Close linkage of the cytochrome P450IIA gene subfamily (Cyp2a) to Cyp2b and Coh on mouse chromosome 7

The cytochrome P450IIB gene subfamily (Cyp2b) has previously been mapped close to the Coh locus encoding a cytochrome P450 with coumarin 7-hydroxylase (COH) activity on mouse chromosome 7. Given this observation, it had been considered that COH was a member of the P450IIB subfamily. However, recent biochemical and cDNA expression experiments indicate that a member of the P450IIA subfamily, rather than of the P450IIB subfamily, encodes COH. We have resolved this apparent anomaly between the genetic and biochemical data by showing that genes from the P450IIA subfamily (Cyp2a) are closely linked to Coh and to Cyp2b on mouse chromosome 7.     

Direct Detection of Fungal Siderophores on Bats with White-Nose Syndrome via Fluorescence Microscopy-Guided Ambient Ionization Mass Spectrometry

White-nose syndrome (WNS) caused by the pathogenic fungus Pseudogymnoascus destructans is decimating the populations of several hibernating North American bat species. Little is known about the molecular interplay between pathogen and host in this disease. Fluorescence microscopy ambient ionization mass spectrometry was used to generate metabolic profiles from the wings of both healthy and diseased bats of the genus Myotis. Fungal siderophores, molecules that scavenge iron from the environment, were detected on the wings of bats with WNS, but not on healthy bats. This work is among the first examples in which microbial molecules are directly detected from an infected host and highlights the ability of atmospheric ionization methodologies to provide direct molecular insight into infection.     

The Presence of HLA-E-Restricted,CMV-Specific CD8+ T Cells in the Blood of Lung Transplant Recipients Correlates with Chronic Allograft Rejection

The human cytomegalovirus (CMV) immune evasion protein, UL40, shares an identical peptide sequence with that found in the leader sequence of many human leukocyte antigen (HLA)-C alleles and when complexed with HLA-E, can modulate NK cell functions via interactions with the CD94-NKG2 receptors. However the UL40-derived sequence can also be immunogenic, eliciting robust CD8+ T cell responses. In the setting of solid organ transplantation these T cells may not only be involved in antiviral immunity but also can potentially contribute to allograft rejection when the UL40 epitope is also present in allograft-encoded HLA. Here we assessed 15 bilateral lung transplant recipients for the presence of HLA-E-restricted UL40 specific T cells by tetramer staining of peripheral blood mononuclear cells (PBMC). UL40-specific T cells were observed in 7 patients post-transplant however the magnitude of the response varied significantly between patients. Moreover, unlike healthy CMV seropositive individuals, longitudinal analyses revealed that proportions of such T cells fluctuated markedly. Nine patients experienced low-grade acute cellular rejection, of which 6 also demonstrated UL40-specific T cells. Furthermore, the presence of UL40-specific CD8⁺ T cells in the blood was significantly associated with allograft dysfunction, which manifested as Bronchiolitis Obliterans Syndrome (BOS). Therefore, this study suggests that minor histocompatibility antigens presented by HLA-E can represent an additional risk factor following lung transplantation.     

Binding of the molecular chaperone αB-crystallin to Aβ amyloid fibrils inhibits fibril elongation

The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ₄₂ fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ₄₂. Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aβ fibrils, both in solution and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing molecular chaperone protection against amyloid fibril formation.     

An assessment of the effect of harvest time and harvest method on biomass loss for Miscanthus × giganteus

This study examines the amount of biomass loss occurring in Miscanthus × giganteus crop at harvest. The study assesses loss incurred as a direct result of the harvest systems employed to collect the material along with examining how the time of harvest effects the amount of loss occurring over the spring harvest window. Pre harvest losses of 4.8–5.1% were measured prior to harvest. There was no significant difference between pre harvest loss and post harvest loss when a self‐propelled forage harvester fitted with a maize harvesting header was used to harvest the crop. The use of a conditioner mower and baler significantly increased crop losses to 9.4–14.1%. This demonstrates that correct selection of the harvest system can significantly increase biomass recovery. Additional losses were measured at headlands when the mower/baler system was used, but headland losses will not occur when self‐propelled forage harvesters are utilized. Losses were significantly greater in the area beside the swath after the baler pass when compared to prior to baling. This study has shown that correct selection of harvest systems can significantly increase biomass recovery, with no significant difference in pre harvest loss or harvest loss occurring as a result of cutting the M. × giganteus crop at different dates during the harvest window (March 1st, March 25th, April 21st).     

Impact of Commonly Used Transplant Immunosuppressive Drugs on Human NK Cell Function Is Dependent upon Stimulation Condition

Lung transplantation is a recognised treatment for patients with end stage pulmonary disease. Transplant recipients receive life-long administration of immunosuppressive drugs that target T cell mediated graft rejection. However little is known of the impact on NK cells, which have the potential to be alloreactive in response to HLA-mismatched ligands on the lung allograft and in doing so, may impact negatively on allograft survival. NK cells from 20 healthy controls were assessed in response to Cyclosporine A, Mycophenolic acid (MPA; active form of Mycophenolate mofetil) and Prednisolone at a range of concentrations. The impact of these clinically used immunosuppressive drugs on cytotoxicity (measured by CD107a expression), IFN-γ production and CFSE proliferation was assessed in response to various stimuli including MHC class-I negative cell lines, IL-2/IL-12 cytokines and PMA/Ionomycin. Treatment with MPA and Prednisolone revealed significantly reduced CD107a expression in response to cell line stimulation. In comparison, addition of MPA and Cyclosporine A displayed reduced CD107a expression and IFN-γ production following PMA/Ionomycin stimulation. Diminished proliferation was observed in response to treatment with each drug. Additional functional inhibitors ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, PD98059, Rottlerin, Rapamycin) were used to elucidate intracellular pathways of NK cell activation in response to stimulation with K562 or PMA-I. CD107a expression was significantly decreased with the addition of PD98059 following K562 stimulation. Similarly, CD107a expression significantly decreased following PMA-I stimulation with the addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, PD98059 and Rottlerin. Ten lung transplant patients, not receiving immunosuppressive drugs pre-transplant, were assessed for longitudinal changes post-transplant in relation to the administration of immunosuppressive drugs. Individual patient dynamics revealed different longitudinal patterns of NK cell function post-transplantation. These results provide mechanistic insights into pathways of NK cell activation and show commonly administered transplant immunosuppression agents and clinical rejection/infection events have differential effects on NK cell function that may impact the immune response following lung transplantation.     

Iron promotes the toxicity of amyloid beta peptide by impeding its ordered aggregation

We have previously shown that overexpressing subunits of the iron-binding protein ferritin can rescue the toxicity of the amyloid β (Aβ) peptide in our Drosophila model system. These data point to an important pathogenic role for iron in Alzheimer disease. In this study, we have used an iron-selective chelating compound and RNAi-mediated knockdown of endogenous ferritin to further manipulate iron in the brain. We confirm that chelation of iron protects the fly from the harmful effects of Aβ. To understand the pathogenic mechanisms, we have used biophysical techniques to see how iron affects Aβ aggregation. We find that iron slows the progression of the Aβ peptide from an unstructured conformation to the ordered cross-β fibrils that are characteristic of amyloid. Finally, using mammalian cell culture systems, we have shown that iron specifically enhances Aβ toxicity but only if the metal is present throughout the aggregation process. These data support the hypothesis that iron delays the formation of well ordered aggregates of Aβ and so promotes its toxicity in Alzheimer disease.     

In situ B cell-mediated immune responses and tubulointerstitial inflammation in human lupus nephritis

The most prevalent severe manifestation of systemic lupus erythematosus is nephritis, which is characterized by immune complex deposition, inflammation, and scarring in glomeruli and the tubulointerstitium. Numerous studies indicated that glomerulonephritis results from a systemic break in B cell tolerance, resulting in the local deposition of immune complexes containing Abs reactive with ubiquitous self-Ags. However, the pathogenesis of systemic lupus erythematosus tubulointerstitial disease is not known. In this article, we demonstrate that in more than half of a cohort of 68 lupus nephritis biopsies, the tubulointerstitial infiltrate was organized into well-circumscribed T:B cell aggregates or germinal centers (GCs) containing follicular dendritic cells. Sampling of the in situ-expressed Ig repertoire revealed that both histological patterns were associated with intrarenal B cell clonal expansion and ongoing somatic hypermutation. However, in the GC histology, the proliferating cells were CD138(-)CD20(+) centroblasts, whereas they were CD138(+)CD20(low/-) plasmablasts in T:B aggregates. The presence of GCs or T:B aggregates was strongly associated with tubular basement membrane immune complexes. These data implicate tertiary lymphoid neogenesis in the pathogenesis of lupus tubulointerstitial inflammation.     

LEDGF dominant interference proteins demonstrate prenuclear exposure of HIV-1 integrase and synergize with LEDGF depletion to destroy viral infectivity

Target cell overexpression of the integrase binding domain (IBD) of LEDGF/p75 (LEDGF) inhibits HIV-1 replication. The mechanism and protein structure requirements for this dominant interference are unclear. More generally, how and when HIV-1 uncoating occurs postentry is poorly defined, and it is unknown whether integrase within the evolving viral core becomes accessible to cellular proteins prior to nuclear entry. We used LEDGF dominant interference to address the latter question while characterizing determinants of IBD antiviral activity. Fusions of green fluorescent protein (GFP) with multiple C-terminal segments of LEDGF inhibited HIV-1 replication substantially, but minimal chimeras of either polarity (GFP-IBD or IBD-GFP) were most effective. Combining GFP-IBD expression with LEDGF depletion was profoundly antiviral. CD4(+) T cell lines were rendered virtually uninfectable, with single-cycle HIV-1 infectivity reduced 4 logs and high-input (multiplicity of infection = 5.0) replication completely blocked. We restricted GFP-IBD to specific intracellular locations and found that antiviral activity was preserved when the protein was confined to the cytoplasm or directed to the nuclear envelope. The life cycle block triggered by the cytoplasm-restricted protein manifested after nuclear entry, at the level of integration. We conclude that integrase within the viral core becomes accessible to host cell protein interaction in the cytoplasm. LEDGF dominant interference and depletion impair HIV-1 integration at distinct postentry stages. GFP-IBD may trigger premature or improper integrase oligomerization.