A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.     

Varietal effects of eight paired lines of transgenic Bt maize and near-isogenic non-Bt maize on soil microbial and nematode community structure

A glasshouse experiment was undertaken to provide baseline data on the variation between conventional maize (Zea mays L.) varieties and genetically modified maize plants expressing the insecticidal Bacillus thuringiensis protein (Bt, Cry1Ab). The objective was to determine whether the variation in soil parameters under a range of conventional maize cultivars exceeded the differences between Bt and non-Bt maize cultivars. Variations in plant growth parameters (shoot and root biomass, percentage carbon, percentage nitrogen), Bt protein concentration in shoots, roots and soil, soil nematode abundance and soil microbial community structure were determined. Eight paired varieties (i.e. varieties genetically modified to express Bt protein and their near-isogenic control varieties) were investigated, together with a Bt variety for which no near-isogenic control was available (NX3622, a combined transformant expressing both Bt and herbicide tolerance) and a conventional barley (Hordeum vulgare L.) variety which was included as a positive control. The only plant parameter which showed a difference between Bt varieties and near-isogenic counterparts was the shoot carbon to nitrogen ratio; this was observed for only two of the eight varieties, and so was not attributable to the Bt trait. There were no detectable differences in the concentration of Bt protein in plant or soil with any of the Bt-expressing varieties. There were significant differences in the abundance of soil nematodes, but this was not related to the Bt trait. Differences in previously published soil nematode studies under Bt maize were smaller than these varietal effects. Soil microbial community structure, as determined by phospholipid fatty acid (PLFA) analysis, was strongly affected by plant growth stage but not by the Bt trait. The experimental addition of purified Cry1Ab protein to soil confirmed that, at ecologically relevant concentrations, there were no measurable effects on microbial community structure.     

Peptide blockers of the inhibition of neuronal nicotinic acetylcholine receptors by amyloid beta

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.     

Characterization of novel SF3b and 17S U2 snRNP proteins,including a human Prp5p homologue and an SF3b DEAD-box protein

Mass spectrometry was used to identify novel proteins associated with the human 17S U2 snRNP and one of its stable subunits, SF3b. Several additional proteins were identified, demonstrating that 17S U2 snRNPs are significantly more complex than previously thought. Two of the newly identified proteins, namely the DEAD-box proteins SF3b125 and hPrp5 (a homologue of Saccharomyces cerevisiae Prp5p) were characterized further. Immunodepletion experiments with HeLa nuclear extract indicated that hPrp5p plays an important role in pre-mRNA splicing, acting during or prior to prespliceosome assembly. The SF3b-associated protein SF3b125 dissociates at the time of 17S U2 formation, raising the interesting possibility that it might facilitate the assembly of the 17S U2 snRNP. Finally, immunofluorescence/FISH studies revealed a differential subnuclear distribution of U2 snRNA, hPrp5p and SF3b125, which were enriched in Cajal bodies, versus SF3b155 and SF3a120, which were not; a model for 17S U2 snRNP assembly based on these findings is presented. Taken together, these studies provide new insight into the composition of the 17S U2 snRNP and the potential function of several of its proteins.     

Detection of Borrelia burgdorferi and Anaplasma phagocytophilum in the black‐legged tick,Ixodes scapularis,within southwestern Pennsylvania

Prevalence studies of Borrelia burgdorferi and Anaplasma phagocytophilum have been rare for ticks from southwestern Pennsylvania. We collected 325 Ixodes scapularis ticks between 2011 and 2012 from four counties in southwestern Pennsylvania. We tested for the presence of Borrelia burgdorferi and Anaplasma phagocytophilum using PCR. Of the ticks collected from Pennsylvania, B. burgdorferi (causative agent of Lyme disease) was present in 114/325 (35%) and Anaplasma phagocytophilum (causative agent of Human Granulocytic Anaplasmosis) was present in 48/325 (15%) as determined by PCR analysis.     

Nitrogen mineralization and denitrification as influenced by crop residue particle size

Managing the crop residue particle size has the potential to affect N conservation in agricultural systems. We investigated the influence of barley (Hordeum vulgare) and pea (Pisum sativum) crop residue particle size on N mineralization and denitrification in two laboratory experiments. Experiment 1: ¹⁵N-labelled ground (3 mm) and cut (25 mm) barley residue, and microcrystalline cellulose+glucose were mixed into a sandy loam soil with additional inorganic N. Experiment 2: inorganic¹⁵ N and C₂H₂ were added to soils with barley and pea material after 3, 26, and 109 days for measuring gross N mineralization and denitrification.Net N immobilization over 60 days in Experiment 1 cumulated to 63 mg N kg^-1 soil (ground barley), 42 (cut barley), and 122 (cellulose+glucose). More N was seemingly net mineralized from ground barley (3.3 mg N kg^-1 soil) than from cut barley (2.7 mg N kg^-1 soil). Microbial biomass peaked at day 4 with the barley treatments and at day 14 with the cellulose+glucose whereafter the biomass leveled out at values 79 mg C kg^-1 (ground), 104 (cut), and 242 (cellulose+glucose) higher than for the control soil. Microbial growth yields were similar for the two barley treatments, ca. 60 mg C g^-1 substrate C added, which was lower than the 142 mg C g^-1 C added with cellulose+glucose. This suggests that the 75% (w/w) holocelluloses and sugars contained with the barley material remained physically protected despite grinding. In Experiment 2 gross mineralization on day 3 was 4.8 mg N kg^-1 d^-1 with ground pea, twice as much as for all other treatments. On day 26 the treatment with ground barley had the greatest gross N mineralization. In static cores ground barley denitrified 11-fold more than did cut barley, whereas denitrification was similar for the two pea treatments. In suspensions denitrification was similar for the two treatments both with barley and pea residue.We conclude that the higher microbial activity associated with the initial decomposition of ground plant material is due to a more intimate plant residue-soil contact. On the long term, grinding the plant residues has no significant effect on N dynamics.     

Mortality of lemmings, Lemmus lemmus, at peak density in a mountainous area of Norway

We estimated carcass density and determined cause of death in a peak-density lemming, Lemmus lemmus , population at Finse, Norway. Of 74 recovered carcasses, 80% had subcutaneous haematoma (blood underneath the skin) in the head region, though the skin on the skull most often appeared unharmed. Thirty percent were apparently killed by a mustelid predator, while 51% were probably killed by corvids. Carcass density was 26.6 and 9.4ha^-1 in good and poor lemming habitats, respectively.     

Unique Gene Expression and MR T2 Relaxometry Patterns Define Chronic Murine Dextran Sodium Sulphate Colitis as a Model for Connective Tissue Changes in Human Crohn’s Disease

Introduction

Chronically relapsing inflammation, tissue remodeling and fibrosis are hallmarks of inflammatory bowel diseases. The aim of this study was to investigate changes in connective tissue in a chronic murine model resulting from repeated cycles of dextran sodium sulphate (DSS) ingestion, to mimic the relapsing nature of the human disease.

Materials and Methods

C57BL/6 mice were exposed to DSS in drinking water for 1 week, followed by a recovery phase of 2 weeks. This cycle of exposure was repeated for up to 3 times (9 weeks in total). Colonic inflammation, fibrosis, extracellular matrix proteins and colonic gene expression were studied. In vivo MRI T ₂ relaxometry was studied as a potential non-invasive imaging tool to evaluate bowel wall inflammation and fibrosis.

Results

Repeated cycles of DSS resulted in a relapsing and remitting disease course, which induced a chronic segmental, transmural colitis after 2 and 3 cycles of DSS with clear induction of fibrosis and remodeling of the muscular layer. Tenascin expression mirrored its expression in Crohn’s colitis. Microarray data identified a gene expression profile different in chronic colitis from that in acute colitis. Additional recovery was associated with upregulation of unique genes, in particular keratins, pointing to activation of molecular pathways for healing and repair. In vivo MRI T₂ relaxometry of the colon showed a clear shift towards higher T₂ values in the acute stage and a gradual regression of T₂ values with increasing cycles of DSS.

Conclusions

Repeated cycles of DSS exposure induce fibrosis and connective tissue changes with typical features, as occurring in Crohn’s disease. Colonic gene expression analysis revealed unique expression profiles in chronic colitis compared to acute colitis and after additional recovery, pointing to potential new targets to intervene with the induction of fibrosis. In vivo T₂ relaxometry is a promising non-invasive assessment of inflammation and fibrosis.     

CEREBRAL METABOLIC AND VASCULAR EFFECTS OF BARBITURATE THERAPY FOLLOWING COMPLETE GLOBAL ISCHEMIA

Complete global cerebral ischemia was induced in dogs by temporary ligation of the ascending aorta for 10min. Prior to the ischemic period, half of the animals were given pentobarbital 30-38 mg/kg, a maneuver previously reported to prevent or attenuate cerebral damage in this same model. Cerebral blood flow (CBF) and cerebral metabolic rate (CMR_O2) were followed from prior to the ischemic period to 6 h post-ischemia. At varying time intervals following ischemia, brain biopsies were obtained and analyzed for cerebral metabolites to determine the cerebral energy state. Only a few differences were observed between pentobarbital-treated and untreated animals. Post-ischemic CMR_O2, stabilized at a significantly lower level in treated than in untreated animals. However, CBF was proportionately lower and thus O₂ delivery relative to O₂ needs in the two groups was comparable. Also in both groups, the CBF and CMR_O2 stabilized at levels significantly below pre-ischemia controls. Cerebral energy stores in both groups were depleted after 10min of ischemia but were restored to near normal within 4min post-ischemia. Total restoration of the adenine nucleotide pool and ATP were delayed as was the return of brain lactate to normal. A 10min period of post-ischemic hyperemia was observed in all animals and in the initial 4min post-ischemia CMR_O2 was also increased. The latter is probably accounted for by the O₂ needs for restoration of cerebral energy and O₂ stores. We conclude that cerebral protection as provided by barbiturates following complete global ischemia cannot be accounted for by any measurable effect on CBF, CMR_O2, or the cerebral energy stores during the initial 6 h post-ischemia.