Identification of a PAI-1-binding site within an intrinsically disordered region of vitronectin

© 2019 The Protein Society The serine protease inhibitor, plasminogen activator inhibitor Type-1 (PAI-1) is a metastable protein that undergoes an unusual transition to an inactive conformation with a short half-life of only 1–2 hr. Circulating PAI-1 is bound to a cofactor vitronectin, which stabilizes PAI-1 by slowing this latency conversion. A well-characterized PAI-1-binding site on vitronectin is located within the somatomedin B (SMB) domain, corresponding to the first 44 residues of the protein. Another PAI-1 recognition site has been identified with an engineered form of vitronectin lacking the SMB domain, yet retaining PAI-1 binding capacity (Schar, Blouse, Minor, Peterson. J Biol Chem. 2008;283:28487–28496). This additional binding site is hypothesized to lie within an intrinsically disordered domain (IDD) of vitronectin. To localize the putative binding site, we constructed a truncated form of vitronectin containing 71 amino acids from the N-terminus, including the SMB domain and an additional 24 amino acids from the IDD region. This portion of the IDD is rich in acidic amino acids, which are hypothesized to be complementary to several basic residues identified within an extensive vitronectin-binding site mapped on PAI-1 (Schar, Jensen, Christensen, Blouse, Andreasen, Peterson. J Biol Chem. 2008;283:10297–10309). Steady-state and stopped-flow fluorescence measurements demonstrate that the truncated form of vitronectin exhibits the same rapid biphasic association as full-length vitronectin and that the IDD hosts the elusive second PAI-1 binding site that lies external to the SMB domain of vitronectin

Radiocarbon dating of methane and carbon dioxide evaded from a temperate peatland stream

Streams draining peatlands export large quantities of carbon in different chemical forms and are an important part of the carbon cycle. Radiocarbon (14C) analysis/dating provides unique information on the source and rate that carbon is cycled through ecosystems, as has recently been demonstrated at the air-water interface through analysis of carbon dioxide (CO2) lost from peatland streams by evasion (degassing). Peatland streams also have the potential to release large amounts of methane (CH4) and, though 14C analysis of CH4 emitted by ebullition (bubbling) has been previously reported, diffusive emissions have not. We describe methods that enable the 14C analysis of CH4 evaded from peatland streams. Using these methods, we investigated the 14C age and stable carbon isotope composition of both CH4 and CO2 evaded from a small peatland stream draining a temperate raised mire. Methane was aged between 1617-1987 years BP, and was much older than CO2 which had an age range of 303-521 years BP. Isotope mass balance modelling of the results indicated that the CO2 and CH4 evaded from the stream were derived from different source areas, with most evaded CO2 originating from younger layers located nearer the peat surface compared to CH4. The study demonstrates the insight that can be gained into peatland carbon cycling from a methodological development which enables dual isotope (14C and 13C) analysis of both CH4 and CO2 collected at the same time and in the same way

Is there a cloud in the silver lining for imatinib?

Imatinib mesylate (Gleevec® or Glivec®), a small molecule tyrosine kinase inhibitor for the treatment of chronic myeloid leukaemia, has been said to herald the dawn of a new er-a of rationally designed, molecularly targeted oncotherapy. Lurking on the same new horizon, however, is the age-old spectre of drug resistance. This review sets the intoxicating clinical perspective against the more sobering laboratory evidence of such divergent mechanisms of imatinib resistance as gene amplification and stem cell quiescence. Polychemotherapy has already been considered to combat resistance, but a more innovative, as yet unformulated, approach may be advocated

The C:N:P:S stoichiometry of soil organic matter

The formation and turnover of soil organic matter (SOM) includes the biogeochemical processing of the macronutrient elements nitrogen (N), phosphorus (P) and sulphur (S), which alters their stoichiometric relationships to carbon (C) and to each other. We sought patterns among soil organic C, N, P and S in data for c. 2000 globally distributed soil samples, covering all soil horizons. For non-peat soils, strong negative correlations (p < 0.001) were found between N:C, P:C and S:C ratios and % organic carbon (OC), showing that SOM of soils with low OC concentrations (high in mineral matter) is rich in N, P and S. The results can be described approximately with a simple mixing model in which nutrient-poor SOM (NPSOM) has N:C, P:C and S:C ratios of 0.039, 0.0011 and 0.0054, while nutrient-rich SOM (NRSOM) has corresponding ratios of 0.12, 0.016 and 0.016, so that P is especially enriched in NRSOM compared to NPSOM. The trends hold across a range of ecosystems, for topsoils, including O horizons, and subsoils, and across different soil classes. The major exception is that tropical soils tend to have low P:C ratios especially at low N:C. We suggest that NRSOM comprises compounds selected by their strong adsorption to mineral matter. The stoichiometric patterns established here offer a new quantitative framework for SOM classification and characterisation, and provide important constraints to dynamic soil and ecosystem models of carbon turnover and nutrient dynamics

In vivo isolated kidney perfusion with tumour necrosis factor α (TNF-α) in tumour-bearing rats

Isolated perfusion of the extremities with high-dose tumour necrosis factor α (TNF-α) plus melphalan leads to dramatic tumour response in patients with irresectable soft tissue sarcoma or multiple melanoma in transit metastases. We developed in vivo isolated organ perfusion models to determine whether similar tumour responses in solid organ tumours can be obtained with this regimen. Here, we describe the technique of isolated kidney perfusion. We studied the feasibility of a perfusion with TNF-α and assessed its anti-tumour effects in tumour models differing in tumour vasculature. The maximal tolerated dose (MTD) proved to be only 1 μg TNF-α. Higher doses appeared to induce renal failure and a secondary cytokine release with fatal respiratory and septic shock-like symptoms. In vitro, the combination of TNF-α and melphalan did not result in a synergistic growth-inhibiting effect on CC 531 colon adenocarcinoma cells, whereas an additive effect was observed on osteosarcoma ROS-1 cells. In vivo isolated kidney perfusion, with TNF-α alone or in combination with melphalan, did not result in a significant anti-tumour response in either tumour model in a subrenal capsule assay. We conclude that, because of the susceptibility of the kidney to perfusion with TNF-α, the minimal threshold concentration of TNF-α to exert its anti-tumour effects was not reached. The applicability of TNF-α in isolated kidney perfusion for human tumours seems, therefore, questionable. © 1999 Cancer Research Campaig

Local IL-17 Production Exerts a Protective Role in Murine Experimental Glomerulonephritis

IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of glomerulonephritis and IL-17 deficient mice are protected from nephrotoxic nephritis. However, a regulatory role for IL-17 has recently emerged. We describe a novel protective function for IL-17 in the kidney. Bone marrow chimeras were created using wild-type and IL-17 deficient mice and nephrotoxic nephritis was induced. IL-17 deficient hosts transplanted with wild-type bone marrow had worse disease by all indices compared to wild-type to wild-type bone marrow transplants (serum urea p<0.05; glomerular thrombosis p<0.05; tubular damage p<0.01), suggesting that in wild-type mice, IL-17 production by renal cells resistant to radiation is protective. IL-17 deficient mice transplanted with wild-type bone marrow also had a comparatively altered renal phenotype, with significant differences in renal cytokines (IL-10 p<0.01; IL-1β p<0.001; IL-23 p<0.01), and macrophage phenotype (expression of mannose receptor p<0.05; inducible nitric oxide synthase p<0.001). Finally we show that renal mast cells are resistant to radiation and produce IL-17, suggesting they are potential local mediators of disease protection. This is a novel role for intrinsic cells in the kidney that are radio-resistant and produce IL-17 to mediate protection in nephrotoxic nephritis. This has clinical significance as IL-17 blockade is being trialled as a therapeutic strategy in some autoimmune diseases

Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OFMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding

Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection

BACKGROUND Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients. Recurrences are common after antibiotic therapy. Actoxumab and bezlotoxumab are human monoclonal antibodies against C. difficile toxins A and B, respectively. METHODS We conducted two double-blind, randomized, placebo-controlled, phase 3 trials, MODIFY I and MODIFY II, involving 2655 adults receiving oral standard-of-care antibiotics for primary or recurrent C. difficile infection. Participants received an infusion of bezlotoxumab (10 mg per kilogram of body weight), actoxumab plus bezlotoxumab (10 mg per kilogram each), or placebo; actoxumab alone (10 mg per kilogram) was given in MODIFY I but discontinued after a planned interim analysis. The primary end point was recurrent infection (new episode after initial clinical cure) within 12 weeks after infusion in the modified intention-to-treat population. RESULTS In both trials, the rate of recurrent C. difficile infection was significantly lower with bezlotoxumab alone than with placebo (MODIFY I: 17% [67 of 386] vs. 28% [109 of 395]; adjusted difference, −10.1 percentage points; 95% confidence interval [CI], −15.9 to −4.3; P<0.001; MODIFY II: 16% [62 of 395] vs. 26% [97 of 378]; adjusted difference, −9.9 percentage points; 95% CI, −15.5 to −4.3; P<0.001) and was significantly lower with actoxumab plus bezlotoxumab than with placebo (MODIFY I: 16% [61 of 383] vs. 28% [109 of 395]; adjusted difference, −11.6 percentage points; 95% CI, −17.4 to −5.9; P<0.001; MODIFY II: 15% [58 of 390] vs. 26% [97 of 378]; adjusted difference, −10.7 percentage points; 95% CI, −16.4 to −5.1; P<0.001). In prespecified subgroup analyses (combined data set), rates of recurrent infection were lower in both groups that received bezlotoxumab than in the placebo group in subpopulations at high risk for recurrent infection or for an adverse outcome. The rates of initial clinical cure were 80% with bezlotoxumab alone, 73% with actoxumab plus bezlotoxumab, and 80% with placebo; the rates of sustained cure (initial clinical cure without recurrent infection in 12 weeks) were 64%, 58%, and 54%, respectively. The rates of adverse events were similar among these groups; the most common events were diarrhea and nausea. CONCLUSIONS Among participants receiving antibiotic treatment for primary or recurrent C. difficile infection, bezlotoxumab was associated with a substantially lower rate of recurrent infection than placebo and had a safety profile similar to that of placebo. The addition of actoxumab did not improve efficacy. (Funded by Merck; MODIFY I and MODIFY II ClinicalTrials.gov numbers, NCT01241552 and NCT01513239.