Technetium-99m-labeled ceftizoxime loaded long-circulating and pH-sensitive liposomes used to identify osteomyelitis

Osteomyelitis is an infectious disease located in the bone or bone marrow. Long-circulating and pH-sensitive liposomes containing a technetium-99m-labeled antibiotic, ceftizoxime, (SpHL-(99m)Tc-CF) were developed to identify osteomyelitis foci. Biodistribution studies and scintigraphic images of bone infection or non infection-bearing rats that had been treated with these liposomes were performed. A high accumulation in infectious foci and high values in the target-non target ratio could be observed. These results indicate the potential of SpHL-(99m)Tc-CF as a potential agent for the diagnosis of bone infections.     

Social laughter is correlated with an elevated pain threshold

Although laughter forms an important part of human non-verbal communication, it has received rather less attention than it deserves in both the experimental and the observational literatures. Relaxed social (Duchenne) laughter is associated with feelings of wellbeing and heightened affect, a proximate explanation for which might be the release of endorphins. We tested this hypothesis in a series of six experimental studies in both the laboratory (watching videos) and naturalistic contexts (watching stage performances), using change in pain threshold as an assay for endorphin release. The results show that pain thresholds are significantly higher after laughter than in the control condition. This pain-tolerance effect is due to laughter itself and not simply due to a change in positive affect. We suggest that laughter, through an endorphin-mediated opiate effect, may play a crucial role in social bonding.     

Nitrile hydratase genes are present in multiple eukaryotic supergroups

Background

Nitrile hydratases are enzymes involved in the conversion of nitrile-containing compounds into ammonia and organic acids. Although they are widespread in prokaryotes, nitrile hydratases have only been reported in two eukaryotes: the choanoflagellate Monosiga brevicollis and the stramenopile Aureococcus anophagefferens. The nitrile hydratase gene in M. brevicollis was believed to have arisen by lateral gene transfer from a prokaryote, and is a fusion of beta and alpha nitrile hydratase subunits. Only the alpha subunit has been reported in A. anophagefferens.

Methodology/Principal Findings

Here we report the detection of nitrile hydratase genes in five eukaryotic supergroups: opisthokonts, amoebozoa, archaeplastids, CCTH and SAR. Beta-alpha subunit fusion genes are found in the choanoflagellates, ichthyosporeans, apusozoans, haptophytes, rhizarians and stramenopiles, and potentially also in the amoebozoans. An individual alpha subunit is found in a dinoflagellate and an individual beta subunit is found in a haptophyte. Phylogenetic analyses recover a clade of eukaryotic-type nitrile hydratases in the Opisthokonta, Amoebozoa, SAR and CCTH; this is supported by analyses of introns and gene architecture. Two nitrile hydratase sequences from an animal and a plant resolve in the prokaryotic nitrile hydratase clade.

Conclusions/Significance

The evidence presented here demonstrates that nitrile hydratase genes are present in multiple eukaryotic supergroups, suggesting that a subunit fusion gene was present in the last common ancestor of all eukaryotes. The absence of nitrile hydratase from several sequenced species indicates that subunits were lost in multiple eukaryotic taxa. The presence of nitrile hydratases in many other eukaryotic groups is unresolved due to insufficient data and taxon sampling. The retention and expression of the gene in distantly related eukaryotic species suggests that it plays an important metabolic role. The novel family of eukaryotic nitrile hydratases presented in this paper represents a promising candidate for research into their molecular biology and possible biotechnological applications.     

Substrate Inhibition Competes with Halide Inhibition in Polyphenol Oxidase

Polyphenol oxidase (PPO) is a ubiquitous enzyme important in the food industry. Although PPO activity followed Michaelis?CMenten kinetics at catechol concentrations of up to 1?mM, it slowly decreased at catechol concentrations above 2?mM. This result indicated that in addition to the active site (site A), the enzyme possesses a second catechol-binding site (site B) that exerts an inhibitory effect on PPO activity. Halides inhibit PPO activity in such a way that substrate inhibition is lessened when halide concentration is increased. Furthermore, elevated concentrations of catechol diminished the degree of inhibition by halides. These findings suggest that halides also bind to site B to inhibit PPO activity. A steady-state kinetic analysis demonstrated that the dissociation constant between catechol and PPO depended on the binding of halides to site B. The dissociation constants were greatest when chloride bound to the site. Bromide and iodide yielded lower dissociation constants, in that order. These data indicate that the binding of halide to site B modulated the structure of site A, thereby exerting an inhibitory effect.     

Quantification of isotope encoded proteins in 2-D gels using surface enhanced resonance Raman

A strategy for quantification of multiple protein isoforms from a complex sample background is demonstrated, combining isotopomeric rhodamine 6G (R6G) labels and surface-enhanced Raman in polyacrylamide matrix. The procedure involves isotope-encoding by lysine-labeling with (R6G) active ester reagents, isoform separation by 2-DGE, fluorescence quantification using internal standardization to water, and silver nanoparticle deposition followed by surface-enhanced Raman detection. R6G sample encoding and standardization enabled the determination of total protein concentration and the distribution of specific isoforms using the combined detection approach of water-referenced fluorescence spectral imaging and ratiometric quantification. A detection limit of approximately 13.5 picomolar R6G-labeled protein was determined for the surface-enhanced Raman in a gel matrix (15-fold lower than fluorescence). High quantification accuracies for small differences in protein populations at low nanogram abundance were demonstrated for human GMP synthetase (hGMPS) either as purified protein samples in a single-point determination mode (3% relative standard deviation, RSD%) or as HCT116 human cancer cellular lysate in an imaging application (with 16% RSD%). These results represent a prototype for future applications of isotopic surface-enhanced resonance Raman scatter to quantification of protein distributions.     

Sequence of ontogenetic development and atrophy of the oral apparatus of six anuran tadpoles

The development of the oral structures of six species of anuran tadpoles with four different types of mouth parts and the metamorphic atrophy of these structures in two species with different mouth parts are described. The oral labia of typical tadpoles, oral flaps of microhylids, and lateral oral folds of Rhinophrynus are assumed to be homologous. We also suggest that the barbels of the tadpoles of Rhinophrynus are homologs of the marginal papillae of species with an oral disc. Developmental patterns and sequences of the oral structures of all tadpoles examined follow a common pattern: stomodeal invagination, oral pad development, jaw sheeth delimitation, tooth row ridge development, jaw sheath keratinization, and labial tooth keratinization. Developmental patterns remain constant, while interspecific differences are apparent because of truncations of ontogeny at specific stages. Metamorphic atrophy of oral structures occurs roughly in the reverse order of development, although the procedure is rapid and more haphazard than development.     

Profiling the changes in signaling pathways in ascorbic acid/β‐glycerophosphate‐induced osteoblastic differentiation

Despite numerous reports on the ability of ascorbic acid and β‐glycerophosphate (AA/β‐GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential substrates) for protein kinases and traditional biochemical techniques to examine the signaling pathways modulated during AA/β‐GP‐induced osteoblast differentiation. The kinomic profile obtained after 7 days of treatment with AA/β‐GP identified 18 kinase substrates with significantly enhanced or reduced phosphorylation. Peptide substrates for Akt, PI3K, PKC, BCR, ABL, PRKG1, PAK1, PAK2, ERK1, ERBB2, and SYK showed a considerable reduction in phosphorylation, whereas enhanced phosphorylation was observed in substrates for CHKB, CHKA, PKA, FAK, ATM, PKA, and VEGFR‐1. These findings confirm the potential usefulness of peptide microarrays for identifying kinases known to be involved in bone development in vivo and in vitro and show that this technique can be used to investigate kinases whose function in osteoblastic differentiation is poorly understood. J. Cell. Biochem. 112: 71–77, 2011. © 2010 Wiley‐Liss, Inc.     

Using DNA barcode to relate landscape attributes to small vertebrate roadkill

Large vertebrates are the main focus of roadkill studies because their greater size facilitates taxonomic identification and the collection of statistical data. However, these studies fail to effectively include and identify small vertebrates and correlate roadkill events with the surrounding landscape. Here we showed the effectiveness of molecular data to identify small vertebrate roadkill, and we correlated landscape structure attributes with the location of roadkill for functional groups of varying mobility. The extraction of DNA from roadkilled individuals was followed by the amplification of two mitochondrial genes. We compared each DNA sequence to a database and used the highest similarity values for species identification. The species were classified according to their taxa and degree of mobility: birds, reptilia and amphibia with low and intermediate movement capability. After calculating the landscape attributes for each roadkill point, we used a competing model approach based on Akaike Information Criteria to determine which landscape variable best explained the occurrence of roadkills. Combining molecular and morphological characteristics, we identified 82.93% of the roadkilled animals. DNA barcoding allowed the identification of 310% more specimens than by morphological characteristics alone. Roadkilled birds with intermediate movement capability were strongly influenced by dominated areas by agriculture and sugar cane monocultures. Roadkilled reptiles with low movement capability were positively correlated with the presence of forest remnants, while those with intermediate movement capability seemed to be more frequent in heavily anthropized landscapes. We showed that molecular data is a powerful tool for precisely identifying small-sized roadkilled animals. Our results also highlight that different landscape structure attributes enable the prediction of roadkill occurrence along roads, which in turn allows us to identify roadkill hotspots and plan appropriate mitigation actions.