The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein

CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of beta-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CPalpha and CPbeta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPalpha is phosphorylated by protein kinase CK2 in vitro, that CPalpha is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPalpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.     

High-selenium yeast supplementation in free-living North American men: no effect on thyroid hormone metabolism or body composition.

In a prior study, we observed decreased serum 3,3',5-triiodothyronine (T(3)), increased serum thyrotropin and increased body weight in five men fed 297 microg/d of selenium (Se) in foods naturally high in Se while confined in a metabolic research unit. In an attempt to replicate and confirm those observations, we conducted a randomized study of high-Se yeast supplements (300 microg/d) or placebo yeast administered to 42 healthy free-living men for 48 weeks. Serum thyroxine, T(3) and thyrotropin did not change in supplemented or control subjects. Body weight increased in both groups during the 48-week treatment period and remained elevated for the 48-week follow-up period. Body fat increased by 1.2 kg in both groups. Energy intake and voluntary activity levels were not different between the groups and remained unchanged during the treatment period. Dietary intakes of Se, macronutrients and micronutrients were not different between groups and remained unchanged during the treatment period. These results suggest that our previous observation of a hypothyroidal response to high-Se foods was confounded by some aspect of the particular foods used, or were merely chance observations. Because of the high dose and long administration period, the present study suggests that the effects of Se supplements on thyroid hormone metabolism and energy metabolism in healthy North American men with adequate Se status do not represent a significant risk for unhealthy weight gain.     

Kidneys extract BNP and NT-proBNP in healthy young men.

Renal metabolism of the cardiac marker NH2-terminal-pro-brain natriuretic peptide (NT-proBNP) has been suggested. Therefore, we determined the renal extraction ratios of NT-proBNP and its bioactive coproduct brain natriuretic peptide (BNP) at rest and during exercise. In addition, the cerebral ratios were evaluated. Ten young healthy men were investigated at baseline, during moderate cycle exercise (heart rate: 140, Borg scale: 14-15), and in the recovery with BNP and NT-proBNP measured from the brachial artery and the jugular and renal veins, and the renal and cerebral extraction ratios (Ext-Ren and Ext-Cer, respectively) were calculated. Cardiac output, stroke volume, heart rate, mean arterial pressures, and estimated glomerular filtration were determined. BNP and NT-proBNP were extracted by the kidneys but not by the brain. We observed no effect of exercise. The mean values (+/- SE) of Ext-Ren of NT-proBNP were similar (0.19 +/- 0.05, 0.21 +/- 0.06, and 0.12 +/- 0.03, respectively) during the three sessions (P > 0.05). Also the Ext-Ren of BNP were similar (0.18 +/- 0.07, 0.15 +/- 0.11, and 0.14 +/- 0.06, respectively; P > 0.05). There were no significant differences between Ext-Ren of BNP and NT-proBNP during the three sessions (P > 0.05). The Ext-Cer of both peptides varied insignificantly between -0.21 +/- 0.15 and 0.11 +/- 0.08. The renal extraction ratio of both BNP and NT-proBNP is approximately 0.15-0.20. There is no cerebral extraction, and short-term moderate exercise does not affect these values. Our findings suggest that the kidneys extract BNP and NT-proBNP to a similar extent in healthy young men.     

Drosophila Myt1 is a Cdk1 inhibitory kinase that regulates multiple aspects of cell cycle behavior during gametogenesis

The metazoan Wee1-like kinases Wee1 and Myt1 regulate the essential mitotic regulator Cdk1 by inhibitory phosphorylation. This regulatory mechanism, which prevents Cdk1 from triggering premature mitotic events, is also induced during the DNA damage response and used to coordinate cell proliferation with crucial developmental events. Despite the previously demonstrated role for Myt1 regulation of Cdk1 during meiosis, relatively little is known of how Myt1 functions at other developmental stages. To address this issue, we have undertaken a functional analysis of Drosophila Myt1 that has revealed novel developmental roles for this conserved cell cycle regulator during gametogenesis. Notably, more proliferating cells were observed in myt1 mutant testes and ovaries than controls. This can partly be attributed to ectopic division of germline-associated somatic cells in myt1 mutants, suggesting that Myt1 serves a role in regulating exit from the cell cycle. Moreover, mitotic index measurements suggested that germline stem cells proliferate more rapidly, in myt1 mutant females. In addition, male myt1 germline cells occasionally undergo an extra mitotic division, resulting in meiotic cysts with twice the normal numbers of cells. Based on these observations, we propose that Myt1 serves unique Cdk1 regulatory functions required for efficient coupling of cell differentiation with cell cycle progression.     

IL-28A,IL-28B,and IL-29: Promising cytokines with type I interferon-like properties

IL-28A, IL-28B and IL-29 (also designated type III interferons) constitute a new subfamily within the IL-10–interferon family. They are produced by virtually any nucleated cell type, particularly dendritic cells, following viral infection or activation with bacterial components, and mediate their effects via the IL-28R1/IL-10R2 receptor complex. Although IL-28/IL-29 are closer to the IL-10-related cytokines in terms of gene structure, protein structure, and receptor usage, they display type I interferon-like anti-viral and cytostatic activities. Unlike type I interferons, the target cell populations of IL-28/IL-29 are restricted and mainly include epithelial cells and hepatocytes. These properties suggest that IL-28/IL-29 are potential therapeutic alternatives to type I interferons in terms of viral infections and tumors. This review describes the current knowledge about these cytokines.     

Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition

Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.     

Crystal structure of alkaline phosphatase from the Antarctic bacterium TAB5

Alkaline phosphatases (APs) are non-specific phosphohydrolases that are widely used in molecular biology and diagnostics. We describe the structure of the cold active alkaline phosphatase from the Antarctic bacterium TAB5 (TAP). The fold and the active site geometry are conserved with the other AP structures, where the monomer has a large central beta-sheet enclosed by alpha-helices. The dimer interface of TAP is relatively small, and only a single loop from each monomer replaces the typical crown domain. The structure also has typical cold-adapted features; lack of disulfide bridges, low number of salt-bridges, and a loose dimer interface that completely lacks charged interactions. The dimer interface is more hydrophobic than that of the Escherichia coli AP and the interactions have tendency to pair with backbone atoms, which we propose to result from the cold adaptation of TAP. The structure contains two additional magnesium ions outside of the active site, which we believe to be involved in substrate binding as well as contributing to the local stability. The M4 site stabilises an interaction that anchors the substrate-coordinating R148. The M5 metal-binding site is in a region that stabilises metal coordination in the active site. In other APs the M5 binding area is supported by extensive salt-bridge stabilisation, as well as positively charged patches around the active site. We propose that these charges, and the TAP M5 binding, influence the release of the product phosphate and thus might influence the rate-determining step of the enzyme.     

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1β replicon-specific sequences detected in total community DNA. The isolation of IncP-1β plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1β plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1β plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTΔP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1β plasmids R751 and pB8. However, this is the first example of an IncP-1β plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

The effect of implantation on scaffoldless three-dimensional engineered bone constructs

Our laboratory has previously developed scaffoldless engineered bone constructs (EBC). Bone marrow stromal cells (BMSC) were harvested from rat femur and cultured in medium that induced osteogenic differentiation. After reaching confluence, the monolayer of cells contracted around two constraint points forming a cylinder. EBCs were placed in small diameter (0.5905 × 0.0625 in.) or large diameter (0.5905 × 0.125 in.) silicone tubing and implanted intramuscularly in the hind limb of a rat. Bone mineral content (BMC) of the EBC was analyzed before implantation and at 1 and 2 mo following implantation and compared to that of native femur bone at different stages of development. Negligible BMC was observed in E-20 femur or EBCs prior to implantation. One-month implantation in both small and large tubing increased BMC in the EBC. BMC of EBC from large tubing was greater than in 14 d rat neonatal femurs, but was 2% and 3% of BMC content in adult bone after 1 and 2 mo of implantation, respectively. Alizarine Red and osteopontin staining of the EBCs before and after implantation confirmed increased bone mineralization in the implanted EBCs. Implanted EBCs also had extensive vascularization. Our data suggest that BMSC can be successfully used for the generation of scaffoldless EBC, and this model can be potentially used for the generation of autologous bone transplants in humans.