A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Purification and characterization of CMP-N-acetylneuraminic acid hydroxylase from pig submandibular glands

N-Glycoloylneuraminic acid (Neu5Gc) is synthesized as its CMP-giycosideby the action of CMPN-acetylneuramlnic acid (CMP-Neu5Ac) hydroxylase.This enzyme is a soluble cytochrome bs-dependent monooxygenaseand has been purified to apparent homogeneity from pig submandibularglands by precipitation with N-cetyN,N,N-trimethylam-moniumbromide and fractionation on Q-Sepharose, Cibacron Blue 3GA-Agarose,Reactive Brown 10-Agarose, Hexyl-Agarose and Superose S.12.This procedure resulted in an 8960-fold purification of thehydroxylase with a recovery of 0.8%. The molecular mass of thisprotein was shown to be 65 kDa on SDS-PAGE and 60 kDa as determinedby gel filtration on Superose S.12, which suggests that theenzyme is a monomer. The purified CMP-Neu5Ac hydroxylase isactivated by FeSO₄ and inhibited by iron-binding reagents suchas o-phenanthroline, KCN, Tiron and ferro-zine. An apparentKm of 11 µM was determined for the substrate CMP-Neu5Acusing purified hydroxylase in the presence of Triton X-100-solubilizedmicrosomes. In a reconstituted system consisting of purifiedhydroxylase, cytochrome b₅, cytochrome b₅ reductase and catalase,an apparent K_m of 3 µM was measured. The apparent K_mforcytochrome b₅ in this system was 0.24 µM. Immunizationof a rabbit with enriched and purified hydroxylase led to anantiserum that inhibited CMP-Neu5Ac hydroxylase activity andreacted with the purified 65 kDa protein on a Western blot afterSDS-PAGE. Antibodies specific for this 65 kDa protein were isolatedand showed a strong reaction with the purified CMP-Neu5Ac hydroxylasefrom mouse liver after immunoblotting. Initial experiments withthis monospecific antibody suggest that the activity of thehydroxylase in a particular tissue correlates with the amountof immuno-reactive protein. cytochrome b₅ N-glcoloylneuraminic acid hydroxylase pig submandibular gland mucin sialic acid     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Separation of dissociated thyroid follicular and parfollicular cells: association of serotonin binding protein with parafollicular cells

Parafollicular cells (PC) of the sheep thyroid gland are neural crest derivatives that synthesize and release the biogenic amine serotonin (5-HT) as well as the hormone calcitonin. The thyroid also contains a highly specific serotonin-binding protein (SBP). Separation of dissociated thyroid cells was done to study the cellular localization of SBP and to develop a means of isolating PC for study. Various methods were used to obtain an enriched and purified population of PC. Minced thyroid glands were enzymatically dissociated and the cells were layered on a Ficoll linear density gradient. Fractions obtained from the gradient were examined for cell number, viability, 5-HT concentration, SBP activity, and morphology by electron microscopy. One of the fractions was found to be enriched in PC. High levels of 5-HT and SBP were also found in this fraction, whereas these levels were low where the majority of cells were found. This PC-rich fraction, however, contained numerous follicular cells (FC); therefore, additional approaches to cell separation were used. FC can be stimulated in vitro with thyroid stimulating hormone (TSH) to become intensely phagocytic. When stimulated cells were incubated in the presence of silica microspheres, the FC engulfed the microspheres, which were toxic to them. PC did not become phagocytic and were unharmed by the microspheres. Suspended cells, after incubation with microspheres, were centrifuged on a discontinuous gradient, and a PC-rich fraction was obtained. Silica, however, interfered with analysis of SBP. Another method to take advantage of the phagocytic potential of FC was therefore used. TSH-stimulated cell suspensions were passed through a column of sepharose to which thyroglobulin had been coupled. Stimulated FC apparently adhered to the beads and were retained by the columns. Fractions eluting from the columns were greatly enriched with PC. These fractions contained high levels of 5-HT and SBP, and considerably reduced FC contamination was found by quantitative electron microscopy. It is concluded that SBP is localized to PC in the sheep thyroid. The idea that these cells resemble serotonergic neurons in their mechanisms of 5-HT storage is supported.     

Single and combined effects of Drosophila suzukii and Drosophila melanogaster on sour rot development in viticulture

Sour rot is a disease complex that causes serious damage in viticulture. The common vinegar fly Drosophila melanogaster (Diptera: Drosophilidae) is associated with sour rot in overripe or otherwise damaged grapes. Drosophila suzukii (Diptera: Drosophilidae) is an invasive species, which is suspected to induce sour rot in previously undamaged grapes due to the flies' ability to infest healthy, undamaged soft fruits with its serrated ovipositor. As a consequence, infection of healthy grapes by D. suzukii may facilitate the colonization by D. melanogaster. We investigated the single and combined effects of D. suzukii and D. melanogaster on sour rot development by measuring volatile acidity under near-natural conditions in the vineyard, along with laboratory experiments under controlled climate. In 2017, the combined field and laboratory experiments suggested that the presence of D. suzukii and D. melanogaster increased the volatile acidity levels at a similar rate. In 2018, the field experiments showed an only marginal increase in sour rot development in treatments with both Drosophila species. Under more favourable laboratory conditions, the presence of D. suzukii, but not D. melanogaster triggered sour rot emergence. A facilitating effect of D. suzukii infestation for D. melanogaster was not detectable. These findings suggest that D. suzukii does in fact have the potential to trigger sour rot, but will probably rarely do so under field conditions in the vineyard, at least in the studied region. Instead, our study showed that D. melanogaster can have a similar impact on sour rot development as D. suzukii, emphasizing the need of comparative studies.     

ArchbotLit—the archaeobotanical literature database: an update of the search engine for literature on archaeological remains of cultivated plants since 1981

Vegetation History and Archaeobotany - The online Archaeobotanical Literature Database (ArchbotLit) is an important tool for getting targeted access to archaeobotanical publications. It offers the...     

Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte–Neuron Communication

Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²⁺ entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.     

Repeated H2O2 exposure drives cell cycle progression in an in vitro model of ulcerative colitis

The production of hydrogen peroxide (H₂O₂) drives tumourigenesis in ulcerative colitis (UC). Recently, we showed that H₂O₂ activates DNA damage checkpoints in human colonic epithelial cells (HCEC) through c‐Jun N‐terminal Kinases (JNK) that induces p21^WAF1. Moreover, caspases circumvented the G1/S and intra‐S checkpoints, and cells accumulated in G2/M. The latter observation raised the question of whether repeated H₂O₂ exposures alter JNK activation, thereby promoting a direct passage of cells from G2/M arrest to driven cell cycle progression. Here, we report that increased proliferation of repeatedly H₂O₂‐exposed HCEC cells (C‐cell cultures) was associated with (i) increased phospho‐p46 JNK, (ii) decreased total JNK and phospho‐p54 JNK and (iii) p21^WAF1 down‐regulation. Altered JNK activation and p21^WAF1 down‐regulation were accompanied by defects in maintaining G2/M and mitotic spindle checkpoints through adaptation, as well as by apoptosis resistance following H₂O₂ exposure. This may cause increased proliferation of C‐cell cultures, a defining initiating feature in the inflammation‐carcinoma pathway in UC. We further suggest that dysregulated JNK activation is attributed to a non‐apoptotic function of caspases, causing checkpoint adaptation in C‐cell cultures. Additionally, loss of cell‐contact inhibition and the overcoming of senescence, hallmarks of cancer, contributed to increased proliferation. Furthermore, there was evidence that p54 JNK inactivation is responsible for loss of cell‐contact inhibition. We present a cellular model of UC and suggest a sinusoidal pattern of proliferation, which is triggered by H₂O₂‐induced reactive oxygen species generation, involving an interplay between JNK activation/inactivation, p21^WAF1, c‐Fos, c‐Jun/phospho‐c‐Jun, ATF2/phospho‐ATF2, β‐catenin/TCF4‐signalling, c‐Myc, CDK6 and Cyclin D2, leading to driven cell cycle progression.     

Appraisal Tools for Clinical Practice Guidelines: A Systematic Review

Introduction

Clinical practice guidelines can improve healthcare processes and patient outcomes, but are often of low quality. Guideline appraisal tools aim to help potential guideline users in assessing guideline quality. We conducted a systematic review of publications describing guideline appraisal tools in order to identify and compare existing tools.

Methods

Among others we searched MEDLINE, EMBASE and the Cochrane Database of Systematic Reviews from 1995 to May 2011 for relevant primary and secondary publications. We also handsearched the reference lists of relevant publications.On the basis of the available literature we firstly generated 34 items to be used in the comparison of appraisal tools and grouped them into thirteen quality dimensions. We then extracted formal characteristics as well as questions and statements of the appraisal tools and assigned them to the items.

Results

We identified 40 different appraisal tools. They covered between three and thirteen of the thirteen possible quality dimensions and between three and 29 of the possible 34 items. The main focus of the appraisal tools were the quality dimensions “evaluation of evidence” (mentioned in 35 tools; 88%), “presentation of guideline content” (34 tools; 85%), “transferability” (33 tools; 83%), “independence” (32 tools; 80%), “scope” (30 tools; 75%), and “information retrieval” (29 tools; 73%). The quality dimensions “consideration of different perspectives” and “dissemination, implementation and evaluation of the guideline” were covered by only twenty (50%) and eighteen tools (45%) respectively.

Conclusions

Most guideline appraisal tools assess whether the literature search and the evaluation, synthesis and presentation of the evidence in guidelines follow the principles of evidence-based medicine. Although conflicts of interest and norms and values of guideline developers, as well as patient involvement, affect the trustworthiness of guidelines, they are currently insufficiently considered. Greater focus should be placed on these issues in the further development of guideline appraisal tools.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.