4)/Smad4 is an essential factor in transforming growth factor (TGF)-β signaling and is also known as a frequently mutated tumor suppressor gene in human pancreatic and colon cancer. However, considering the fact that TGF-β can contribute to cancer progression through transcriptional target genes, such as Snail, MMPs, and epithelial–mesenchymal transition (EMT)-related genes, loss of Smad4 in human cancer would be required for obtaining the TGF-β signaling-independent advantage, which should be essential for cancer cell survival. Here, we provide the evidences about novel role of Smad4, serum-deprivation-induced apoptosis. Elimination of serum can obviously increase the Smad4 expression and induces the cell death by p53-independent PUMA induction. Instead, Smad4-deficient cells show the resistance to serum starvation. Induced Smad4 suppresses the PAK1, which promotes the PUMA destabilization. We also found that Siah-1 and pVHL are involved in PAK1 destabilization and PUMA stabilization. In fact, Smad4-expressed cancer tissues not only show the elevated expression of PAK1, but also support our hypothesis that Smad4 induces PUMA-mediated cell death through PAK1 suppression. Our results strongly suggest that loss of Smad4 renders the resistance to serum-deprivation-induced cell death, which is the TGF-β-independent tumor suppressive role of Smad4

p21-Activated Kinases Are Required for Transformation in a Cell-Based Model of Neurofibromatosis Type 2

NF2 is an autosomal dominant disease characterized by development of bilateral vestibular schwannomas and other benign tumors in central nervous system. Loss of the NF2 gene product, Merlin, leads to aberrant Schwann cell proliferation, motility, and survival, but the mechanisms by which this tumor suppressor functions remain unclear. One well-defined target of Merlin is the group I family of p21-activated kinases, which are allosterically inhibited by Merlin and which, when activated, stimulate cell cycle progression, motility, and increased survival. Here, we examine the effect of Pak inhibition on cells with diminished Merlin function.Using a specific peptide inhibitor of group I Paks, we show that loss of Pak activity restores normal cell movement in cells lacking Merlin function. In addition, xenografts of such cells form fewer and smaller tumors than do cells without Pak inhibition. However, in tumors, loss of Pak activity does not reduce Erk or Akt activity, two signaling proteins that are thought to mediate Pak function in growth factor pathways.These results suggest that Pak functions in novel signaling pathways in NF2, and may serve as a useful therapeutic target in this disease