Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A₂ receptor

In humans, thromboxane (TX) A₂ signals through the TPα and TPβ isoforms of its G-protein coupled TXA₂ receptor (TP) to mediate a host of (patho)physiologic responses. Herein, angio-associated migratory cell protein (AAMP) was identified as a novel interacting partner of both TPα and TPβ through an interaction dependent on common (residues 312-328) and unique (residues 366-392 of TPβ) sequences within their carboxyl-terminal (C)-tail domains. While the interaction was constitutive in mammalian cells, agonist-stimulation of TPα/TPβ led to a transient dissociation of AAMP from immune complexes which coincided with a transient redistribution of AAMP from its localization in an intracellular fibrous network. Although the GTPase RhoA is a downstream effector of both AAMP and the TPs, AAMP did not influence TP-mediated RhoA or vice versa. Small interfering RNA (siRNA)-mediated disruption of AAMP expression decreased migration of primary human coronary artery smooth muscle cells (1° hCoASMCs). Moreover, siRNA-disruption of AAMP significantly impaired 1° hCoASMC migration in the presence of the TXA₂ mimetic U46619 but did not affect VEGF-mediated cell migration. Given their roles within the vasculature, the identification of a specific interaction between TPα/TPβ and AAMP is likely to have substantial functional implications for vascular pathologies in which they are both implicated.     

Localization of aluminium in tea (Camellia sinensis) leaves using low energy X-ray fluorescence spectro-microscopy

Information on localization of Al in tea leaf tissues is required in order to better understand Al tolerance mechanism in this Al-accumulating plant species. Here, we have used low-energy X-ray fluorescence spectro-microscopy (LEXRF) to study localization of Al and other low Z-elements, namely C, O, Mg, Si and P, in fully developed leaves of the tea plant [Camellia sinensis (L.) O. Kuntze]. Plants were grown from seeds for 3?months in a hydroponic solution, and then exposed to 200?μM AlCl₃ for 2?weeks. Epidermal-mesophyll and xylem phloem regions of 20?μm thick cryo-fixed freeze-dried tea-leaf cross-sections were raster scanned with 1.7 and 2.2?keV excitation energies to reach the Al–K and P–K absorption edges. Al was mainly localized in the cell walls of the leaf epidermal cells, while almost no Al signal was obtained from the leaf symplast. The results suggest that the retention of Al in epidermal leaf apoplast represent the main tolerance mechanism to Al in tea plants. In addition LEXRF proved to be a powerful tool for localization of Al in plant tissues, which can help in our understanding of the processes of Al uptake, transport and tolerance in plants.     

Plant economy at a late Neolithic lake dwelling site in Slovenia at the time of the Alpine Iceman

We present the results of a plant macroremain study of the late Neolithic lakeshore settlement Stare gmajne (SG) at Ljubljansko barje, Slovenia, with cultural horizons that ended around 3330 and 3110 cal. b.c., as obtained by dendrochronological and radiocarbon dating of the most frequent construction timbers of Quercus sp. (oak) and Fraxinus sp. (ash). Fourteen systematically taken samples were investigated, using standard methods for studying waterlogged plant remains, which had been developed during lake dwelling research north of the Alps. Most of the remains were preserved in a waterlogged state, and we identified a total of 93 taxa. The most important cultivated plants were Triticum dicoccum (emmer), Hordeum vulgare (six-rowed naked barley), T. monococcum (einkorn), Linum usitatissimum (flax) and Papaver somniferum (opium poppy). The numerous possibly gathered plants also included Trapa natans (water chestnut) and Vitis vinifera ssp. sylvestris (wild grapevine). Chenopodium album (goosefoot) and Brassica rapa (turnip) with seeds/fruits rich in oil and starch were probably gathered as well. Comparisons of the Stare gmajne results with contemporary north Alpine sites (NA) showed, among other things, that Triticum durum/turgidum (tetraploid naked wheat), frequent at NA, was not found at SG. Trapa natans (water chestnut) was rare and Vitis (grapevine) was not found at NA. The observed differences in the wild plant spectra may have ecological causes, for example a warmer climate south of the Alps, but differences in cultivar spectra are more likely for cultural-historical reasons.     

Photosynthesis-Inhibiting efficiency of 4-chloro-2-(chlorophenylcarbamoyl)phenyl alkylcarbamates

A series of photosynthetic electron transport (PET) inhibitors from the group of salicylanilide alkylcarbamates was investigated. The compounds were analyzed using RP-HPLC to determine lipophilicity, and their PET inhibition was determined in spinach (Spinacia oleracea L.) chloroplasts. The site of action of the studied compounds is situated at the donor site of photosystem 2 (PS 2). Compounds substituted by chlorine in C′-3 and C′-4 of the aniline ring and the optimal length of the alkyl chain pentyl-heptyl in the carbamate moiety provided the most active PET inhibitors (IC₅₀ inhibition <10 μmol/L). Disubstitution in C′-3,4 by chlorine caused significant PET inhibiting activity decrease. Nevertheless, for all three series of C′-3, C′-4, C′-3,4 compounds, the dependence of PET activity on lipophilicity showed to be quasi-parabolic.     

Human plasma glycome in attention-deficit hyperactivity disorder and autism spectrum disorders

Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction.     

A comparative study of DNA binding and cell cycle phase perturbation by the dinuclear complex of Cd(II) with the condensation product of 2-acetylpyridine and malonic acid dihydrazide N',N'(2) -bis[(1E)-1-(2-pyridyl)ethylidene]propanedihydrazide

Organometallic Cd(II) compounds have recently attracted attention for their anticancer activity. The interaction of the dinuclear complex of Cd(II) with the condensation product of 2-acetylpyridine and malonic acid dihydrazide, N',N'(2) -bis[(1E)-1-(2-pyridyl)ethylidene]propanedihydrazide (Cd(II)H(2) L), with calf thymus DNA (CT-DNA) was monitored by blue shift in UV-vis spectra of the complex. The binding constant of Cd(II)H(2) L complex with CT-DNA was determined (K(B) = 1.8 × 10(4) M(-1) ) and was indicative of minor groove binding. Agarose gel electrophoretic changes in mobility of supercoiled and circular forms of pBR322 and pUC18 plasmids in the presence of the complex suggest that conformational changes in the plasmids occur upon binding of the Cd(II)H(2) L complex. The Cd(II)H(2) L complex induced perturbation of the cell cycle phase distribution and an increase in the percentage of cells in the sub-G1 phase of human cervical cancer HeLa cell line and murine melanoma B16 cell line. Immunoblotting analysis showed the overexpression of Bcl-2 protein with the Cd(II)H(2) L complex.     

The in vivo genotoxicity of cisplatin,isoflurane and halothane evaluated by alkaline comet assay in Swiss albino mice

The aim of this study was to evaluate the genotoxicity of repeated exposure to isoflurane or halothane and compare it with the genotoxicity of repeated exposure to cisplatin. We also determined the genotoxicity of combined treatment with inhalation anaesthetics and cisplatin on peripheral blood leucocytes (PBL), brain, liver and kidney cells of mice. The mice were divided into six groups as follows: control, cisplatin, isoflurane, cisplatin–isoflurane, halothane and cisplatin–halothane, and were exposed respectively for three consecutive days. The mice were treated with cisplatin or exposed to inhalation anaesthetic; the combined groups were exposed to inhalation anaesthetic after treatment with cisplatin. The alkaline comet assay was performed. All drugs had a strong genotoxicity (P < 0.05 vs. control group) in all of the observed cells. Isoflurane caused stronger DNA damage on the PBL and kidney cells, in contrast to halothane, which had stronger genotoxicity on brain and liver cells. The combination of cisplatin and isoflurane induced lower genotoxicity on PBL than isoflurane alone (P < 0.05). Halothane had the strongest effect on brain cells, but in the combined treatment with cisplatin, the effect decreased to the level of cisplatin alone. Halothane also induced the strongest DNA damage of the liver cells, while the combination with cisplatin increased its genotoxicity even more. The genotoxicity of cisplatin and isoflurane on kidney cells were nearly at the same level, but halothane caused a significantly lower effect. The combinations of inhalation anaesthetics with cisplatin had stronger effects on kidney cells than inhalation anaesthetics alone. The observed drugs and their combinations induced strong genotoxicity on all of the mentioned cells.     

Alimentary prion infections: Touchdown in the intestine

Neurodegenerative diseases are caused by proteinaceous aggregates, usually consisting of misfolded proteins which are often typified by a high proportion of β-sheets that accumulate in the central nervous system. These diseases, including Morbus Alzheimer, Parkinson disease and Transmissible Spongiform Encephalopathies (TSEs)—also termed prion disorders—afflict a substantial proportion of the human population and, as such, the etiology and pathogenesis of these diseases has been the focus of mounting research. Although many of these diseases arise from genetic mutations or are sporadic in nature, the possible horizontal transmissibility of neurodegenerative diseases poses a great threat to population health. In this article we discuss recent studies that suggest that the “non-transmissible” status bestowed upon Alzheimer and Parkinson diseases may need to be revised as these diseases have been successfully induced through tissue transplants. Furthermore, we highlight the importance of investigating the “natural” mechanism of prion transmission including peroral and perenteral transmission, proposed routes of gastrointestinal uptake and neuroinvasion of ingested infectious prion proteins. We examine the multitude of factors which may influence oral transmissibility and discuss the zoonotic threats that Chronic Wasting disease (CWD), Bovine Spongiform Encephalopathy (BSE) and Scrapie may pose resulting in vCJD or related disorders. In addition, we suggest that the 37 kDa/67 kDa laminin receptor on the cell surface of enterocytes, a major cell population in the intestine, may play an important role in the intestinal pathophysiology of alimentary prion infections.Key words: prion, 37 kDa/67 kDa laminin receptor, CJD, BSE, CWD, scrapie, Alzheimer disease, Parkinson disease, intestine, enterocytesMany different mechanisms exist which underlie the etiology of the numerous neurodegenerative diseases affecting the human population. Amongst the most prominent are Morbus Alzheimer, prion disorders, Parkinson disease, Chorea Huntington, frontotemporal dementia and amylotrophic lateral sclerosis. The molecular mechanisms underlying these diseases vary; however, all neurodegenerative diseases share a common feature: they are caused by protein aggregation. The only neurodegenerative diseases proven to be transmissible are prion disorders. In contrast to frontotemporal dementia, recent evidence suggests that Alzheimer and Parkinson diseases may also be transmissible. Pre-symptomatic Alzheimer disease (APP23) mice exhibited an increase in the Alzheimer phenotype when brain homogenate of autopsied human Alzheimer disease patients and older, amyloid beta- (Aβ-) laden APP23 mice was injected into their hippocampi.¹ These findings suggest that the Aβ-abundant brain homogenate of Alzheimer disease patients may possess the ability to induce or supplement the overproduction of Aβ, possibly leading to the onset of Alzheimer disease.The pathological feature associated with Parkinson disease is the formation of Lewy bodies in cell bodies and neuronal processes in the brain.² The main component of these protein aggregates is α-synuclein (reviewed in ref. ²). Autopsies of Parkinson disease patients revealed that Lewy bodies had formed on healthy embryonic neurons that had been grafted onto the brain tissue of the patients several years before (prior to said examination).^3–5 It may thus be proposed that α-synuclein transmission is possible from diseased to healthy neurons, suggesting that Parkinson disease may be transmissible from a Parkinson disease patient to a healthy individual. These findings imply that Alzheimer and Parkinson diseases may be transmissible through tissue transplants and the use of contaminated surgical tools.⁶Prion disorders, also termed Transmissible Spongiform Encephalopathies (TSEs), are fatal neurodegenerative diseases that affect the central nervous system (CNS) of multiple animal species. In lieu of the social, economic and political ramifications of such infections, as well as the possible intra- and interspecies transmissibility of such disorders, various routes of experimental transmission have been investigated including intracerebral, intraperitoneal, intraventricular, intraocular, intraspinal and subcutaneous injections (reviewed in ref. ^7–9). However, such routes of transmission are not representative of the “natural” mechanism as the majority of prion disorders are contracted through ingestion of infectious prion (PrP^Sc) containing material. Thus, the peroral and perenteral prion transmission is of greatest consequence with respect to TSE disease establishment. Moreover, the presence of PrP^Sc in the buccal cavity of scrapie-infected sheep¹⁰ (reviewed in ref. ¹¹) and the possible horizontal transfer as a result hereof, as may be similarly proposed for animals suffering from other TSEs, may further contribute to the oral transmissibility of TSEs.A number of model systems have been employed to study TSE transmissibility. Owing to ethical constraints, TSE transmissibility to humans via the oral route may not be directly investigated and as a result hereof, alternative model systems are needed. These may include the use of transgenic mice, cell lines which are permissive to infection¹² and experimental animals such as sheep, calves, goats, minks, ferrets and non-human primates (reviewed in ref. ⁹).Intestinal entry of PrP^Sc has been proposed to occur via two pathways, the membranous (M) cell-dependent and M cell-independent pathways (Fig. 1).^13,14 The former involves endocytic M (microfold)-cells, which cover the intestinal lymphoid follicles (Peyer''s patches)¹⁴ and may take up prions and thereby facilitate the translocation of these proteins across the intestinal epithelium into the lymphoid tissues (reviewed in ref. ⁹) as has been demonstrated in a cellular model.¹³ Following such uptake by the M cells, the prions may subsequently pass to the dendritic cells and follicular dendritic cells (FDCs) (Fig. 1), which allow for prion transport to the mesenteric lymph nodes and replication, respectively.¹⁵ The prion proteins may subsequently gain access to the enteric nervous system (ENS) and ultimately the central nervous system (CNS).¹⁵Open in a separate windowFigure 1Proposed routes of gastrointestinal entry of ingested infectious prions (PrP^Sc) as well as possible pathways of amplification and transport to the central nervous system.However, prion intestinal translocation has been observed in the absence of M cells and has been demonstrated to be as a result of the action of polar, 37 kDa/67 kDa LRP/LR (non-integrin laminin receptor; reviewed in ref. ^16–18) expressing enterocytes. Enterocytes are the major cell population of the intestinal epithelium and due to their ability to endocytose pathogens, nutrients and macromolecules,¹⁹ it has been proposed that these cells may represent a major entry site for alimentary prions (Fig. 1).Since enterocyte prion uptake has been demonstrated to be dependent on the presence of LRP/LR on the apical brush border of the cells,^14,20 the interaction between varying prion protein strains and the receptor^21–23 may be employed as a model system to study possible oral transmissibility of prion disorders across species as well as the intestinal pathophysiology of alimentary prion infections.²⁴ Moreover, the blockage of such interactions through the use of anti-LRP/LR specific antibodies has been reported to reduce PrP^Sc endocytosis¹⁹ and thus these antibodies may serve as potential therapeutics to prevent infectious prion internalization and thereby prevent prion infections. It must be emphasized that the adhesion of prion proteins to cells is not solely dependent on the LRP/LR-PrP^Sc interactions;²⁴ however, this interaction is of importance with regards to internalization and subsequent pathogenesis.We applied the aforementioned cell model to study the possible oral transmission of PrP^BSE, PrP^CWD and ovine PrP^Sc to cervids, cattle, swine and humans.²⁴ The direct transmission of the aforementioned animal prion disorders to humans as a result of dietary exposure and the possible establishment of zoonotic diseases is of great public concern. It must however be emphasized that the study investigated the co-localization of LRP/LR and various prion strains and not the actual internalization process.PrP^BSE was shown to co-localize with LRP/LR on human enterocytes²⁴, thereby suggesting that PrP^BSE is transmissible to humans via the oral route which is widely accepted as the manner by which variant CJD originated. This suspicion was previously investigated using a macaque model, which was successfully perorally infected by BSE-contaminated material and subsequently lead to the development of a prion disorder that resembles vCJD.²⁵ These results, due to the evolutionary relatedness between macaques and humans, allowed researchers to confirm the oral transmissibility of PrP^BSE to humans. PrP^BSE may also potentially lead to prion disorder establishment in swine,²⁴ livestock of great economic and social importance.The prion disorder affecting elk, mule deer and white-tailed deer is termed CWD. Cases of the disease are most prevalent in the US but are also evident in Canada and South Korea.^26,27 As the infectious prion isoform is reported to be present in the blood²⁸ and skeletal muscle,²⁹ hunting, consumption of wild venison and contact with other animal products derived from CWD-infected elk and deer may thereby pose a public health risk. Our studies demonstrate that PrP^CWD co-localizes with LRP/LR on human enterocytes²⁴ thereby suggesting a possible oral transmissibilty of this TSE to humans. This is, however, inconsistent with results obtained during intra-cerebral inoculation of the brains and spinal cords of transgenic mice overexpressing the human cellular prion protein (PrP^c),^26,27 which is essential for TSE disease establishment and progression. Further, discrepancies have also been reported with respect to non-human primates, as squirrel monkeys have been successfully intracerebrally inoculated with mule-deer prion homogenates,³⁰ while cynolmolgus macaques were resistant to infection.³¹ CWD has been transmitted to ferrets, minks and goats³² and as these animals may serve as domestic animals or livestock, secondary transmission from such animals to humans, through direct contact or ingestion of infected material, may be an additional risk factor that merits further scientific investigation.Ovine PrP^Sc co-localization with LRP/LR on human and bovine enterocytes may be indicative of the infectious agents'' ability to effect cross-species infections. The oral transmissibility of Scrapie has been confirmed in hamsters fed with sheep-scrapie-infected material.³³The discrepancies with regards to the transmissibility of certain infectious prion proteins when assessed by different model systems may be due to the experimental transmission route employed. Oral exposure often results in significantly prolonged incubation times when compared to intracerebral inoculation techniques and thus failure of transgenic mice and normal experimental animals to develop disease phenotypes after being fed TSE-contaminated material may not necessarily indicate that the infection process failed.¹⁴ Apart from the route of infection, numerous other factors may influence transmission between species, including dose, PrP polymorphisms and genetic factors, the prion strain employed as well as the efficacy of prion transport to the CNS.³⁴ The degree of homology between the PrP^c protein in the animals serving as the infectious prion source and recipient has also been described as a feature limiting cross-species transmission.³⁴ The negative results, as referred to above, obtained upon prion-protein inoculation of animal models may have resulted due to the slow rate at which the infectious prion induces conformational conversion of the endogenous PrP^c in the animal cells and this in turn results in low levels of infectious prion replication and symptom development.²⁷Furthermore, even in the event that certain prion disorders are not directly transmissible to humans, most are transmissible to at least a single species of domestic animal or livestock. The infectious agents properties may be altered in the secondary host such that it becomes transmissible to humans (reviewed in ref. ³⁵). Thus, interspecies transmission between animals may indirectly influence human health.It is noteworthy to add that although the oral route of PrP^Sc transmission may result in prolonged incubation times, it may broaden the range of susceptible hosts. A common constituent of food is ferritin, a protein that is resistant to digestive enzyme hydrolysis and, due to its homology across species, it may serve as co-transporter of PrP^Sc and facilitate enterocyte internalization of the infectious prion.³⁶ It may thus be proposed that prion internalization may occur via a ferritin-PrP^Sc complex even in the absence of co-localization between the infectious agent and LRP/LR such that many more cross-species infections (provided that the other infection factors are favorable) may be probable.³⁷ In addition, digestive enzymes in the gastrointestinal tract facilitate PrP^Sc binding to the intestinal epithelium and subsequent intestinal uptake³⁶ and thus depending on the individuals'' digestive processes, the susceptibility to infection and the rate of disease development may vary accordingly. As a result hereof, though laboratory experiments in cell-culture and animal models may render a particular prion disorder non-infectious to humans, this may not be true for all individuals.In lieu of the above statements, with particular reference to inconsistencies in reported results and the multiple factors influencing oral transmissibility of TSEs, further transmission studies are required to evaluate the zoonotic threat which CWD, BSE and Scrapie may pose through ingestion.