Retinol-binding protein 4 is associated with components of the metabolic syndrome, but not with insulin resistance, in men with type 2 diabetes or coronary artery disease

AIMS/HYPOTHESIS: Retinol-binding protein 4 (RBP4) has recently been reported to be associated with insulin resistance and the metabolic syndrome. This study tested the hypothesis that RBP4 is a marker of insulin resistance and the metabolic syndrome in patients with type 2 diabetes or coronary artery disease (CAD) or in non-diabetic control subjects without CAD. METHODS: Serum RBP4 was measured in 365 men (126 with type 2 diabetes, 143 with CAD and 96 control subjects) and correlated with the homeostasis model assessment of insulin resistance index (HOMA-IR), components of the metabolic syndrome and lipoprotein metabolism. RBP4 was detected by ELISA and validated by quantitative Western blotting. RESULTS: RBP4 concentrations detected by ELISA were shown to be strongly associated with the results gained in quantitative Western blots. There were no associations of RBP4 with HOMA-IR or HbA(1c) in any of the groups studied. In patients with type 2 diabetes there were significant positive correlations of RBP4 with total cholesterol, LDL-cholesterol, VLDL-cholesterol, plasma triacylglycerol and hepatic lipase activity. In patients with CAD, there were significant associations of RBP4 with VLDL-cholesterol, plasma triacylglycerol and hepatic lipase activity, while non-diabetic control subjects without CAD showed positive correlations of RBP4 with VLDL-cholesterol and plasma triacylglycerol. CONCLUSIONS/INTERPRETATION: RBP4 does not seem to be a valuable marker for identification of the metabolic syndrome or insulin resistance in male patients with type 2 diabetes or CAD. Independent associations of RBP4 with pro-atherogenic lipoproteins and enzymes of lipoprotein metabolism indicate a possible role of RBP4 in lipid metabolism.

Multidrug resistance protein 4 (MRP4) expression in prostate cancer is associated with androgen signaling and decreases with tumor progression

Multidrug resistance protein 4 (MRP4) is a transmembrane transport protein found in many cell types and is involved in substrate-specific transport of endogenous and exogenous substrates. Recently, it has shown to be expressed in prostate cancer cell lines and to be among the most commonly upregulated transcripts in prostate cancer, although a comprehensive expression analysis is lacking so far. We aimed to investigate its expression by immunohistochemistry in a larger cohort of neoplastic and nonneoplastic prostate tissues (n = 441) and to correlate its expression with clinicopathological parameters including PSA-free survival times and molecular correlates of androgen signaling (androgen receptor (AR), prostate-specific antigen (PSA), and forkhead box A (FoxA)). MRP4 is widely expressed in benign and neoplastic prostate epithelia, but its expression gradually decreases during tumor progression towards castrate-resistant disease. Concordantly, it correlated with conventional prognosticators of disease progression and-within the group of androgen-dependent tumors-with AR and FoxA expression. Moreover, lower levels of MRP4 expression were associated with shorter PSA relapse-free survival times in the androgen-dependent group. In benign tissues, we found zone-dependent differences of MRP4 expression, with the highest levels in the peripheral and central zones. Although MRP4 is known to be regulated in prostate cancer, this study is the first to demonstrate a gradual downregulation of MRP4 protein during malignant tumor progression and a prognostic value of this loss of expression.

Tocopherol-associated protein-1 accelerates apoptosis induced by alpha-tocopheryl succinate in mesothelioma cells

Alpha-tocopheryl succinate (alpha-TOS), a redox-silent analogue of vitamin E, induces apoptosis in multiple cell lines in a selective manner, by activating the intrinsic pathway. Since it is a highly hydrophobic compound, it may require a carrier protein for its trafficking to intracellular targets like mitochondria. We studied the role of the ubiquitous tocopherol-associated protein-1 (TAP1 or sec14-like 2) in apoptosis induction by alpha-TOS in malignant mesothelioma (MM) cells. Over-expression of TAP1 in MM cells sensitised them to apoptosis by low doses of alpha-TOS which were sub-apoptotic for the parental cells. Apoptosis induced in TAP1-over-expressing cells was mitochondria- and caspase-dependent, as suggested by dissipation of mitochondrial trans-membrane potential and inhibition by zVAD-fmk, respectively. Binding assays showed affinity of alpha-TOS for TAP1. Finally, TAP1 over-expressing cells accumulated alpha-TOS at higher levels compared to their normal counterparts. We suggest that TAP1 may act as an intracellular shuttle for alpha-TOS, promoting apoptosis initiated by this vitamin E analogue, as shown here for MM cells.

Immunization with a circumsporozoite epitope fused to Bordetella pertussis adenylate cyclase in conjunction with cytotoxic T-lymphocyte-associated antigen 4 blockade confers protection against Plasmodium berghei liver-stage malaria

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.

Epigallocatechin-3-gallate induces cell death in acute myeloid leukaemia cells and supports all-trans retinoic acid-induced neutrophil differentiation via death-associated protein kinase 2

Acute promyelocytic leukaemia (APL) patients are successfully treated with all-trans retinoic acid (ATRA). However, concurrent chemotherapy is still necessary and less toxic therapeutic approaches are needed. Earlier studies suggested that in haematopoietic neoplasms, the green tea polyphenol epigallocatechin-3-gallate (EGCG) induces cell death without adversely affecting healthy cells. We aimed at deciphering the molecular mechanism of EGCG-induced cell death in acute myeloid leukaemia (AML). A significant increase of death-associated protein kinase 2 (DAPK2) levels was found in AML cells upon EGCG treatment paralleled by increased cell death that was significantly reduced upon silencing of DAPK2. Moreover, combined ATRA and EGCG treatment resulted in cooperative DAPK2 induction and potentiated differentiation. EGCG toxicity of primary AML blasts correlated with 67 kDa laminin receptor (67LR) expression. Pretreatment of AML cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG-mediated cell death. In summary, it was found that (i) DAPK2 is essential for EGCG-induced cell death in AML cells, (ii) ATRA and EGCG cotreatment significantly boosted neutrophil differentiation, and 67LR expression correlates with susceptibility of AML cells to EGCG. We thus suggest that EGCG, by selectively targeting leukaemic cells, may improve differentiation therapies for APL and chemotherapy for other AML subtypes.

Congenital syndactyly in cattle: four novel mutations in the low density lipoprotein receptor-related protein 4 gene (LRP4)

BACKGROUND: Isolated syndactyly in cattle, also known as mulefoot, is inherited as an autosomal recessive trait with variable penetrance in different cattle breeds. Recently, two independent mutations in the bovine LRP4 gene have been reported as the primary cause of syndactyly in the Holstein and Angus cattle breeds. RESULTS: We confirmed the previously described LRP4 exon 33 two nucleotide substitution in most of the affected Holstein calves and revealed additional evidence for allelic heterogeneity by the identification of four new LRP4 non-synonymous point mutations co-segregating in Holstein, German Simmental and Simmental-Charolais families. CONCLUSION: We confirmed a significant role of LRP4 mutations in the pathogenesis of congenital syndactyly in cattle. The newly detected missense mutations in the LRP4 gene represent independent mutations affecting different conserved protein domains. However, the four newly described LRP4 mutations do still not explain all analyzed cases of syndactyly.

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.

Plasma levels of mannan-binding lectin (MBL)-associated serine proteases (MASPs) and MBL-associated protein in cardio- and cerebrovascular diseases

Growing evidence suggests a prominent role of the complement system in the pathogenesis of cardio- and cerebrovascular diseases (CVD). Mannan-binding lectin-associated serine proteases (MASPs) MASP-1 and MASP-2 of the complement lectin pathway contribute to clot formation and may represent an important link between inflammation and thrombosis. MBL-associated protein MAp44 has shown cardioprotective effects in murine models. However, MAp44 has never been measured in patients with CVD and data on MASP levels in CVD are scarce. Our aim was to investigate for the first time plasma levels of MAp44 and MASP-1, -2, -3 concomitantly in patients with CVD. We performed a pilot study in 50 healthy volunteers, in stable coronary artery disease (CAD) patients with one-vessel (n = 51) or three-vessel disease (n = 53) and age-matched controls with normal coronary arteries (n = 53), 49 patients after myocardial infarction (MI) and 66 patients with acute ischaemic stroke. We measured MAp44 and MASP-1 levels by in-house time-resolved immunofluorometric assays. MASP-2 and MASP-3 levels were measured using commercial enzyme-linked immunosorbent assay kits. MASP-1 levels were highest in subacute MI patients and lowest in acute stroke patients. MASP-2 levels were lower in MI and stroke patients compared with controls and CAD patients. MASP-3 and MAp44 levels did not differ between groups. MASP or MAp44 levels were not associated with severity of disease. MASP and MAp44 levels were associated with cardiovascular risk factors including dyslipidaemia, obesity and hypertension. Our results suggest that MASP levels may be altered in vascular diseases. Larger studies are needed to confirm our results and elucidate the underlying mechanisms.

Induction of the autophagy-associated gene MAP1S via PU.1 supports APL differentiation

The PU.1 transcription factor is essential for myeloid development. We investigated if the microtubule-associated protein 1S (MAP1S) is a novel PU.1 target with a link to autophagy, a cellular recycling pathway. Comparable to PU.1, MAP1S expression was significantly repressed in primary AML blasts as compared to mature neutrophils. Accordingly, MAP1S expression was induced during neutrophil differentiation of CD34(+) progenitor and APL cells. Moreover, PU.1 bound to the MAP1S promoter and induced MAP1S expression during APL differentiation. Inhibiting MAP1S resulted in aberrant neutrophil differentiation and autophagy. Taken together, our findings implicate the PU.1-regulated MAP1S gene in neutrophil differentiation and autophagy control.

Cardiac-specific ablation of synapse-associated protein SAP97 in mice decreases potassium currents but not sodium current

BACKGROUND Membrane-associated guanylate kinase (MAGUK) proteins are important determinants of ion channel organization in the plasma membrane. In the heart, the MAGUK protein SAP97, encoded by the DLG1 gene, interacts with several ion channels via their PDZ domain-binding motif and regulates their function and localization. OBJECTIVE The purpose of this study was to assess in vivo the role of SAP97 in the heart by generating a genetically modified mouse model in which SAP97 is suppressed exclusively in cardiomyocytes. METHODS SAP97(fl/fl) mice were generated by inserting loxP sequences flanking exons 1-3 of the SAP97 gene. SAP97(fl/fl) mice were crossed with αMHC-Cre mice to generate αMHC-Cre/SAP97(fl/fl) mice, thus resulting in a cardiomyocyte-specific deletion of SAP97. Quantitative reverse transcriptase-polymerase chain reaction, western blots, and immunostaining were performed to measure mRNA and protein expression levels, and ion channel localization. The patch-clamp technique was used to record ion currents and action potentials. Echocardiography and surface ECGs were performed on anesthetized mice. RESULTS Action potential duration was greatly prolonged in αMHC-Cre/SAP97(fl/fl) cardiomyocytes compared to SAP97(fl/fl) controls, but maximal upstroke velocity was unchanged. This was consistent with the decreases observed in IK1, Ito, and IKur potassium currents and the absence of effect on the sodium current INa. Surface ECG revealed an increased corrected QT interval in αMHC-Cre/SAP97(fl/fl) mice. CONCLUSION These data suggest that ablation of SAP97 in the mouse heart mainly alters potassium channel function. Based on the important role of SAP97 in regulating the QT interval, DLG1 may be a susceptibility gene to be investigated in patients with congenital long QT syndrome.

Dendritic spine morphology determines membrane-associated protein exchange between dendritic shafts and spine heads.

The purpose of this study was to examine whether variability in the shape of dendritic spines affects protein movement within the plasma membrane. Using a combination of confocal microscopy and the fluorescence loss in photobleaching technique in living hippocampal CA1 pyramidal neurons expressing membrane-linked GFP, we observed a clear correlation between spine shape parameters and the diffusion and compartmentalization of membrane-associated proteins. The kinetics of membrane-linked GFP exchange between the dendritic shaft and the spine head compartment were slower in dendritic spines with long necks and/or large heads than in those with short necks and/or small heads. Furthermore, when the spine area was reduced by eliciting epileptiform activity, the kinetics of protein exchange between the spine compartments exhibited a concomitant decrease. As synaptic plasticity is considered to involve the dynamic flux by lateral diffusion of membrane-bound proteins into and out of the synapse, our data suggest that spine shape represents an important parameter in the susceptibility of synapses to undergo plastic change.

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation.

Death-associated protein kinase 2 (DAPK2) is a Ca(2+)/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein's subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Stem Cells From Umbilical Cord Wharton's Jelly From Preterm Birth Have Neuroglial Differentiation Potential

Objective:The aim of the study is to determine the neuroglial differentiation potential of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from preterm birth when compared to term delivery.Study Design:The WJ-MSCs from umbilical cords of preterm birth and term controls were isolated and induced into neural progenitors. The cells were analyzed for neuroglial markers by flow cytometry, real-time polymerase chain reaction, and immunocytochemistry. Results:Independent of gestational age, a subset of WJ-MSC displayed the neural progenitor cell markers Nestin and Musashi-1 and the mature neural markers microtubule-associated protein 2, glial fibrillary acidic protein, and myelin basic protein. Neuroglial induction of WJ-MSCs from term and preterm birth resulted in the enhanced transcription of Nestin and Musashi-1.Conclusions:Undifferentiated WJ-MSCs from preterm birth express neuroglial markers and can be successfully induced into neural progenitors similar to term controls. Their potential use as cellular graft in neuroregenerative therapy for peripartum brain injury in preterm birth has to be tested.