Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission

Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.     

Isolation, purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hydrolyzate

Sarcoplasmic proteins isolated from bovine livers were hydrolyzed using the enzyme thermolysin at 37 ?C for 2 h. The hydrolyzates were filtered through molecular weight cut off membranes (MWCO) and filtrates were obtained. The water activity (a_w) of unhydrolysed sarcoplasmic protein, full hydrolyzates, 10-kDa and 3-kDa filtrates were below the limit necessary for microbial growth. The antioxidant activities of both filtrates and fractions were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe²⁺ chelating ability assay. RP-HPLC was used for purification of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates. The peptidic content of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates were assessed using the Dumas method and peptide contents of each fraction were characterized using electrospray quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry with the resultant spectrum analysed using the software programmes Protein Lynx Global Server 2.4. and TurboSEQUEST. Similarities between the amino acid composition of characterized peptides from each fraction and previously reported antioxidant peptides were found. This study demonstrates that meat by-product such as liver can be utilised as raw material for the generation of bioactive peptides with demonstrated antioxidant activities in vitro using the enzyme thermolysin. It is significant as it presents a potential opportunity for meat processors to use their waste streams for the generation of bioactive peptides for potential functional food use.     

Amino-terminal phosphorylation of activation-induced cytidine deaminase suppresses c-myc/IgH translocation

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates class switch recombination and somatic hypermutation of immunoglobulin genes (Ig) in B lymphocytes. However, AID also produces off-target DNA damage, including mutations in oncogenes and double-stranded breaks that can serve as substrates for oncogenic chromosomal translocations. AID is strictly regulated by a number of mechanisms, including phosphorylation at serine 38 and threonine 140, which increase activity. Here we show that phosphorylation can also suppress AID activity in vivo. Serine 3 is a novel phospho-acceptor which, when mutated to alanine, leads to increased class switching and c-myc/IgH translocations without affecting AID levels or catalytic activity. Conversely, increasing AID phosphorylation specifically on serine 3 by interfering with serine/threonine protein phosphatase 2A (PP2A) leads to decreased class switching. We conclude that AID activity and its oncogenic potential can be downregulated by phosphorylation of serine 3 and that this process is controlled by PP2A.     

The CD40 agonist antibody CP-870,893 enhances dendritic cell and B-cell activity and promotes anti-tumor efficacy in SCID-hu mice

CD40 is a member of the TNF family of receptors that has been shown to play a crucial role in enhancing dendritic cell activity and fostering anti-tumor immune responses. In this study, we demonstrate the in vitro properties and in vivo efficacious activity of the CD40 agonist antibody, CP-870,893. CP-870,893 is a fully human, IgG2 antibody that selectively interacts with CD40 at a site distinct from its ligand-binding region with a KD of 0.4?nM. It enhances the expression of MHC class II, CD54, CD86, and CD23 on human B cells in vitro. CP-870,893 also enhances dendritic cell activity as evidenced by cytokine secretion (IL-12, IL-23, IL-8), the upregulation of CD86 and CD83, and the ability to prime T cells to secrete IFNγ. In SCID-beige mice, a single parenteral injection of CP-870,893 was therapeutically effective against several CD40(pos) human tumors (B-cell lymphoma, breast, colon, and prostate) indicating direct effects on tumor cell survival and/or growth. When mice were co-implanted with human T cells and dendritic cells, the activity of CP-870,893 against CD40(pos) tumors increased, and efficacy was also observed against CD40(neg) and CD40(low) tumors demonstrating the ability of CP-870,893 to enhance anti-tumor immune function in vivo. These studies suggest that CP-870,893 has the potential to be efficacious against a wide range of tumor types through both direct and immune-mediated effects.     

Substance P signaling mediates BMP-dependent heterotopic ossification

Heterotopic ossification (HO) is a disabling condition associated with neurologic injury, inflammation, and overactive bone morphogenetic protein (BMP) signaling. The inductive factors involved in lesion formation are unknown. We found that the expression of the neuro-inflammatory factor Substance P (SP) is dramatically increased in early lesional tissue in patients who have either fibrodysplasia ossificans progressiva (FOP) or acquired HO, and in three independent mouse models of HO. In Nse-BMP4, a mouse model of HO, robust HO forms in response to tissue injury; however, null mutations of the preprotachykinin (PPT) gene encoding SP prevent HO. Importantly, ablation of SP(+) sensory neurons, treatment with an antagonist of SP receptor NK1r, deletion of NK1r gene, or genetic down-regulation of NK1r-expressing mast cells also profoundly inhibit injury-induced HO. These observations establish a potent neuro-inflammatory induction and amplification circuit for BMP-dependent HO lesion formation, and identify novel molecular targets for prevention of HO.     

A role for zinc in regulating hypoxia-induced contractile events in pulmonary endothelium

We previously reported that zinc thiolate signaling contributes to hypoxic contraction of small, nonmuscularized arteries of the lung. The present studies were designed to investigate mechanisms by which hypoxia-released zinc induces contraction in isolated pulmonary endothelial cells and to delineate the signaling pathways involved in zinc-mediated changes in the actin cytoskeleton. We used fluorescence-based imaging to show that hypoxia induced time-dependent increases in actin stress fibers that were reversed by the zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). We further showed that hypoxia-induced phosphorylation of the contractile protein myosin light chain (MLC) and assembly of actin stress fibers were each TPEN sensitive. Hypoxia and zinc-induced inhibition of MLC phosphatase (MLCP) were independent of the regulatory subunit (MYPT1) of MLCP, and therefore hypoxia-released zinc likely inhibits MLCP at its catalytic (PP1) subunit. Inhibition of PKC by Ro-31-8220 and a dominant-negative construct of PKC-ε attenuated hypoxia-induced contraction of isolated pulmonary endothelial cells. Furthermore, zinc-induced phosphorylation of MLC (secondary to inhibition of MLCP) was PKC dependent, and hypoxia-released zinc promoted the phosphorylation of the PKC substrate, CPI-17. Collectively, these data suggest a link between hypoxia, elevations in labile zinc, and activation of PKC, which in turn acts through CPI-17 to inhibit MLCP activity and promote MLC phosphorylation, ultimately inducing stress fiber formation and endothelial cell contraction.     

Essential roles of BCCIP in mouse embryonic development and structural stability of chromosomes

BCCIP is a BRCA2- and CDKN1A(p21)-interacting protein that has been implicated in the maintenance of genomic integrity. To understand the in vivo functions of BCCIP, we generated a conditional BCCIP knockdown transgenic mouse model using Cre-LoxP mediated RNA interference. The BCCIP knockdown embryos displayed impaired cellular proliferation and apoptosis at day E7.5. Consistent with these results, the in vitro proliferation of blastocysts and mouse embryonic fibroblasts (MEFs) of BCCIP knockdown mice were impaired considerably. The BCCIP deficient mouse embryos die before E11.5 day. Deletion of the p53 gene could not rescue the embryonic lethality due to BCCIP deficiency, but partially rescues the growth delay of mouse embryonic fibroblasts in vitro. To further understand the cause of development and proliferation defects in BCCIP-deficient mice, MEFs were subjected to chromosome stability analysis. The BCCIP-deficient MEFs displayed significant spontaneous chromosome structural alterations associated with replication stress, including a 3.5-fold induction of chromatid breaks. Remarkably, the BCCIP-deficient MEFs had a ～20-fold increase in sister chromatid union (SCU), yet the induction of sister chromatid exchanges (SCE) was modestly at 1.5 fold. SCU is a unique type of chromatid aberration that may give rise to chromatin bridges between daughter nuclei in anaphase. In addition, the BCCIP-deficient MEFs have reduced repair of irradiation-induced DNA damage and reductions of Rad51 protein and nuclear foci. Our data suggest a unique function of BCCIP, not only in repair of DNA damage, but also in resolving stalled replication forks and prevention of replication stress. In addition, BCCIP deficiency causes excessive spontaneous chromatin bridges via the formation of SCU, which can subsequently impair chromosome segregations in mitosis and cell division.     

Critical Interaction Domains between Bloom Syndrome Protein and RAD51

The American Cancer Society’s 2009 statistics estimate that 1 out of every 4 deaths is cancer related. Genomic instability is a common feature of cancerous states, and an increase in genomic instability is the diagnostic feature of Bloom Syndrome. Bloom Syndrome, a rare disorder characterized by a predisposition to cancer, is caused by mutations of the BLM gene. This study focuses on the partnerships of BLM protein to RAD51, a Homologous Recombination repair protein essential for survival. A systematic set of BLM deletion fragments were generated to refine the protein binding domains of BLM to RAD51 and determine interacting regions of BLM and ssDNA. Results show that RAD51 and ssDNA interact in overlapping regions; BLM_100–214 and BLM_1317–1367. The overlapping nature of these regions suggests a preferential binding for one partner that could function to regulate homologous recombination and therefore helps to clarify the role of BLM in maintaining genomic stability.     

Water turnover assessment in overweight adolescents

Adequate intake (AI) standards for water in adolescents range between 2.4-3.3 l/day for males and 2.1-2.3 l/day for females, independent of obesity status. Water intakes and excretions of this population are not well documented. The purposes of this study were to assess water turnover, inputs, and outputs in overweight adolescents, compare these parameters between males and females, and evaluate the reproducibility of water turnover. Eighteen girls (BMI 31.7 ± 4 kg/m(2); mean ± s.d.) and nine boys (BMI 26.3 ± 3 kg/m(2)) aged 12-15 years completed two 3-week metabolic balance trials. Rate of water turnover (rH(2)O) was measured by tracking the decline of deuterated water from the body over 14 days. Water inputs (diet*, ad libitum(#), metabolic(#)) and outputs (urine*, feces*, insensible(#)) were assessed (*measured, #estimated). rH(2)O was lower (P = 0.002) in girls vs. boys (3,742 ± 536 vs. 4,537 ± 623 g/day). Per kg body weight, rH(2)O was 28% lower in girls vs. boys (46 ± 7 vs. 64 ± 9 g·kg(-1)·day(-1)). Water input from food and beverages provided and metabolic production were 44 and 28% lower, respectively, in girls vs. boys. Urine and insensible water losses were 21 and 17% lower in girls vs. boys. BMI was positively associated with water turnover in both sexes (girls P = 0.037; boys P = 0.014). The intraclass correlation of rH(2)O between trials was 0.981 (P < 0.001). In conclusion, these overweight adolescents consumed water well in excess of sex-specific AI standards. The lower rH(2)O in girls compared to boys is consistent with adult females and males.