Immuno-spin trapping of DNA radicals

The detection of DNA radicals by immuno-spin trapping (IST) is based on the trapping of radicals with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), forming stable nitrone adducts that are then detected using an anti-DMPO serum. DNA radicals are very reactive species, and because they are paramagnetic they have previously been detected only by electron spin resonance (ESR) with or without spin trapping, which is not available in most bioresearch laboratories. IST combines the simplicity, reliability, specificity and sensitivity of spin trapping with heterogeneous immunoassays for the detection of DNA radicals, and complements existing methods for the measurement of oxidatively generated DNA damage. Here we have used IST to demonstrate that DMPO traps Cu(II)-H(2)O(2)-induced DNA radicals in situ and in real time, forming DMPO-DNA nitrone adducts, but preventing both 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) formation and DNA fragmentation. We also applied IST to detect DNA radicals in rat hepatocytes exposed to Cu(II) and H(2)O(2) under nonlethal conditions.     

A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57

Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6xHis-Ki-1/57(122-413) and 6xHis-Ki-1/57(264-413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein beta subunit as template. Our model suggests the family-typical seven-bladed beta-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.     

Non-invasive characterization of single motor unit electromyographic and mechanomyographic activities in the biceps brachii muscle.

The aim of the study was to investigate amplitude and frequency content of single motor unit (MU) electromyographic (EMG) and mechanomyographic (MMG) responses. Multi-channel surface EMG and MMG signals were detected from the dominant biceps brachii muscle of 10 volunteers during isometric voluntary contractions at 20%, 50%, and 80% of the maximal voluntary contraction (MVC) force. Each contraction was performed three times in the experimental session which was repeated in three non-consecutive days. Single MU action potentials were identified from the surface EMG signals and their times of occurrence used to trigger the averaging of the MMG signal. At each contraction level, the MUs with action potentials of highest amplitude were identified. Single MU EMG and MMG amplitude and mean frequency were estimated with normalized standard error of the mean within subjects (due to repetition of the measure in different trials and experimental sessions) smaller than 15% and 7%, respectively, in all conditions. The amplitude of the action potentials of the detected MUs increased with increasing force (mean +/- SD, 244 +/- 116 microV at 20% MVC, and 1426 +/- 638 microV at 80% MVC; P < 0.001) while MU MMG amplitude increased from 20% to 50% MVC (40.5 +/- 20.9 and 150 +/- 88.4 mm/s(2), respectively; P<0.001) and did not change significantly between 50% and 80% MVC (129 +/ -82.7 mm/s(2) at 80% MVC). MU EMG mean frequency decreased with contraction level (20% MVC: 97.2 +/- 13.9 Hz; 80% MVC: 86.2 +/- 11.4 Hz; P < 0.001) while MU MMG mean frequency increased (20% MVC: 33.2 +/- 6.8 Hz; 80% MVC: 40.1 +/- 6.1 Hz; P < 0.001). EMG peak-to-peak amplitude and mean frequency of individual MUs were not correlated with the corresponding variables of MMG at any contraction level.     

Higher-level phylogeny and morphological evolution of tyrant flycatchers, cotingas, manakins, and their allies (Aves: Tyrannida)

Despite increased understanding of higher-level relationships in passerine birds in the last 15 years, the taxonomic boundaries and phylogenetic interrelationships of the major groups of the Tyrannida (including the cotingas, manakins, tityrines, and tyrant flycatchers) remain unclear. Here, we present an analysis of DNA sequence data obtained from two nuclear exons, three introns, and one mitochondrial gene for 26 genera of Tyrannida and 6 tracheophone outgroups. The analysis resulted in well-supported hypotheses about the earliest evolution within Tyrannida. The Cotingidae, Pipridae, Tityrinae (sensu) [Prum, R.O., Rice, N.H., Mobley, J.A., Dimmick, W.W., 2000. A preliminary phylogenetic hypothesis for the cotingas (Cotingidae) based on mitochondrial DNA. Auk 117, 236-241], Tyrannidae, and the tyrannid subfamiles Tyranninae and Pipromorphinae (sensu) [Sibley, C.G., Monroe, B. L. Jr., 1990. Distribution and Taxonomy of Birds of the World. Yale University Press, New Haven, CT] were all found to be reciprocally monophyletic (given the present taxon sampling). The Cotingidae and Pipridae form a clade that is the sister group to a well-supported clade including Oxyruncus, the Tityrinae, Piprites, and the Tyrannidae. Oxyruncus is the sister group to the Tityrinae, and Piprites is placed as the sister group to the Tyrannidae. The tyrannid subfamilies Tyranninae and Pipromorphinae are monophyletic sister taxa, but the relationships of Platyrinchus mystaceus to these two clades remains ambiguous. The presence of medial (=internal) cartilages in the syrinx is a synapomorphy for the Oxyruncus-Tityrinae-Piprites-Tyrannidae clade. Although morphological synapomorphies currently support the monophyly of both the Pipridae and the Cotingidae, convergences and/or reversals in morphological character states are common in Tyrannida. The relationship between Oxyruncus and the Tityrinae is congruent with additional syringeal synapomorphies and allozyme distance data. Accordingly, we propose the recognition the family Tityridae within the Tyrannida to include the genera Schiffornis, Laniisoma, Laniocera, Iodopleura, Xenopsaris, Pachyramphus, Tityra, and Oxyruncus.     

Cytotoxic effect of Shiga toxin-2 holotoxin and its B subunit on human renal tubular epithelial cells

Shiga toxin-producing Escherichia coli produces watery diarrhea, hemorrhagic colitis and hemolytic-uremic syndrome (HUS). In Argentina, HUS is the most common cause of acute renal failure in children. The purpose of the present study was to examine the cytotoxicity of Stx type 2 (Stx2 holotoxin) and its B subunit (Stx2 B subunit) on human renal tubular epithelial cells (HRTEC), in the presence and absence of inflammatory factors. Cell morphology, cell viability, protein synthesis and apoptosis were measured. HRTEC are sensitive to both Stx2 holotoxin and Stx2 B subunit in a dose- and time-dependent manner. IL-1, LPS and butyrate but not TNF, IL-6 and IL-8, increased the Stx mediated cytotoxicity. The effects of Stx2 B subunit appear at doses higher than those used for Stx2 holotoxin. Although the physiological importance of these effects is not clear, it is important to be aware of any potentially toxic activity in the B subunit, given that it has been proposed for use in a vaccine.     

Purification and characterization of two recombinant human granulocyte colony-stimulating factor glycoforms

Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line. The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133. The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GalNAc. The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM). In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli. The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively. The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination. The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life. Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c. route either singularly or as a mixture. Detectable levels of rhG-CSF persisted for more than 8 h in the i.v. and more than 24 h in the s.c. route of administration. All three substances induced early neutrophilia in mice. All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection. Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v. and s.c. administration, respectively.     

Ca increase in secretory granules of stimulated mast cells

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray micro-analysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components. In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines: It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue. Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.     

Snake communities of moist rainforest and derived savanna sites of Nigeria: biodiversity patterns and conservation priorities

The taxonomic composition and the abundance of two communities of snakes were studied in two different areas of southern Nigeria. One community was studied in a derived savanna area (environs of Ejule, 06°54N, 07°23E), and one community was studied in a moist rainforest area (environs of Eket, 04°50N, 07°59E). Both the specific diversity and the mean frequency of observation of snakes were significantly higher in the forest area than in the savanna area, and the dominance index was higher in the savanna than in the forest site. However, most of the species were found in only one of the two areas, depending on their habitat requirements (e.g. Gastropyxis smaragdina, Dispholidus typus, Thelotornis kirtlandii, Dendroaspis jamesoni, Bitis nasicornis, Causus maculatus, etc). The forest community housed a significant number of arboreal and semiaquatic taxa, but in both sites most of the species were terrestrial. The commonest species in the forest area was an oviparous, semiaquatic, batracophagous natricine snake (Afronatrix anoscopus), whereas the commonest species in the savanna area was an oviparous terrestrial lacertophagous psammophine snake (Psammophis phillipsi). Some conservation implications of our biodiversity analyses are presented. It is suggested that the moist rainforest represents a critically endangered habitat, and should deserve special attention by the international scientific community. Oil industry activity is especially dangerous for snake communities, especially in the southernmost regions of Nigeria.     

Photochemistry in everyday life: the effect of spontaneous emulsification on the photochemistry of trans-anethole.

The photochemical behaviour of spontaneously formed microemulsions obtained upon dilution of ethanolic solutions of trans-anethole (E-1-(4-methoxyphenyl)propene, t-A) with water is compared to that of homogeneous ethanolic t-A solutions. Significant differences in reactivity reflect the confined nature of the aggregated t-A which leads to reduced yields of isomerization and dimerization products. In contrast to homogeneous solutions, where a photostationary state enriched in the Z-isomer (c-A) is rapidly reached, the proportion of c-A formed upon irradiation of t-A microemulsions remains below 15%. In the presence of oxygen the formation of trans-anethole oxide is observed which, when formed in non-homogeneous environments, undergoes polymerization.