Antimicrobial Protegrin-1 Forms Ion Channels: Molecular Dynamic Simulation, Atomic Force Microscopy, and Electrical Conductance Studies

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among β-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a β-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the β-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.     

Evaluation of TFAM and FABP4 gene polymorphisms in three lines of Nellore cattle selected for growth

We analyzed the polymorphisms TFAM HaeIII, TFAM MboI and FABP4 MspA1I in three Nellore lines selected for growth in order to evaluate how selection affects the frequencies of these polymorphisms and evaluate their association with growth and carcass traits in Zebu cattle. Birth, weaning and yearling weights, rump height, longissimus muscle area, backfat thickness, and rump fat thickness were analyzed. The sample was constituted of animals from two lines selected for yearling weight (NeS and NeT), and a control line (NeC), established in 1980, at the S?o Paulo Instituto de Zootecnia. Two hundred and seventy-two heifers were genotyped for TFAM gene SNPs, and 325 heifers were genotyped for the FABP4 SNP. High frequencies were observed for the alleles A (TFAM HaeIII), C (TFAM MboI) and C (FABP4 MspA1I). Significant differences in allele frequencies between NeS and NeT were observed for the TFAM HaeIII, and between the line NeT and lines NeC and NeS for the FABP4 MspA1I SNP. Five haplotypes were observed for the two polymorphisms in the TFAM gene, haplotype AACC being the most frequent. None of the markers separately or according to haplotype was significantly associated with the growth and carcass traits. The low frequencies of alleles that are associated with high marbling scores and thick subcutaneous fat in taurine breeds might explain the low means for these traits in Nellore cattle.     

Identification of the pattern of heterochromatin distribution in Passiflora species with C-banding

We tested four C-banding protocols to obtain heterochromatic bands in the passion fruit species Passiflora edulis and P. cacaoensis (Passifloraceae). Three of these protocols had been previously described. The three published protocols were not adequate to obtain C-bands in these species. An adapted protocol demonstrated heterochromatin distribution in metaphasic chromosomes of species of Passiflora for the first time. The differentiated coloration for C-bands was obtained with immersion of the slides in 99% ethanol, 45% acetic acid (additional step), 0.2 N hydrochloric acid, hydroxide of barium, 45% acetic acid, and 2X standard saline citrate at four different temperatures. The C-bands were observed in the satellites and in the telomere and centromere regions of all chromosomes, both in P. edulis and in P. cacaoensis.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Bioimaging of copper alterations in the aging mouse brain by autoradiography, laser ablation inductively coupled plasma mass spectrometry and immunohistochemistry

Copper may play an important role in the brain in aging and neurodegenerative diseases. We compare the active Cu uptake, Cu-containing enzyme levels, and total Cu distribution in the brains of young and aging mice. (67)Cu was administered intravenously to 2, 7-9, and 14 month old mice. Active uptake of (67)Cu in the brain was measured at 24 h by digital phosphor autoradiography. Cerebral superoxide dismutase-1 (SOD-1) and cytochrome-C oxidase subunit-1 (CCO-1) levels were analyzed by immunohistochemistry. The total Cu distribution in brain section was determined by imaging laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). In aging mice, active (67)Cu uptake and SOD-1 levels were significantly decreased in the brain, whereas blood (67)Cu and CCO-1 levels were similar for all mice, irrespective of age. Paradoxically, global Cu cerebral content was increased in aged mice, suggesting that regulation of active Cu uptake by the brain may be linked to total Cu levels in an attempt to maintain Cu homeostasis. However, focal areas of both decreased Cu uptake and Cu content were noted in the striatum and ventral cortex in aging mice. These focal areas of Cu deficit correspond to the regions of greatest reduction in SOD-1 in the aged mice. In aging, dysregulated Cu homeostasis may result in decreased SOD-1 levels, which may contribute to oxidative vulnerability of the aging brain. This study illustrates the importance of a multi-modality approach in studying the biodistribution and homeostasis of Cu in the brain.     

An abelisaurid humerus from the Upper Cretaceous of India

The Lameta Formation (Upper Cretaceous, Maastrichtian) of India has yielded abundant fossils of abelisaurid theropods, including bones from the cranium, vertebral column, pectoral and pelvic girdles, and hindlimb. However, the forelimbs of Indian abelisaurids remain unknown. Here we describe an abelisaurid humerus from exposure of the Lameta Formation near the village of Rahioli in northwestern India. This new material exhibits derived traits that are distinctive of Abelisauridae, for example an articular head that is hemispherical in proximal view, thus establishing the specimen as the first abelisaurid humerus from India.     

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H₂O₂ system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H₂O₂ was 86-times lower than that of HRP (K_m = 23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H₂O₂ as electron donor was 2838-times lower than that of HRP (k_cat/K_m = 15.8 and 44 833 mM⁻¹ min⁻¹, respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H₂O₂ concentration, and that it poorly protects recombinant Escherichia coli from H₂O₂ stress. These observations indicate that rice Hb1 inefficiently scavenges H₂O₂ as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.     

Climate's role in the distribution of the Cretaceous terrestrial Crocodyliformes throughout Gondwana

A high diversity of terrestrial crocodyliform species has been found in the continental Cretaceous deposits of Gondwana. They are widespread in the sedimentary basins of Brazil, Uruguay, Argentina, Bolivia, Morocco, Cameroon, Niger, Malawi, Madagascar and Pakistan, from alluvial, fluvial and lacustrine deposits. A peculiar aspect of these terrestrial crocodyliforms is that only some of them are cosmopolitan. They comprise distinct groups as the basal Notosuchia, baurusuchids, sphagesaurids and Sebecian peirosaurids. There is a distribution pattern of the terrestrial Crocodyliformes faunas throughout the Cretaceous. The oldest are composed of the small and probably omnivorous Notosuchia and Araripesuchus, found in Early Cretaceous deposits. In the Late Cretaceous this fauna was enriched by the medium-sized to large-size baurusuchids, sphagesaurids, peirosaurids and larger Araripesuchus which present specializations as active terrestrial predators. The distribution analysis of the terrestrial Crocodyliformes from Early and Late Cretaceous palaeogeographic and palaeoclimatic maps indicates that temperature was the principal influence on their Gondwanan distribution. Although expressed seasonality, aridity is a limiting factor for the distribution of extant crocodilians. The Cretaceous basal Notosuchia, baurusuchids, sphagesaurids, Araripesuchus, Sebecian peirosaurids, are found in arid climatic belts during Early and Late Cretaceous. To live in a hot and arid climate they have presumably developed ecological strategies that allowed such habits. The aridity or seasonal warm and cyclic dry and wet climate periods plays a role, that have not yet been analyzed, that may explain the domain of bizarre Crocodyliformes in Gondwana during the Cretaceous.