Tumor necrosis factor-alpha-converting enzyme mediates the inducible cleavage of fractalkine

Fractalkine (FK, CX3CL1) is a novel multidomain protein expressed on the surface of endothelial cells. As a full-length transmembrane protein, FK binds cells expressing CX3CR1, its cognate receptor, with high affinity. Proteolytic cleavage of FK releases a soluble form that is a potent chemoattractant for monocytes, T cells, and natural killer cells. Activation of protein kinase C dramatically increases the rate of this cleavage. Regulation of FK cleavage is critical for maintaining the balance between the immobilized and soluble forms, but the protease responsible has not been identified. Here we report that tumor necrosis factor-alpha-converting enzyme (TACE) is primarily responsible for the inducible cleavage of FK. After transfection into host cells, the proteolytic cleavage of FK was blocked by TACE-specific inhibitors and was not detected in cells genetically altered to remove TACE activity. In contrast, the constitutive cleavage of FK was not mediated by TACE and proceeded normally in TACE-null fibroblasts. We conclude that TACE is primarily responsible for the inducible cleavage of FK. These studies identify a potentially important link between local generation of potent cytokines and control of the balance between the cell adhesion and chemotactic properties of FK.     

Small B cell lymphocytic lymphoma presenting as obstructive sleep apnea

Background  

Most lymphomas that involve the tonsil are large B cell lymphomas. Large B-cell lymphoma is a high grade malignancy which progresses rapidly. Tonsillar lymphoma usually presents as either a unilaterally enlarged palatine tonsil or as an ulcerative and fungating lesion over the tonsillar area. Small lymphocytic lymphomas (SLL) of the Waldeyer's ring are uncommon.     

Effects of single nucleotide polymorphisms in the 5'-flanking region of heat shock protein 70.2 gene on semen quality in boars

This study aims to elucidate the effects of single nucleotide polymorphisms (SNPs) in the 5'-flanking region of porcine heat shock protein 70.2 gene (HSP70.2) on semen quality in boars. Genomic DNA isolated from 55 boars (41 Duroc, nine Landrace, and five Yorkshire) was subjected to PCR amplification of the 5'-flanking region of HSP70.2. The nucleotide sequences were determined by automated sequencing. Five SNPs (sites 44, 232, 250, 345, and 393) were detected in this region. Semen quality was evaluated in terms of sperm motility, percentage of normal sperm, percentage of sperm with proximal plasma droplet, percentage of abnormal sperm, sperm concentration, semen volume per ejaculate and total sperm number per ejaculate. The effect of the SNPs on semen quality was evaluated based on breed-corrected data within a season. During the cool season, the sperm motility of boars with AA genotype at the 232 site was significantly higher than that of boars with CC genotype (P<0.05). Meanwhile, boars with AC genotype at the 232 site had higher total sperm number per ejaculate than did those with CC genotype. In the hot season, heterozygotes at both the 232 and 250 sites had significantly higher total sperm number of per ejaculate than AA homozygotes (P<0.05). Semen volume of boars with TT and TC genotypes at the 345 site was significantly larger than that of those with CC genotype (P<0.05). Meanwhile, semen quality for boars with TT genotype at the 345 site was significantly higher than that of boars with TC or CC genotype (P<0.05), that is the semen contained higher percentages of normal sperm and lower percentages of abnormal sperm or sperm with proximal plasma droplets. Results herein suggest that the SNPs in the 5'-flanking region of porcine HSP70.2 are associated with semen quality traits in the hot season.     

Integrin activation is required for VEGF and FGF receptor protein presence on human microvascular endothelial cells

Endothelial cell proliferation and migration is initiated by growth factors including FGF and VEGF that bind to specific transmembrane receptor tyrosine kinases. Mechanisms that regulate in vivo expression of fibroblast growth factor receptors (FGFR) and vascular endothelial growth factor receptors (VEGFR) are not well understood. Since it is well known that different matrices influence the proliferation and migration of endothelial cells in culture, we hypothesized that changes in the extracellular matrix environment can regulate growth factor receptors on endothelial cells. We cultured human microvascular endothelial cells on different matrices (vitronectin, laminin, fibronectin, fibrin, and collagen IV) and examined for the presence of growth factor receptors (FGFR-1, FGFR-2, VEGFR-1, and VEGFR-2). We show that vitronectin increased the presence of all four growth factor receptors and most notably, VEGFR-1. In contrast, fibrin decreased all four receptors, especially FGFR-1 and FGFR-2. Inhibiting phosphotyrosine signaling abolished immunostaining for all four receptors, regardless of the matrix, but was not dependent on activating the Fyn-Shc pathway. Cells plated on vitronectin in the presence of blocking antibodies to integrins _v3 and _v5 similarly decreased presence of these growth factor receptors. Our data suggests a possible mechanism of how matrix-integrin interactions regulate endothelial cell responsiveness to growth factors and anchorage-dependent cell growth.     

Dissecting the EGFR-PI3K-AKT pathway in oral cancer highlights the role of the EGFR variant III and its clinical relevance

Background

Dysregulated epidermal growth factor receptor (EGFR)-phosphoinositide-3-kinase (PI3K)-AKT signaling is considered pivotal for oral cancer, and the pathway is a potential candidate for therapeutic targeting.

Results

A total of 108 archival samples which were from surgically resected oral cancer were examined. Immunohistochemical staining showed the protein expression of membranous wild-type EGFR and cytoplasmic phosphorylated AKT was detected in 63.9% and 86.9% of the specimens, respectively. In 49.1% of the samples, no phosphatase and tensin homolog (PTEN) expression was detected. With regard to the EGFR variant III (EGFRvIII), 75.0% of the samples showed positive expression for moderate to severe staining, 31.5% of which had high expression levels. Real-time polymerase chain reaction assays for gene copy number assessment of PIK3CA revealed that 24.8% of the samples had alterations, and of EGFR showed that 49.0% had amplification. Direct sequencing of PIK3CA gene showed 2.3% of the samples had a hotspot point mutation. Statistical assessment showed the expression of the EGFRvIII correlated with the T classification and TNM stage. The Kaplan-Meier analyses for patient survival showed that the individual status of phosphorylated AKT and EGFRvIII led to significant differences in survival outcome. The multivariate analysis indicated that phosphorylated AKT, EGFRvIII expression and disease stage were patient survival determinants.

Conclusions

Aberrations in the EGFR-PI3K-AKT pathway were frequently found in oral cancers. EGFRvIII and phosphorylated AKT were predictors for the patient survival and clinical outcome.     

Chemical genetic screen for AMPKα2 substrates uncovers a network of proteins involved in mitosis

The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in?vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.     

Fixed Dystonia in Complex Regional Pain Syndrome: a Descriptive and Computational Modeling Approach

Background  

Complex regional pain syndrome (CRPS) may occur after trauma, usually to one limb, and is characterized by pain and disturbed blood flow, temperature regulation and motor control. Approximately 25% of cases develop fixed dystonia. Involvement of dysfunctional GABAergic interneurons has been suggested, however the mechanisms that underpin fixed dystonia are still unknown. We hypothesized that dystonia could be the result of aberrant proprioceptive reflex strengths of position, velocity or force feedback.     

Alkynyl-farnesol reporters for detection of protein S-prenylation in cells

Protein S-prenylation is a lipid modification that regulates membrane-protein and protein-protein interactions in cell signaling. Though sites of protein S-prenylation can be predicted based upon conserved C-terminal CaaX or CC/CXC motifs, biochemical detection of protein S-prenylation in cells is still challenging. Herein, we report an alkynyl-isoprenol chemical reporter (alk-FOH) as an efficient substrate for prenyltransferases in mammalian cells that enables sensitive detection of S-farnesylated and S-geranylgeranylated proteins using bioorthogonal ligation methods. Fluorescent detection alleviates the need to deplete cellular isoprenoids for biochemical analysis of S-prenylated proteins and enables robust characterization of S-prenylated proteins, such as effectors that are injected into host cells by bacterial pathogens. This alkynyl-prenylation reporter provides a sensitive tool for biochemical analysis and rapid profiling of prenylated proteins in cells.     

Ubiquitination Regulates the Neuroprotective Function of the Deubiquitinase Ataxin-3 in Vivo

Deubiquitinases (DUBs) are proteases that regulate various cellular processes by controlling protein ubiquitination. Cell-based studies indicate that the regulation of the activity of DUBs is important for homeostasis and is achieved by multiple mechanisms, including through their own ubiquitination. However, the physiological significance of the ubiquitination of DUBs to their functions in vivo is unclear. Here, we report that ubiquitination of the DUB ataxin-3 at lysine residue 117, which markedly enhances its protease activity in vitro, is critical for its ability to suppress toxic protein-dependent degeneration in Drosophila melanogaster. Compared with ataxin-3 with only Lys-117 present, ataxin-3 that does not become ubiquitinated performs significantly less efficiently in suppressing or delaying the onset of toxic protein-dependent degeneration in flies. According to further studies, the C terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase that ubiquitinates ataxin-3 in vitro, is dispensable for its ubiquitination in vivo and is not required for the neuroprotective function of this DUB in Drosophila. Our work also suggests that ataxin-3 suppresses degeneration by regulating toxic protein aggregation rather than stability.