Mutagenesis of surfactant protein D informed by evolution and x-ray crystallography enhances defenses against influenza A virus in vivo

The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.     

A peptide corresponding to the C-terminal region of pleiotrophin inhibits angiogenesis in vivo and in vitro

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both α(ν) β(3) integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN(112-136) ) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN(112-136) inhibited binding of PTN to α(ν) β(3) integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β(3) . Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing α(ν) β(3) -selective antibody. PTN(112-136) did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β(3) Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN(112-136) may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN(112-136) was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123-132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN(112-136) induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to α(ν) β(3) integrin.     

Urinary Calprotectin and Posttransplant Renal Allograft Injury

Objective

Current methods do not predict the acute renal allograft injury immediately after kidney transplantation. We evaluated the diagnostic performance of urinary calprotectin for predicting immediate posttransplant allograft injury.

Methods

In a multicenter, prospective-cohort study of 144 incipient renal transplant recipients, we postoperatively measured urinary calprotectin using an enzyme-linked immunosorbent assay and estimated glomerular filtration rate (eGFR) after 4 weeks, 6 months, and 12 months.

Results

We observed a significant inverse association of urinary calprotectin concentrations and eGFR 4 weeks after transplantation (Spearman r = −0.33; P<0.001). Compared to the lowest quartile, patients in the highest quartile of urinary calprotectin had an increased risk for an eGFR less than 30 mL/min/1.73 m² four weeks after transplantation (relative risk, 4.3; P<0.001; sensitivity, 0.92; 95% CI, 0.77 to 0.98; specificity, 0.48; 95% CI, 0.31 to 0.66). Higher urinary calprotectin concentrations predicted impaired kidney function 4 weeks after transplantation, as well as 6 months and 12 months after transplantation. When data were analyzed using the urinary calprotectin/creatinine-ratio similar results were obtained. Urinary calprotectin was superior to current use of absolute change of plasma creatinine to predict allograft function 12 months after transplantation. Urinary calprotectin predicted an increased risk both in transplants from living and deceased donors. Multivariate linear regression showed that higher urinary calprotectin concentrations and older donor age predicted lower eGFR four weeks, 6 months, and 12 months after transplantation.

Conclusions

Urinary calprotectin is an early, noninvasive predictor of immediate renal allograft injury after kidney transplantation.     

Interaction of nitric oxide with the oxygen evolving complex of photosystem II and manganese catalase: a comparative study

Thermus thermophilus catalase. Flash fluorescence studies indicate that the S₃ state of the OEC in the presence of ca. 0.6 mM NO is reduced to the S₁ with an apparent halftime of ca. 0.4 s at about 18 °C, compared with a biphasic decay, with approximate halftimes of 28 s for S₃ to S₂ and 140 s for S₂ to S₁ in the absence of NO. Under similar conditions the S₂ state is reduced by NO to the S₁ state with an approximate halftime of 2 s. These results extend a recent study indicating a slow reduction of the S₁ state at −30°C, via the S₀ and S₋₁ states, to a Mn(II)-Mn(III) state resembling the corresponding state in catalase. The reductive mode of action of NO is repeated with the di-Mn cluster of catalase: the Mn(III)-Mn(III) redox state is reduced to the Mn(II)-Mn(II) state via the intermediate Mn(II)-Mn(III) state. The kinetics of this reduction suggest a decreasing reduction potential with decreasing oxidation state, similar to what is observed with the active states of the OEC. What is unique about the OEC is the rapid interaction of NO with the S₃ state of the OEC, which is compatible with a metalloradical character of this state. Received: 16 June 1999 / Accepted: 28 February 2000     

Rehabilitation management in two siblings with Von Hippel-Lindau syndrome: A case series

Von Hippel Lindau (VHL) is a hereditary multiple neoplasia syndrome. We report a case series of two siblings with Von Hippel Lindau (VHL) disease admitted to the rehabilitation department after surgical excision of Central Nervous System (CNS) haemangioblastomas. These clinical cases present rehabilitation challenges in VHL disease. We present a 39-year-old brother and his 45-year-old sister, with the diagnosis of incomplete spinal cord injury (SCI) associated with VHL syndrome lesions. The female patient was diagnosed with chronic motor incomplete cervical SCI and the male patient with acute motor incomplete thoracic SCI. Our target was to increase their functionality and improve their quality of life. Both underwent a comprehensive inpatient rehabilitation program. Programs were individualized as the female patient was admitted 15 years after her spinal cord surgical intervention, while the male patient’s admission was after 4 months of his surgery.     

Intrafamilial spread of Helicobacter pylori: a genetic analysis

Background. A high incidence of Helicobacter pylori among family members of children with H. pylori gastritis has previously been documented on biopsy material. The main objective of this study was the genetic clarification of H. pylori strains involved in intrafamilial dispersion. Materials and Methods. Formalin‐fixed, paraffin‐embedded material of antral mucosa from 32 members of 11 families was studied for the presence of genetic homogeneity. To achieve this goal, the entire genome of H. pylori was studied by the polymerase chain reaction (PCR)‐based random amplified polymorphic DNA (RAPD) fingerprinting method. Furthermore, the Urease A gene was analyzed using a multiplex PCR‐assay and an alternative mutation detection method based on the Hydrolink? analysis. Results. RAPD fingerprinting confirmed that closely related H. pylori strains were involved in the intrafamilial dispersion. Mutations and small deletions in Urease A gene were found in 22 out of 32 individuals. Conclusions. The homology of the H. pylori genome in members of the same family strongly supports the hypothesis of transmission of H. pylori from person‐to‐person or from a common source.     

The influence of the location of a nest box in an individually ventilated cage on the preference of mice to use it

The improvement of housing conditions for mice by using environmental enrichment materials is of high concern for the scientific community. Plastic, autoclavable nest boxes are commercially available and ready to use for specific cases such as in individually ventilated cages, metabolic cages, or during toxicological studies. The aim of this study was to see if the location of the nest box within the cage could influence the mice to prefer and use it. Located on the cage floor or hung from the cage lid, a nest box (MPLEX, Otto Environmental, Milwaukee, Wisconsin), enriched the cages. The study concluded that the location of the nest boxes in the individually ventilated cage plays a significant role in the mice preferring to use it or to avoid it. It is also important to use environmental enrichment items that provide animals with the possibility of expressing their preferences and manipulating them in a way to cope better with their environmental conditions.     

Putrescine stimulates chemiosmotic ATP synthesis

Putrescine is a main polyamine found in animals, plants and microbes, but the molecular mechanism underlying its mode of action is still obscure. In vivo chlorophyll a fluorescence in tobacco leaf discs indicated that putrescine treatment affects the energization of the thylakoid membrane. Molecular dissection of the electron transport chain by biophysical and biochemical means provided new evidence that putrescine can play an important bioenergetic role acting as a cation and as a permeant natural buffer. We demonstrate that putrescine increases chemiosmotic ATP synthesis more than 70%. Also a regulation of the energy outcome by small changes in putrescine pool under the same photonic environment (i.e., photosynthetically active radiation) is shown. The proposed molecular mechanism has at least four conserved features: (i) presence of a membrane barrier, (ii) a proton-driven ATPase, (iii) a DeltapH and (iv) a pool of putrescine.