A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas.

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.

Educational professionals’ understanding of childhood traumatic brain injury

Primary objectives: To determine the understanding of educational professionals around the topic of childhood brain injury and explore the factor structure of the Common Misconceptions about Traumatic Brain Injury Questionnaire (CM-TBI).

Research design: Cross sectional postal survey.

Methods and procedures: The CM-TBI was posted to all educational establishments in one region of the United Kingdom. One representative from each school was asked to complete and return the questionnaire (N = 388).

Main outcomes and results: Differences were demonstrated between those participants who knew someone with a brain injury and those who did not, with a similar pattern being shown for those educators who had taught a child with brain injury. Participants who had taught a child with brain injury demonstrated greater knowledge in areas such as seatbelts/prevention, brain damage, brain injury sequelae, amnesia, recovery, and rehabilitation. Principal components analysis suggested the existence of four factors and the discarding of half the original items of the questionnaire.

Conclusions: In the first European study to explore this issue, we highlight that teachers are ill prepared to cope with children who have sustained a brain injury. Given the importance of a supportive school environment in return to life following hospitalisation, the lack of understanding demonstrated by teachers in this research may significantly impact on a successful return to school.

Novel Protective Properties of IGFBP-3 Result in Enhanced Pericyte Ensheathment, Reduced Microglial Activation, Increased Microglial Apoptosis, and Neuronal Protection after Ischemic Retinal Injury

This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3-mediated response was examined using green fluorescent protein-positive (GFP(+)) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3-expressing plasmid resulted in increased differentiation of GF(P)+ hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP(+) HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity. (Am J Pathol 2011, 178:1517-1524; DOI: 10.1016/j.ajpath.2010.12.031)

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury:efficacy and mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P <0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P <0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.

Proteolytic cleavage of elafin by 20S proteasome may contribute to inflammation in acute lung injury

RATIONALE:
We hypothesise that elafin levels in acute lung injury (ALI) decrease over time due, in part, to proteolytic degradation as observed in other lung diseases.
OBJECTIVES:
The aim of this study was to characterise temporal changes in elafin concentration in patients with ALI and to evaluate whether a decrease in elafin levels is due to elevated protease activity.
METHODS:
Bronchoalveolar lavage fluid (BALF) was obtained from patients with ALI within 48 h of onset of ALI (day 0), at day 3 and at day 7. Elafin levels were quantified by ELISA. Elafin susceptibility to proteolytic cleavage by ALI BALF was assessed by Western blot and by high-performance liquid chromatography-mass spectrometry.
MEASUREMENTS AND MAIN RESULTS:
Elafin levels were found to be significantly increased at the onset of ALI compared with healthy volunteers and fell significantly by day 7 compared with day 0. In contrast, levels of secretory leukocyte protease inhibitor did not decrease over time. This decrease in elafin was due to cleavage by the 20S proteasome which was significantly increased in ALI BALF. Incubation of ALI BALF with the proteasome inhibitor epoxomicin confirmed that 20S proteasome protease activity was responsible for proteolytic cleavage of elafin, resulting in diminished anti-elastase activity. In addition, free neutrophil elastase activity significantly increased in ALI BALF from day 0 to day 7.
CONCLUSIONS:
Elafin concentrations fall within the pulmonary compartment over the course of ALI as a result of proteolytic degradation. This loss of elafin may predispose people, in part, to excessive inflammation in ALI.

Phloem transport of arsenic species from flag leaf to grain during grain filling

Strategies to reduce arsenic (As) in rice grain, below concentrations that represent a serious human health concern, require that the mechanisms of As accumulation within grain be established. Therefore, retranslocation of As species from flag leaves into filling rice grain was investigated.

Arsenic species were delivered through cut flag leaves during grain fill. Spatial unloading within grains was investigated using synchrotron X-ray fluorescence (SXRF) microtomography. Additionally, the effect of germanic acid (a silicic acid analog) on grain As accumulation in arsenite-treated panicles was examined.

Dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) were extremely efficiently retranslocated from flag leaves to rice grain; arsenate was poorly retranslocated, and was rapidly reduced to arsenite within flag leaves; arsenite displayed no retranslocation. Within grains, DMA rapidly dispersed while MMA and inorganic As remained close to the entry point. Germanic acid addition did not affect grain As in arsenite-treated panicles. Three-dimensional SXRF microtomography gave further information on arsenite localization in the ovular vascular trace (OVT) of rice grains.

These results demonstrate that inorganic As is poorly remobilized, while organic species are readily remobilized, from leaves to grain. Stem translocation of inorganic As may not rely solely on silicic acid transporters.

Risk of fatal injury in older adult drivers, passengers, and pedestrians

Objectives To compare risk of fatal injury in elderly road users (drivers, passengers, pedestrians) with that of younger age groups and to assess the contribution of elderly road users to the number of reported fatalities in the population. Design Fatality age was categorized as 21 to 29, 30 to 39, 40 to 49, 50 to 59, 60 to 69, or 70 and older, and road user was categorized as driver, passenger, or pedestrian. Estimated number of trips made by each age group was used to adjust for exposure and to measure individual risk. Setting Fatalities recorded in Britain between 1989 and 2009. Participants Population-wide fatal injury counts in Britain. Measurements Age of fatally injured drivers, passengers, and pedestrians. Estimated number of trips made per year by drivers, passengers, and pedestrians. Results Risk of fatal injury, but not fatality numbers in the population, were higher for older adult (=70) drivers than for younger age groups. Risk of fatal injury was also high for older adult passengers and pedestrians, who represented the majority of older adult fatalities. Conclusion Previous emphasis on driver impairment in older age has unduly focussed attention on elderly drivers, who represent a minority of all driver fatalities. Older adults represent a much larger proportion of passenger and pedestrian fatalities. Additional policy schemes and initiatives should be targeted at safeguarding older adult passengers and making the road environment safer for elderly pedestrians. © 2012, Copyright the Authors Journal compilation © 2012, The American Geriatrics Society.

PsT-1: A new Tryptophyllin peptide from the skin secretion of Waxy Monkey Leaf Frog, Phyllomedusa sauvagei

The Waxy Monkey Leaf Frog, Phyllomedusa sauvagei, has been extensively-studied for many years, and a broad spectrum of bioactive peptides has been found in its skin secretions. Here we report the discovery of a novel tryptophyllin (TPH) peptide, named PsT-1, from this frog species. Skin secretions from specimens of P. sauvagei were collected by mild electrical stimulation. Peptides were identified and characterized by transcriptome cloning, and the structure was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This novel peptide was encoded by a single precursor of 61 amino acid residues, whose primary structure was deduced from cloned skin cDNA. Analysis of different amphibian tryptophyllins revealed that PsT-1 exhibited a high degree of primary structural similarity to its homologues, PdT-1 and PdT-2, from the Mexican giant leaf frog, Pachymedusa dacnicolor. A synthetic replicate of PsT-1 was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle. It was also found that PsT-1 had an anti-proliferative effect on three different human prostate cancer cell lines (LNCaP/PC3/DU145), by use of an MTT assay coupled with direct cell counting as measures of cell growth. These data indicate that PsT-1 is a likely bradykinin receptor antagonist and its biological effects are probably mediated through bradykinin receptors. As a BK antagonist, PST-1, with antagonistic effects on BK in artery smooth muscle, inhibition of proliferation in prostate cancer cells and lack of undesirable side effects, may have potential in cardiovascular, inflammatory and anticancer therapy.

Does parenchymal brain injury affect biobehavioral pain responses in very low birth weight infants at 32 weeks' postconceptional age?

Children with neurologic impairments have shown diminished pain response compared with control subjects; however, it remains unclear what mechanisms underlie this response or when it develops. If this were also true with premature infants who undergo neonatal intensive care, then infants with parenchymal brain injury (PBI) would be at increased risk of underrecognition and undertreatment of procedural pain. The purpose of this study was to determine whether infants with PBI display altered responses to acute procedural pain at 32 weeks' postconceptional age (PCA), compared with control subjects.