Peak plasma interleukin-6 and other peripheral markers of inflammation in the first week of ischaemic stroke correlate with brain infarct volume, stroke severity and long-term outcome

Background

Cerebral ischaemia initiates an inflammatory response in the brain and periphery. We assessed the relationship between peak values of plasma interleukin-6 (IL-6) in the first week after ischaemic stroke, with measures of stroke severity and outcome.

Methods

Thirty-seven patients with ischaemic stroke were prospectively recruited. Plasma IL-6, and other markers of peripheral inflammation, were measured at pre-determined timepoints in the first week after stroke onset. Primary analyses were the association between peak plasma IL-6 concentration with both modified Rankin score (mRS) at 3 months and computed tomography (CT) brain infarct volume.

Results

Peak plasma IL-6 concentration correlated significantly (p < 0.001) with CT brain infarct volume (r = 0.75) and mRS at 3 months (r = 0.72). It correlated similarly with clinical outcome at 12 months or stroke severity. Strong associations were also noted between either peak plasma C-reactive protein (CRP) concentration or white blood cell (WBC) count, and all outcome measures.

Conclusions

These data provide evidence that the magnitude of the peripheral inflammatory response is related to the severity of acute ischaemic stroke, and clinical outcome.     

Selective cytotoxicity of 3-amino-l-tyrosine correlates with peroxidase activity

Summary In the presence of 3-amino-l-tyrosine (3-AT), abundant brown pigment forms in human HL-60 cells, but not in a variety of other cell lines, which are reported to be lower in mean myeloperoxidase (MPO) content than HL-60. Cells were assessed for peroxidase activity with an ABTS-based colorimetric assay and compared to values obtained with known amounts of human myeloperoxidase. HL-60 cells were estimated to contain the equivalent of 37.1 ng myeloperoxidase/10⁶ cells versus 26.1 and 5.0 ng/10⁶ cells for human K562 and murine RAW 264.7 cell lines, respectively. HL-60 cells exhibited a nearly 60% inhibition of proliferation and >70% reduction in cell viability after 4 d of culture in the presence of 100 μg 3-AT per ml. Higher concentrations of 3-AT (up to 400 μg/ml) for 4 d reduced HL-60 proliferation by 80% and decreased viability to 1–3%. Comparable levels of cytotoxicity were achieved in KG-1 cells after 7 d with 200 or 400 μg 3-AT per ml. K562 cells exhibited a 40% reduction in cell number after 7 d with 400 μg 3-AT per ml, but concentrations less than 400 μg/ml did not significantly affect K562 proliferation. K562 viability remained unchanged with doses of 3-AT up to 400 μg/ml. RAW 264.7 cells exhibited unchanged viability and proliferation in the presence of 3-AT at concentrations up to 400 μg 3-AT per ml. K562, KG-1, and RAW 264.7 cells exhibited no evidence of brown pigment formation in the presence of 3-AT and medium containing 10% fetal bovine serum. However, RAW 264.7 cells that were converted to protein-free medium and exposed to 3-AT exhibited intense brown pigment in some cell nuclei. A high percentage of HL-60 cells treated with 3-AT exhibited membrane blebbing, pyknosis, and nuclear fragmentation, which was not observed among other 3-AT-treated cell lines. A mechanism involving toxic intermediates of peroxidase-mediated “aminomelanin” formation is hypothesized.     

Characterization of the Hansenula polymorpha PUR7 gene and its use as selectable marker for targeted chromosomal integration

The Hansenula polymorpha genes encoding the putative functional homologs of the enzymes involved in the seventh and eighth step in purine biosynthesis, HpPUR7 and HpPUR8, were cloned and sequenced. An overexpression vector designated pHIPA4 was constructed, which contains the HpPUR7 gene as selectable marker and allows expression of genes of interest via the strong, inducible alcohol oxidase promoter. An ade11 auxotrophic mutant that is affected in the activity of the HpPUR7 gene product was used to construct strain NCYC495 ade11.1 leu1.1 ura3. This strain grew on methanol at wild-type rates (doubling time of approximately 4 h) and is suitable for independent introduction of four expression cassettes, each using one of the markers for selection, in addition to the zeocin resistance marker. It was subsequently used as a host for overproduction of two endogenous peroxisomal matrix proteins, amine oxidase and catalase. Efficient site-specific integration of pHIPA4 and overproduction of amine oxidase and catalase is demonstrated. The expression cassette appeared to be pre-eminently suited to mediate moderate protein production levels. The advantages of pHIPA4 and the new triple auxotrophic strain in relation to the use of H. polymorpha as a versatile cell factory or as a model organism for fundamental studies on the principles of peroxisome homeostasis is discussed.     

TNF receptor 1, IL-1 receptor, and iNOS genetic knockout mice are not protected from anthrax infection

Anthrax produces at least two toxins that cause an intense systemic inflammatory response, edema, shock, and eventually death. The relative contributions of various elements of the immune response to mortality and course of disease progression are poorly understood. We hypothesized that knockout mice missing components of the immune system will have an altered response to infection. Parent strain mice and knockouts were challenged with LD95 of anthrax spores (5 x 10(6)) administered subcutaneously. Our results show that all genetic knockouts succumbed to anthrax infection at the same frequency as the parent. TNF antibody delayed death but TNF receptor 1 knockout had no effect. IL-1 receptor or iNOS knockouts died sooner. Anthrax was more abundant in the injection site of TNF-alpha and iNOS knockouts compared to parent suggesting that attenuated cellular response increases rate of disease progression. With the exception of edema and necrosis at the injection site pathological changes in internal organs were not observed.     

Structural fingerprints of the Ras-GTPase activating proteins neurofibromin and p120GAP

Ras specific GTPase activating proteins (GAPs), neurofibromin and p120GAP, bind GTP bound Ras and efficiently complement its active site. Here we present comparative data from mutations and fluorescence-based assays of the catalytic domains of both RasGAPs and interpret them using the crystal structures. Three prominent regions in RasGAPs, the arginine-finger loop, the phenylalanine-leucine-arginine (FLR) region and alpha7/variable loop contain structural fingerprints governing the GAP function. The finger loop is crucial for the stabilization of the transition state of the GTPase reaction. This function is controlled by residues proximal to the catalytic arginine, which are strikingly different between the two RasGAPs. These residues specifically determine the orientation and therefore the positioning of the arginine finger in the Ras active site. The invariant FLR region, a hallmark for RasGAPs, indirectly contributes to GTPase stimulation by forming a scaffold, which stabilizes Ras switch regions. We show that a long hydrophobic side-chain in the FLR region is crucial for this function. The alpha7/variable loop uses several conserved residues including two lysine residues, which are involved in numerous interactions with the switch I region of Ras. This region determines the specificity of the Ras-RasGAP interaction.     

Comparative effects of extremely high power microwave pulses and a brief CW irradiation on pacemaker function in isolated frog heart slices

The existence of specific bioeffects due to high peak power microwaves and their potential health hazards are among the most debated but least explored problems in microwave biology. The present study attempted to reveal such effects by comparing the bioeffects of short trains of extremely high power microwave pulses (EHPP, 1 micros width, 250-350 kW/g, 9.2 GHz) with those of relatively low power pulses (LPP, 0.5-10 s width, 3-30 W/g, 9.2 GHz). EHPP train duration and average power were made equal to those of an LPP; therefore both exposure modalities produced the same temperature rise. Bioeffects were studied in isolated, spontaneously beating slices of the frog heart. In most cases, a single EHPP train or LPP immediately decreased the inter-beat interval (IBI). The effect was proportional to microwave heating, fully reversible, and easily reproducible. The magnitude and time course of EHPP- and LPP-induced changes always were the same. No delayed or irreversible effects of irradiation were observed. The same effect could be repeated in a single preparation numerous times with no signs of adaptation, sensitization, lasting functional alteration, or damage. A qualitatively different effect, namely, a temporary arrest of preparation beats, could be observed when microwave heating exceeded physiologically tolerable limits. This effect also did not depend on whether the critical temperature rise was produced by LPP or EHPP exposure. Within the studied limits, we found no indications of EHPP-specific bioeffects. EHPP- and LPP-induced changes in the pacemaker rhythm of isolated frog heart preparation were identical and could be entirely attributed to microwave heating.     

The methylotrophic yeast Hansenula polymorpha: a versatile cell factory

The development of heterologous overexpression systems for soluble proteins has greatly advanced the study of the structure/function relationships of these proteins and their biotechnological and pharmaceutical applications. In this paper we present an overview on several aspects of the use of the methylotrophic yeast Hansenula polymorpha as a host for heterologous gene expression. H. polymorpha has been successfully exploited as a cell factory for the large-scale production of such components. Stable, engineered strains can be obtained by site-directed integration of expression cassettes into the genome, for which various constitutive and inducible promoters are available to control the expression of the foreign genes. New developments have now opened the way to additional applications of H. polymorpha, which are unprecedented for other organisms. Most importantly, it may be the organism of choice for reliable, large-scale production of heterologous membrane proteins, using inducible intracellular membranes and targeting sequences to specifically insert these proteins stably into these membranes. Furthermore, the use of H. polymorpha offers the possibility to accumulate the produced components into specific compartments, namely peroxisomes. These organelles are massively induced during growth of the organism on methanol and may occupy up to 80% of the cell volume. Accumulation inside peroxisomes prevents undesired modifications (e.g. proteolytic processing or glycosylation) and is also in particular advantageous when proteins are produced which are toxic or harmful for the host.     

Identification of immuno-reactive proteins from a sheep gastrointestinal nematode, Trichostrongylus colubriformis, using two-dimensional electrophoresis and mass spectrometry

Gastrointestinal nematode infections of livestock animals are prevalent and costly problems worldwide. Currently, infections are controlled by anthelmintic chemicals but increasing drug resistance has prompted research interest to shift towards alternative methods of control such as vaccine development and selection of worm-resistant animals. The present study analyses proteins from Trichostrongylus colubriformis infective L3s that are recognised by IgG of immune sheep. Following protein separation via two-dimensional electrophoresis and Western blot probing with plasma from sheep resistant to T. colubriformis, mass spectrometry-based proteomic analyses were used to identify immuno-reactive protein spots. We were able to identify 28 immune targets, including aspartyl protease inhibitor, enolase, chaperone proteins, galectin, glycolytic enzymes, kinase, phosphatase and structural muscle proteins such as myosin, paramyosin, calponin and DIM-1. The data suggest that immune responses to T. colubriformis are dispersed over a relatively large number of parasite antigens, including several cytoplasmically expressed proteins. The results have new implications for understanding the molecular mechanisms that underpin host-parasite interaction during gastrointestinal nematode infections.     

1H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

NSD3s, the proline-tryptophan-tryptophan-proline (PWWP) domain-containing, short isoform of the human oncoprotein NSD3, displays high transforming properties. Overexpression of human NSD3s or the yeast protein Pdp3 in Saccharomyces cerevisiae induces similar metabolic changes, including increased growth rate and sensitivity to oxidative stress, accompanied by decreased oxygen consumption. Here, we set out to elucidate the biochemical pathways leading to the observed metabolic phenotype by analyzing the alterations in yeast metabolome in response to NSD3s or Pdp3 overexpression using ¹H nuclear magnetic resonance (NMR) metabolomics. We observed an increase in aspartate and alanine, together with a decrease in arginine levels, on overexpression of NSD3s or Pdp3, suggesting an increase in the rate of glutaminolysis. In addition, certain metabolites, including glutamate, valine, and phosphocholine were either NSD3s or Pdp3 specific, indicating that additional metabolic pathways are adapted in a protein-dependent manner. The observation that certain metabolic pathways are differentially regulated by NSD3s and Pdp3 suggests that, despite the structural similarity between their PWWP domains, the two proteins act by unique mechanisms and may recruit different downstream signaling complexes. This study establishes for the first time a functional link between the human oncoprotein NSD3s and cancer metabolic reprogramming.     

Cretaceous and living colloniidae of the redefined subfamily Petropomatinae,with two new genera and one new species,with notes on opercular evolution in turbinoideans,and the fossil record of Liotiidae (Vetigastropoda: Turbinoidea)

Opercular characters are of major importance to understanding the living and fossil groups in the vetigastropod superfamily TurbinoideaRafinesque. Here we evaluate current knowledge of fossil turbinoid opercula in the light of a newly discovered Recent colloniid with an unfamiliar opercular morphology. The operculum ofLiotipoma n. gen. has a multispiral and conical inner surface; the outer surface is rugose with a central pit. Similar opercula are known from the Late CretaceousSohlipoma n. gen. and the Early CretaceousPetropoma Gabb. These genera are assigned to the here redefined colloniid subfamily PetropomatinaeCox inKnight et al., which was based originally onGabb’s misinterpretation of the inner and outer sides of the operculum ofPetropoma peruana Gabb. In contrast, opercula of most living members of Colloniinae s.S., have a flat inner side with a multispiral pattern that becomes paucispiral. In addition, opercula typical for living Turbinidae are here traced to the Late Cretaceous. The new genusLiotipoma has lamellar sculpture like that of Liotiidae H. & A.Adams, showing that such sculpture is not restricted to the Liotiidae. We also reject previous allocations of the Permian DichostasiinaeYochelson and Jurassic Cross-ostomatinaeCox inKnight et al. to Liotiidae, and find an undisputed earliest known Jurassic occurrence for the Liotiidae. The recent suggestion that the change from corneous to calcareous opercula among turbinids occurred concurrently with a change in pattern from multispiral to paucispiral is not supported by our data.