An amphipathic cyclic tetrapeptide scaffold containing halogenated β2,2‐amino acids with activity against multiresistant bacteria

The present study describes the synthesis and biological studies of a small series of head‐to‐tail cyclic tetrapeptides of the general structure c(Lys‐β^2,2‐Xaa‐Lys) containing one lipophilic β^2,2‐amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram‐positive and gram‐negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum β‐lactamase—carbapenemase (ESBL‐CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram‐positive clinical isolates with minimum inhibitory concentrations of 4–8 μg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic β^2,2‐amino acids form a valuable scaffold for designing novel antimicrobial agents.     

Implications for in vitro studies of the autoxidation of ferrous ion and the iron-catalyzed autoxidation of dithiothreitol

The influences of buffers and iron chelators on the rate of autoxidation of Fe²⁺ were examined in the pH range 6.0–7.4. The catalysis by Fe²⁺ and Fe³⁺ of the autoxidation of dithiothreitol was also investigated. In buffers which are non- or poor chelators of iron, 0.25 mM Fe²⁺, and 0.3 mM dithiothreitol when present with iron, oxidize within minutes at pH 7.4 and 30°C. The stability of each increases as the pH is decreased and more than 90% of each remains after 1 h at pH 6.0. In the presence of buffers or oxy-ligands which preferentially and strongly chelate Fe³⁺ over Fe²⁺, Fe²⁺ autoxidizes rapidly in the pH range 6.0–7.4 while dithiothreitol is protected. Ligands which preferentially bind strongly to Fe²⁺ stabilize both Fe²⁺ and dithiothreitol at pH 7.4. Dithiothreitol readily reduces Fe³⁺ in non-chelating buffers or in the presence of strong chelators of Fe²⁺, however, the ferrous ions produced are prone to reoxidation at higher pH values. These results show that Fe²⁺ and dithiothreitol are very susceptible to autoxidation in the neutral pH range, and that the rates are strongly influenced by the presence of chelators of Fe²⁺ and Fe³⁺. The rapid autoxidations of these species need to be taken into account when designing and interpreting experiments involving Fe²⁺ or both dithiothreitol and iron.     

White matter injury following systemic endotoxemia or asphyxia in the fetal sheep

White matter injury is the most frequently observed brain lesion in preterm infants. The etiology remains unclear, however, both cerebral hypoperfusion and intrauterine infections have been suggested as risk factors. We compared the neuropathological outcome, including the effect on oligodendrocytes, astrocytes, and microglia, following either systemic asphyxia or endotoxemia in fetal sheep at midgestation. Fetal sheep were subjected to either 25 minutes of umbilical cord occlusion or systemic endotoxemia by administration of Escherichia coli lipopolysaccharide (LPS O111:B4, 100 ng/kg, IV). Periventricular white matter lesions were observed in 2 of 6 asphyxiated fetuses, whereas the remaining animals showed diffuse injury throughout the subcortical white matter and neuronal necrosis in subcortical regions, including the striatum and hippocampus. LPS-treatment resulted in focal inflammatory infiltrates and cystic lesions in periventricular white matter in 2 of 5 animals, but with no neuron specific injury. Both experimental paradigms resulted in microglia activation in the white matter, damaged astrocytes, and loss of oligodendrocytes. These results show that the white matter at midgestation is sensitive to injury following both systemic asphyxia and endotoxemia. Asphyxia induced lesions in both white and subcortical grey matter in association with microglia activation, and endotoxemia resulted in selective white matter damage and inflammation.     

Phylogeny of Passerida (Aves: Passeriformes) based on nuclear and mitochondrial sequence data

Passerida is a monophyletic group of oscine passerines that includes almost 3500 species (about 36%) of all bird species in the world. The current understanding of higher-level relationships within Passerida is based on DNA-DNA hybridizations [C.G. Sibley, J.E. Ahlquist, Phylogeny and Classification of Birds, 1990, Yale University Press, New Haven, CT]. Our results are based on analyses of 3130 aligned nucleotide sequence data obtained from 48 ingroup and 13 outgroup genera. Three nuclear genes were sequenced: c-myc (498-510 bp), RAG-1 (930 bp), and myoglobin (693-722 bp), as well one mitochondrial gene; cytochrome b (879 bp). The data were analysed by parsimony, maximum-likelihood, and Bayesian inference. The African rockfowl and rockjumper are found to constitute the deepest branch within Passerida, but relationships among the other taxa are poorly resolved--only four major clades receive statistical support. One clade corresponds to Passeroidea of [C.G. Sibley, B.L. Monroe, Distribution and Taxonomy of Birds of the World, 1990, Yale University Press, New Haven, CT] and includes, e.g., flowerpeckers, sunbirds, accentors, weavers, estrilds, wagtails, finches, and sparrows. Starlings, mockingbirds, thrushes, Old World flycatchers, and dippers also group together in a clade corresponding to Muscicapoidea of Sibley and Monroe [op. cit.]. Monophyly of their Sylvioidea could not be corroborated--these taxa falls either into a clade with wrens, gnatcatchers, and nuthatches, or one with, e.g., warblers, bulbuls, babblers, and white-eyes. The tits, penduline tits, and waxwings belong to Passerida but have no close relatives among the taxa studied herein.     

Sillem's Mountain Finch Leucosticte sillemi is a valid species of rosefinch (Carpodacus,Fringillidae)

Sillem's Mountain Finch Leucosticte sillemi was described in 1992 on the basis of an adult and an immature specimen collected in western Tibet in September 1929, but its taxonomic validity and phylogenetic position have been unclear. Based on phylogenetic analysis of mitochondrial DNA from the holotype, we show that L. sillemi is not a colour morph of Brandt's Mountain Finch Leucosticte brandti but represents a valid, previously overlooked species of rosefinch (Carpodacus) that has secondarily acquired a pale plumage convergent on that of Leucosticte. Sillem's Mountain Finch is one of the least known species of bird and represents the only known species of rosefinch in which males have no reddish plumage coloration. This species and its sister taxon, the Tibetan Rosefinch Carpodacus roborowskii, are likely to be the world's highest‐altitude sister‐species pair of birds.     

Immunocytochemical localization of aminopeptidase N on the cell surface of isolated porcine thyroid follicles

Summary The ultrastructural location of aminopeptidase N on the cell surface of isolated porcine thyroid follicle cells was studied with immunocytochemistry using antibodies against intestinal aminopeptidase N and protein A-colloidal gold. Gold particles, indicating immunoreactivity, were selectively attached to the apical cell surface. Occasionally, there was a sparse labelling of the basal cell surface. In follicles kept at 4° C most gold particles at the apical cell surface appeared as clusters, with each gold particle situated at a constant distance of about 20 nm from the membrane surface. The gold particles were concentrated on the membranes of microvilli, in comparison to the smooth (intermicrovillar) portions of the apical plasma membrane. In follicles incubated at 37° C for 5–180 min gold particles were slowly internalized by predominantly smooth-surfaced micropinocytic vesicles and subsequently appeared in colloid droplets and lysosomes. Gold particles were not observed in Golgi cisternae. TSH did not appear to influence the rate of internalization. TSH-induced pseudopods were unlabelled.Our electron-microscopic observations confirm previous immunofluorescence-microscopic evidence that aminopeptidase N is selectively expressed in the apical plasma membrane domain in the thyroid follicle cell. Furthermore, aminopeptidase N appears to be distributed in microdomains within the apical plasma membrane. Earlier indications of molecular differences between the pseudopod membrane and the apical plasma membrane proper are further emphasized.This study was supported by Grant No 12X-537 from the Swedish Medical Research Council     

Comprehensive phylogeny of the laughingthrushes and allies (Aves,Leiothrichidae) and a proposal for a revised taxonomy

DNA phylogenies have gradually shed light on the phylogenetic relationships of the large babbler group. We focus in this study on the family Leiothrichidae (laughingthrushes and “song babblers”), which represents the largest clade of babblers in terms of species diversity. Our phylogeny includes all genera and 82% of the recognized species, using mitochondrial and nuclear loci. The sister group to Leiothrichidae is composed of the Pellorneidae (“jungle babblers”) plus the genus Alcippe. Within Leiothrichidae, four strongly supported primary clades (A–D) are recovered. Clade A includes Grammatoptila, Laniellus and Cutia. Clade B includes a large group of laughingthrushes, all of them classified in Trochalopteron. In Clade C, the two laughingthrushes endemic to southern India, T. fairbanki and T. cachinnans, which have recently been proposed to be placed in the newly erected genus Montecincla, form a sister clade to the group comprising the “song babblers” (Lioptila, Leiothrix, Heterophasia, Minla, Liocichla, Actinodura, Chrysominla, Siva, and Sibia). Clade D includes the African babblers (Turdoides, Phyllanthus, Kupeornis), Asian relatives (Argya, Acanthoptila, Chatarrhaea) and all remaining laughingthrushes (Garrulax). The time estimates suggest that the early diversification of the Leiothrichidae occurred in the mid‐Miocene, a period that corresponds to the diversification of many passerine groups in Asia. A revised taxonomic classification of the family is proposed in the light of these results.     

Glycoprotein Synthesis in Plants: III. Interaction between UDP-N-Acetylglucosamine and GDP-Mannose as Substrates

This report presents evidence that enzymes present in crude extracts prepared from developing cotyledons of Phaseolus vulgaris can catalyze the transfer of radioactivity from UDP-N-[¹⁴C]acetylglucosamine into a chitobiosyl-lipid, lipid-oligosaccharide, and glycoprotein. Kinetic evidence supports the concept that the N-acetylglucosamine-containing lipids are precursors to the glycoprotein. Evidence is also presented which shows an interaction between GDP-mannose and UDP-N-acetylglucosamine when used as substrates for the synthesis of lipid-oligosaccharide and glycoprotein. Kinetic evidence, as well as isolation and characterization of the oligosaccharides released from lipid by mild acid hydrolyses, support the conclusion that mannose and N-acetylglucosamine are contained in the same oligosaccharide and that N-acetylglucosamine is present at the reducing end of the oligosaccharide. Ninety-eight per cent of the radioactivity which is incorporated from UDP-N-[¹⁴C]acetylglucosamine into the insoluble residue is solubilized by protease treatment. The glycopeptide released is quite similar in size and composition to the glycopeptide released by proteolytic digestion of vicilin, the major storage protein of Phaseolus vulgaris.     

Piliation of Lactobacillus rhamnosus GG Promotes Adhesion,Phagocytosis, and Cytokine Modulation in Macrophages

Recently, spaCBA-encoded pili on the cell surface of Lactobacillus rhamnosus GG were identified to be key molecules for binding to human intestinal mucus and Caco-2 intestinal epithelial cells. Here, we investigated the role of the SpaCBA pilus of L. rhamnosus GG in the interaction with macrophages in vitro by comparing the wild type with surface mutants. Our results show that SpaCBA pili play a significant role in the capacity for adhesion to macrophages and also promote bacterial uptake by these phagocytic cells. Interestingly, our data suggest that SpaCBA pili also mediate anti-inflammatory effects by induction of interleukin-10 (IL-10) mRNA and reduction of interleukin-6 (IL-6) mRNA in a murine RAW 264.7 macrophage cell line. These pili appear to mediate these effects indirectly by promoting close contact with the macrophages, facilitating the exertion of anti-inflammatory effects by other surface molecules via yet unknown mechanisms. Blockage of complement receptor 3 (CR3), previously identified to be a receptor for streptococcal pili, significantly decreased the uptake of pilus-expressing strains in RAW 264.7 cells, while the expression of IL-10 and IL-6 mRNA by these macrophages was not affected by this blocking. On the other hand, blockage of Toll-like receptor 2 (TLR2) significantly reduced the expression of IL-6 mRNA irrespective of the presence of pili.