Macrophage inhibitory cytokine‐1 is associated with cognitive impairment and predicts cognitive decline – the Sydney Memory and Aging Study

Higher levels of macrophage inhibitory cytokine‐1, also known as growth differentiation factor 15 (MIC‐1/GDF15), are associated with adverse health outcomes and all‐cause mortality. The aim of this study was to examine the relationships between MIC‐1/GDF15 serum levels and global cognition, five cognitive domains, and mild cognitive impairment (MCI), at baseline (Wave 1) and prospectively at 2 years (Wave 2), in nondemented participants aged 70–90 years. Analyses were controlled for age, sex, education, Framingham risk score, history of cerebrovascular accident, acute myocardial infarction, angina, cancer, depression, C‐reactive protein, tumor necrosis factor‐α, interleukins 6 and 12, and apolipoprotein ε4 genotype. Higher MIC‐1/GDF15 levels were significantly associated with lower global cognition at both waves. Cross‐sectional associations were found between MIC‐1/GDF15 and all cognitive domains in Wave 1 (all P < 0.001) and between processing speed, memory, and executive function in Wave 2 (all P < 0.001). Only a trend was found for the prospective analyses, individuals with high MIC‐1/GDF15 at baseline declined in global cognition, executive function, memory, and processing speed. However, when categorizing MIC‐1/GDF15 by tertiles, prospective analyses revealed statistically significant lower memory and executive function in Wave 2 in those in the upper tertile compared with the lower tertile. Receiver operating characteristics (ROC) analysis was used to determine MIC‐1/GDF15 cutoff values associated with cognitive decline and showed that a MIC‐1/GDF15 level exceeding 2764 pg/ml was associated with a 20% chance of decline from normal to MCI or dementia. In summary, MIC‐1/GDF15 levels are associated with cognitive performance and cognitive decline. Further research is required to determine the pathophysiology of this relationship.     

Design and characterization of a protein superagonist of IL‐15 fused with IL‐15Rα and a high‐affinity T cell receptor

To avoid high systemic doses, strategies involving antigen‐specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer‐associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross‐presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high‐affinity single‐chain T cell receptor Vα‐Vβ (scTv), to deliver cytokines to intracellular tumor‐associated antigens presented as pepMHC. As typical wild‐type T cell receptors (TCRs) exhibit low affinity (K_d = 1–100 μM or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2‐K^b (SIY/K^b) with nanomolar affinity (K_d = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL‐2), interleukin 15 (IL‐15), or IL‐15/IL‐15Rα (IL‐15 linked to IL‐15Rα sushi domain, called “superfusion”). The fusions were purified with good yields and bound specifically to SIY/K^b with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine‐dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Growth differentiation factor‐15 (GDF‐15) suppresses in vitro angiogenesis through a novel interaction with connective tissue growth factor (CCN2)

Growth differentiation factor‐15 (GDF‐15) and the CCN family member, connective tissue growth factor (CCN2), are associated with cardiac disease, inflammation, and cancer. The precise role and signaling mechanism for these factors in normal and diseased tissues remains elusive. Here we demonstrate an interaction between GDF‐15 and CCN2 using yeast two‐hybrid assays and have mapped the domain of interaction to the von Willebrand factor type C domain of CCN2. Biochemical pull down assays using secreted GDF‐15 and His‐tagged CCN2 produced in PC‐3 prostate cancer cells confirmed a direct interaction between these proteins. To investigate the functional consequences of this interaction, in vitro angiogenesis assays were performed. We demonstrate that GDF‐15 blocks CCN2‐mediated tube formation in human umbilical vein endothelial (HUVEC) cells. To examine the molecular mechanism whereby GDF‐15 inhibits CCN2‐mediated angiogenesis, activation of α_Vβ₃ integrins and focal adhesion kinase (FAK) was examined. CCN2‐mediated FAK activation was inhibited by GDF‐15 and was accompanied by a decrease in α_Vβ₃ integrin clustering in HUVEC cells. These results demonstrate, for the first time, a novel signaling pathway for GDF‐15 through interaction with the matricellular signaling molecule CCN2. Furthermore, antagonism of CCN2 mediated angiogenesis by GDF‐15 may provide insight into the functional role of GDF‐15 in disease states. J. Cell. Biochem. 114: 1424–1433, 2013. © 2012 Wiley Periodicals, Inc.     

20‐year follow‐up study of physical morbidity and mortality in relationship to antipsychotic treatment in a nationwide cohort of 62,250 patients with schizophrenia (FIN20)

Antipsychotics are effective in preventing relapses of schizophrenia, but it is generally believed that their long‐term use is harmful for patients’ physical well‐being. However, there are no long‐term studies which have verified this view. This nationwide, register‐based cohort study aimed to assess the risk of hospitalization due to physical health problems, as a marker for severe physical morbidity, and the risk of all‐cause mortality, as well as of cardiovascular and suicidal death, associated with antipsychotic use in all patients treated for schizophrenia in inpatient care between 1972 and 2014 in Finland (N=62,250), with up to 20 years of follow‐up (median: 14.1 years). The use of antipsychotic drugs (i.e., use of any antipsychotic compared with non‐use) and the use of specific antipsychotics were investigated, and outcomes were somatic and cardiovascular hospitalization, and all‐cause, cardiovascular and suicide death. Hospitalization‐based outcomes were analyzed by a within‐individual design to eliminate selection bias, comparing use and non‐use periods in the same individual by stratified Cox model. Mortality outcomes were assessed by traditional between‐individual Cox multivariate models. The adjusted hazard ratios (aHRs) for any somatic hospitalization and cardiovascular hospitalization were 1.00 (95% CI: 0.98‐1.03) and 1.00 (95% CI: 0.92‐1.07) during use of any antipsychotic compared to non‐exposure periods within the same individual. The aHRs were 0.48 (95% CI: 0.46‐0.51) for all‐cause mortality, 0.62 (95% CI: 0.57‐0.67) for cardiovascular mortality, and 0.52 (95% CI: 0.43‐0.62) for suicide mortality during use vs. non‐use of any antipsychotic. The most beneficial mortality outcome was associated with use of clozapine in terms of all‐cause (aHR=0.39, 95% CI: 0.36‐0.43), cardiovascular (aHR=0.55, 95% CI: 0.47‐0.64) and suicide mortality (aHR=0.21, 95% CI: 0.15‐0.29). The cumulative mortality rates during maximum follow‐up of 20 years were 46.2% for no antipsychotic use, 25.7% for any antipsychotic use, and 15.6% for clozapine use. These data suggest that long‐term antipsychotic use does not increase severe physical morbidity leading to hospitalization, and is associated with substantially decreased mortality, especially among patients treated with clozapine.     

Parathyroid hormone inhibits phosphorylation of mitogen‐activated protein kinase (MAPK) ERK1/2 through inhibition of c‐Raf and activation of MKP‐1 in osteoblastic cells

Parathyroid hormone (PTH) regulation of mitogen‐activated protein kinases (MAPK) ERK1/2 contributes to PTH regulation of osteoblast growth and apoptosis. We investigated the mechanisms by which PTH inhibits ERK1/2 activity in osteoblastic UMR 106‐01 cells. Treatment with PTH significantly inhibited phosphorylated ERK1/2 between 5 and 60 min. Transient transfection of cells with a cDNA encoding MAPK phosphatase‐1 (MKP‐1) resulted in 30–40% inhibition of pERK1/2; however MKP‐1 protein levels were only significantly stimulated by PTH after 30 mins, suggesting another mechanism for the early phase of pERK1/2 inhibition. The active upstream kinase c‐Raf phosphorylation at serine 338 (ser³³⁸) was significantly inhibited by PTH treatment within 5 min and transfection of the cells with constitutively‐active c‐Raf blocked PTH inhibition of pERK1/2. Inhibition of pERK1/2 and phosphor‐c‐Raf were seen when cells were treated with PTH(1‐34) or PTH(1‐31) analogues that stimulate cAMP, but not with PTH(3‐34), PTH(7‐34) or PTH(18‐48) that do not stimulate cAMP. Stimulation of the cells with forskolin or 8BrcAMP also inhibited pERK1/2 and c‐Raf.p338. Our results suggest that rapid PTH inhibition of ERK1/2 activity is mediated by PKA dependent inhibition of c‐Raf activity and that stimulation of MKP‐1 may contribute to maintaining pERK1/2 inhibition over prolonged time. Copyright © 2009 John Wiley & Sons, Ltd.     

The impact of Arabidopsis thaliana SNF1‐related‐kinase 1 (SnRK1)‐activating kinase 1 (SnAK1) and SnAK2 on SnRK1 phosphorylation status: characterization of a SnAK double mutant

Arabidopsis thaliana SNF1‐related‐kinase 1 (SnRK1)‐activating kinase 1 (AtSnAK1) and AtSnAK2 have been shown to phosphorylate in vitro and activate the energy signalling integrator, SnRK1. To clarify this signalling cascade in planta, a genetic‐ and molecular‐based approach was developed. Homozygous single AtSnAK1 and AtSnAK2 T‐DNA insertional mutants did not display an apparent phenotype. Crossing of the single mutants did not allow the isolation of double‐mutant plants, whereas self‐pollinating the S1?/? S2+/? sesquimutant specifically gave approximatively 22% individuals in their offspring that, when rescued on sugar‐supplemented media in vitro, were shown to be AtSnAK1 AtSnAK2 double mutants. Interestingly, this was not obtained in the case of the other sesquimutant, S1+/? S2?/?. Although reduced in size, the double mutant had the capacity to produce flowers, but not seeds. Immunological characterization established the T‐loop of the SnRK1 catalytic subunit to be non‐phosphorylated in the absence of both SnAKs. When the double mutant was complemented with a DNA construct containing an AtSnAK2 open reading frame driven by its own promoter, a normal phenotype was restored. Therefore, wild‐type plant growth and development is dependent on the presence of SnAK in vivo, and this is correlated with SnRK1 phosphorylation. These data show that both SnAKs are kinases phosphorylating SnRK1, and thereby they contribute to energy signalling in planta.     

Microglia of medicinal leech (Hirudo medicinalis) express a specific activation marker homologous to vertebrate ionized calcium‐binding adapter molecule 1 (Iba1/alias aif‐1)

The Ionized calcium‐Binding Adapter molecule 1 (Iba1), also known as Allograft Inflammatory Factor 1 (AIF‐1), is a 17 kDa cytokine‐inducible protein, produced by activated macrophages during chronic transplant rejection and inflammatory reactions in Vertebrates. In mammalian central nervous system (CNS), Iba1 is a sensitive marker associated with activated macrophages/microglia and is upregulated following neuronal death or brain lesions. The medicinal leech Hirudo medicinalis is able to regenerate its CNS after injury, leading to a complete functional repair. Similar to Vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. We identified a gene, named Hmiba1, coding a 17.8 kDa protein showing high similarity with Vertebrate AIF‐1. The present work constitutes the first report on an Iba1 protein in the nervous system of an invertebrate. Immunochemistry and gene expression analyses showed that HmIba1, like its mammalian counterpart, is modulated in leech CNS by mechanical injury or chemical stimuli (ATP). We presently demonstrate that most of leech microglial cells migrating and accumulating at the lesion site specifically expressed the activation marker HmIba1. While the functional role of Iba1, whatever species, is still unclear in reactive microglia, this molecule appeared as a good selective marker of activated cells in leech and presents an interesting tool to investigate the functions of these cells during nerve repair events. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 987–1001, 2014     

Solution structure of a novel T‐cell adhesion inhibitor derived from the fragment of ICAM‐1 receptor: Cyclo(1,8)‐Cys‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Cys

This study is aimed at elucidating the structure of a novel T‐cell adhesion inhibitor, cyclo(1,8)‐CPRGGSVC using one‐ and two‐dimensional (2D) ¹H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)‐PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule‐1 (ICAM‐1). Our previous results show that the cyclo(1,8)‐CPRGGSVC peptide binds to the LFA‐1 I‐domain and inhibits heterotypic T‐cell adhesion, presumably by blocking the LFA‐1/ICAM‐1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type‐I β‐turn conformation at the Pro2‐Arg3‐Gly4‐Gly5 (PRGG) sequence. The β‐turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM‐1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)‐CPRGGSVC peptide. Meanwhile, the Gly5‐Ser6‐Val7‐Cys8‐Cys1 (GSVCC) sequence forms a “turn‐like” random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)‐CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA‐1 I‐domain. The conserved β‐turn conformation of the PRGG motif in ICAM‐1, cIBR, and cyclo(1,8)‐CPRGGSVC peptides can potentially be used to design peptidomimetics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 633–641, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Porcine proximal tubular cells (LLC‐PK1) are able to tolerate high levels of lithium chloride in vitro: assessment of the influence of 1–20 mM LiCl on cell death and alterations in cell biology and biochemistry

Lithium, a prophylactic drug for the treatment of bipolar disorder, is prescribed with caution due to its side effects, including renal damage. In this study porcine LLC‐PK₁ renal tubular cells were used to establish the direct toxicity of lithium on proximal cells and gain insights into the molecular mechanisms involved. In the presence of LiCl, cell proliferation exhibited insignificant decreases in a concentration‐dependent manner, but once confluent, constant cell numbers were observed. Cell cycle studies indicated a small dose‐dependant accumulation of cells in the G₂/M stage after 24 h, as well as an increase in cells in the G₀/G₁ phase after treatment with 1–10 mM LiCl, but not at 20 mM LiCl. No evidence of apoptosis was observed based on cell morphology or DNA fragmentation studies, or evidence of protein expression changes for Bax, Bcl‐2, and p53 proteins using immunocytochemistry. In addition caspases 3, 8 and 9 activity remained unaltered between control and lithium‐treated cultures. To conclude, exposure to high concentrations of lithium did not result in overt toxic effects to LLC‐PK₁ renal cells, although LiCl did alter some aspects of cell behaviour, which could potentially influence function over time.     

Changing gull diet in a changing world: A 150‐year stable isotope (δ13C, δ15N) record from feathers collected in the Pacific Northwest of North America

The world's oceans have undergone significant ecological changes following European colonial expansion and associated industrialization. Seabirds are useful indicators of marine food web structure and can be used to track multidecadal environmental change, potentially reflecting long‐term human impacts. We used stable isotope (δ¹³C, δ¹⁵N) analysis of feathers from glaucous‐winged gulls (Larus glaucescens) in a heavily disturbed region of the northeast Pacific to ask whether diets of this generalist forager changed in response to shifts in food availability over 150 years, and whether any detected change might explain long‐term trends in gull abundance. Sampled feathers came from birds collected between 1860 and 2009 at nesting colonies in the Salish Sea, a transboundary marine system adjacent to Washington, USA and British Columbia, Canada. To determine whether temporal trends in stable isotope ratios might simply reflect changes to baseline environmental values, we also analysed muscle tissue from forage fishes collected in the same region over a multidecadal timeframe. Values of δ¹³C and δ¹⁵N declined since 1860 in both subadult and adult gulls (δ¹³C, ~ 2–6‰; δ¹⁵N, ~4–5‰), indicating that their diet has become less marine over time, and that birds now feed at a lower trophic level than previously. Conversely, forage fish δ¹³C and δ¹⁵N values showed no trends, supporting our conclusion that gull feather values were indicative of declines in marine food availability rather than of baseline environmental change. Gradual declines in feather isotope values are consistent with trends predicted had gulls consumed less fish over time, but were equivocal with respect to whether gulls had switched to a more garbage‐based diet, or one comprising marine invertebrates. Nevertheless, our results suggest a long‐term decrease in diet quality linked to declining fish abundance or other anthropogenic influences, and may help to explain regional population declines in this species and other piscivores.     

The ABCs of flower development: mutational analysis of AP1/FUL‐like genes in rice provides evidence for a homeotic (A)‐function in grasses

The well‐known ABC model describes the combinatorial interaction of homeotic genes in specifying floral organ identities. While the B‐ and C‐functions are highly conserved throughout flowering plants and even in gymnosperms, the A‐function, which specifies the identity of perianth organs (sepals and petals in eudicots), remains controversial. One reason for this is that in most plants that have been investigated thus far, with Arabidopsis being a remarkable exception, one does not find recessive mutants in which the identity of both types of perianth organs is affected. Here we report a comprehensive mutational analysis of all four members of the AP1/FUL‐like subfamily of MADS‐box genes in rice (Oryza sativa). We demonstrate that OsMADS14 and OsMADS15, in addition to their function of specifying meristem identity, are also required to specify palea and lodicule identities. Because these two grass‐specific organs are very likely homologous to sepals and petals of eudicots, respectively, we conclude that there is a floral homeotic (A)‐function in rice as defined previously. Together with other recent findings, our data suggest that AP1/FUL‐like genes were independently recruited to fulfil the (A)‐function in grasses and some eudicots, even though other scenarios cannot be excluded and are discussed.     

Interaction of β3/β2‐Peptides,Consisting of Val‐Ala‐Leu Segments,with POPC Giant Unilamellar Vesicles (GUVs) and White Blood Cancer Cells (U937) – A New Type of Cell‐Penetrating Peptides,and a Surprising Chain‐Length Dependence of Their Vesicle‐ and Cell‐Lysing Activity

Many years ago, β²/β³‐peptides, consisting of alternatively arranged β²‐ and β³h‐amino‐acid residues, have been found to undergo folding to a unique type of helix, the 10/12‐helix, and to exhibit non‐polar, lipophilic properties (Helv. Chim. Acta 1997 , 80, 2033). We have now synthesized such ‘mixed’ hexa‐, nona‐, dodeca‐, and octadecapeptides, consisting of Val‐Ala‐Leu triads, with N‐terminal fluorescein (FAM) labels, i.e., 1 – 4 , and studied their interactions with POPC (=1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow‐cytometry assay, a membrane‐toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All β³/β²‐peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric β³/β²‐, β³‐, and β²‐nonamers, 2, 5 , and 6 , respectively, the derivatives 5 and 6 consisting exclusively of β³‐ or β²‐amino‐acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the β³/β²‐nonamer and/or the β³/β²‐dodecamer derivative, 2 and/or 3 , respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host‐defense antimicrobial peptides. Possible sources of the chain‐length‐dependent destructive potential of the β³/β²‐nona‐ and β³/β²‐dodecapeptide derivatives, and a possible relationship with the phosphate‐to‐phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of ‘mixed’ β³/β²‐peptides with membranes and to evaluate possible biomedical applications.     

Transgenic expression of n‐3 fatty acid desaturase (fat‐1) in C57/BL6 mice: Effects on glucose homeostasis and body weight

The fat‐1 gene, derived from Caenorhabditis elegans, encodes for a fatty acid n‐3 desaturase. In order to study the potential metabolic benefits of n‐3 fatty acids, independent of dietary fatty acids, we developed seven lines of fat‐1 transgenic mice (C57/BL6) controlled by the regulatory sequences of the adipocyte protein‐2 (aP2) gene for adipocyte‐specific expression (AP‐lines). We were unable to obtain homozygous fat‐1 transgenic offspring from the two highest expressing lines, suggesting that excessive expression of this enzyme may be lethal during gestation. Serum fatty acid analysis of fat‐1 transgenic mice (AP‐3) fed a high n‐6 unsaturated fat (HUSF) diet had an n‐6/n‐3 fatty acid ratio reduced by 23% (P < 0.025) and the n‐3 fatty acid eicosapentaenoic acid (EPA) concentration increased by 61% (P < 0.020). Docosahexaenoic acid (DHA) was increased by 19% (P < 0.015) in white adipose tissue. Male AP‐3‐fat‐1 line of mice had improved glucose tolerance and reduced body weight with no change in insulin sensitivity when challenged with a high‐carbohydrate (HC) diet. In contrast, the female AP‐3 mice had reduced glucose tolerance and no change in insulin sensitivity or body weight. These findings indicate that male transgenic fat‐1 mice have improved glucose tolerance likely due to increased insulin secretion while female fat‐1 mice have reduced glucose tolerance compared to wild‐type mice. Finally the inability of fat‐1 transgenic mice to generate homozygous offspring suggests that prolonged exposure to increased concentrations of n‐3 fatty acids may be detrimental to reproduction. J. Cell. Biochem. 107: 809–817, 2009. © 2009 Wiley‐Liss, Inc.