The Autophagy Inhibitor Bafilomycin Inhibits Antibody-Dependent Natural Killer Cell-Mediated Killing of Breast Carcinoma Cells.

The resistance of breast cancer cells to therapeutic antibodies such as anti-HER2 trastuzumab can be overcome by engaging natural killer (NK) cells for killing antibody-binding tumor cells via antibody-dependent cellular cytotoxicity (ADCC). Here, we investigated how autophagy modulation affects trastuzumab-mediated ADCC in HER2-positive JIMT1 breast cancer cells and NK cells. Autophagy inducers (rapamycin and resveratrol) had no significant impact, but the inhibitor bafilomycin nearly abolished ADCC.

Smoothelin-like protein 1 promotes insulin sensitivity and modulates the contractile properties of endometrial epithelial cells with insulin resistance.

Introduction: The incidence of infertility is significantly higher in women with diseases linked to impaired glucose homeostasis, such as insulin resistance. Defective glucose metabolism interferes with fertilization; however, the molecular mechanism underlying this interference is unclear. Smoothelin-like protein 1 (SMTNL1) was isolated from muscle and steroid hormone-responsive tissues and regulates the contractile functions of various cell types through the inhibition of myosin phosphatase (MP) holoenzyme.

Identification of Bacterial Metabolites Modulating Breast Cancer Cell Proliferation and Epithelial-Mesenchymal Transition.

Breast cancer patients are characterized by the oncobiotic transformation of multiple microbiome communities, including the gut microbiome. Oncobiotic transformation of the gut microbiome impairs the production of antineoplastic bacterial metabolites. The goal of this study was to identify bacterial metabolites with antineoplastic properties.

Migration of Myogenic Cells Is Highly Influenced by Cytoskeletal Septin7.

Septin7 as a unique member of the GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be essential in the formation of hetero-oligomeric septin complexes. As a cytoskeletal component, Septin7 is involved in many important cellular processes. However, its contribution in striated muscle physiology is poorly described.

Adenosine A Receptor Activation Regulates Niemann-Pick C1 Expression and Localization in Macrophages.

Adenosine plays an important role in modulating immune cell function, particularly T cells and myeloid cells, such as macrophages and dendritic cells. Cell surface adenosine A receptors (AR) regulate the production of pro-inflammatory cytokines and chemokines, as well as the proliferation, differentiation, and migration of immune cells. In the present study, we expanded the AR interactome and provided evidence for the interaction between the receptor and the Niemann-Pick type C intracellular cholesterol transporter 1 (NPC1) protein.

Cathepsin D interacts with adenosine A receptors in mouse macrophages to modulate cell surface localization and inflammatory signaling.

Adenosine A receptor (AR)-dependent signaling in macrophages plays a key role in the regulation of inflammation. However, the processes regulating AR targeting to the cell surface and degradation in macrophages are incompletely understood. For example, the C-terminal domain of the AR and proteins interacting with it are known to regulate receptor recycling, although it is unclear what role potential AR-interacting partners have in macrophages.

The voltage-gated proton channel hHv1 is functionally expressed in human chorion-derived mesenchymal stem cells.

The voltage-gated proton channel Hv1 is widely expressed, among others, in immune and cancer cells, it provides an efficient cytosolic Hextrusion mechanism and regulates vital functions such as oxidative burst, migration and proliferation. Here we demonstrate the presence of human Hv1 (hHv1) in the placenta/chorion-derived mesenchymal stem cells (cMSCs) using RT-PCR. The voltage- and pH-dependent gating of the current is similar to that of hHv1 expressed in cell lines and that the current is blocked by 5-chloro-2-guanidinobenzimidazole (ClGBI) and activated by arachidonic acid (AA).

Endogenous single-strand DNA breaks at RNA polymerase II promoters in Saccharomyces cerevisiae.

Molecular combing and gel electrophoretic studies revealed endogenous nicks with free 3'OH ends at ∼100 kb intervals in the genomic DNA (gDNA) of unperturbed and G1-synchronized Saccharomyces cerevisiae cells. Analysis of the distribution of endogenous nicks by Nick ChIP-chip indicated that these breaks accumulated at active RNA polymerase II (RNAP II) promoters, reminiscent of the promoter-proximal transient DNA breaks of higher eukaryotes. Similar periodicity of endogenous nicks was found within the ribosomal rDNA cluster, involving every ∼10th of the tandemly repeated 9.

A adenosine receptors control pancreatic dysfunction in high-fat-diet-induced obesity.

Adenosine, a key extracellular signaling mediator, regulates several aspects of metabolism by activating 4 G-protein-coupled receptors, the A, A, A, and A adenosine receptors (ARs). The role of AARs in regulating high-fat-diet (HFD)-induced metabolic derangements is unknown. To evaluate the role of AARs in regulating glucose and insulin homeostasis in obesity, we fed AAR-knockout (KO) and control mice an HFD for 16 wk to initiate HFD-induced metabolic disorder.

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity.

CD39 improves survival in microbial sepsis by attenuating systemic inflammation.

Sepsis remains the leading cause of morbidity and mortality in critically ill patients. Excessive inflammation is a major cause of organ failure and mortality in sepsis. Ectonucleoside triphosphate diphosphohydrolase 1, ENTPDase1 (CD39) is a cell surface nucleotide-metabolizing enzyme, which degrades the extracellular purines ATP and ADP, thereby regulating purinergic receptor signaling.

Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis.

Adenosine and inflammation: what's new on the horizon?

Adenosine contributes to the maintenance of tissue integrity by modulating the immune system. Encouraging results have emerged with adenosine receptor ligands for the management of several inflammatory conditions in preclinical and clinical settings. However, therapeutic applications of these drugs are sometimes complicated by the occurrence of serious adverse effects.

A2B adenosine receptors prevent insulin resistance by inhibiting adipose tissue inflammation via maintaining alternative macrophage activation.

Obesity causes increased classical and decreased alternative macrophage activation, which in turn cause insulin resistance in target organs. Because A2B adenosine receptors (ARs) are important regulators of macrophage activation, we examined the role of A2B ARs in adipose tissue inflammation and insulin resistance. A2B AR deletion impaired glucose and lipid metabolism in mice fed chow but not a high-fat diet, which was paralleled by dysregulation of the adipokine system, and increased classical macrophage activation and inhibited alternative macrophage activation.

Adenosine augments IL-10-induced STAT3 signaling in M2c macrophages.

The alternatively activated macrophage phenotype induced by IL-10 is called M2c. Adenosine is an endogenous purine nucleoside that accumulates in the extracellular space in response to metabolic disturbances, hypoxia, inflammation, physical damage, or apoptosis. As adenosine is known to regulate classically activated M1 and IL4- and IL-13-activated M2a macrophages, the goal of the present study was to explore its effects on M2c macrophages.

CalpB modulates border cell migration in Drosophila egg chambers.

Background: Calpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration.

Regulation of calpain B from Drosophila melanogaster by phosphorylation.

Calpain B is one of the two catalytically competent calpain (calcium-activated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombinant protein by protein kinase A as well as by the extracellular signal-regulated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca2+ binding region of an EF-hand motif, and Ser240 close to the autocatalytic activation site of calpain B, as being the residues phosphorylated by protein kinase A.

Separation of 1-23-kb complementary DNA strands by urea-agarose gel electrophoresis.

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea-agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of approximately 1-20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes.

Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA.

By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at approximately 50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.

Phosphorylation blocks the activity of tubulin polymerization-promoting protein (TPPP): identification of sites targeted by different kinases.

Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Delta3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes.

Germ line specific expression of a protein phosphatase Y interacting protein (PPYR1) in Drosophila.

PPYR1, the product of the CG15031 gene, was identified as a protein phosphatase Y (PPY) interacting protein in Drosophila melanogaster using a yeast two-hybrid screen. PPYR1 displays a biphasic expression pattern: the maternal protein is abundant in the developing egg chambers and in the early embryos, while the zygotic protein appears later in development and is localized specifically in the testes of the males. The maternal and zygotic gene products differ from each other in their size having apparent molecular masses of 47 and 66 kDa, respectively.

Expression of protein phosphatase 1 during the asexual development of Neurospora crassa.

We cloned and sequenced the cDNA and the gene encoding the catalytic subunit of protein phosphatase 1 from the filamentous fungus Neurospora crassa. The gene, designated ppp-1 (phosphoprotein phosphatase 1), was mapped by restriction fragment length polymorphism to linkage group III, in the vicinity of con-7 and trp-1. The expression of the gene was monitored by reverse transcriptase and polymerase chain reactions, by Western blotting, and by protein phosphatase activity assays in synchronized cultures.