Advantage of using inosine at the 3' termini of 16S rRNA gene universal primers for the study of microbial diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3' termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54 degrees C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50 degrees C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3' termini in studying the microbial diversity of environmental samples.     

Partial purification and MALDI-TOF MS analysis of UN1, a tumor antigen membrane glycoprotein

UN1 is a membrane glycoprotein that is expressed in immature human thymocytes, a subpopulation of peripheral T lymphocytes, the HPB acute lymphoblastic leukemia (ALL) T-cell line and fetal thymus. We previously reported the isolation of a monoclonal antibody (UN1 mAb) recognizing the UN1 protein that was classified as "unclustered" at the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. UN1 was highly expressed in breast cancer tissues and was undetected in non-proliferative lesions and in normal breast tissues, indicating a role for UN1 in the development of a tumorigenic phenotype of breast cancer cells. In this study, we report a partial purification of the UN1 protein from HPB-ALL T cells by anion-exchange chromatography followed by immunoprecipitation with the UN1 mAb and MALDI-TOF MS analysis. This analysis should assist in identifying the amino acid sequence of UN1.     

Rat organic anion transporting protein 1A1 (Oatp1a1): purification and phosphopeptide assignment

Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated or singly or doubly phosphorylated forms and sensitive to alkaline phosphatase treatment. The beta-elimination reaction resulted in a mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosphorylated peptide, while phosphorylation at S634 and S635 accounted for all doubly phosphorylated peptide. These findings identify the site of oatp1a1 phosphorylation and demonstrate that it is an ordered process, in which phosphorylation at S634 precedes that at S635. The mechanism by which phosphorylation results in loss of transport activity in hepatocytes remains to be established. Whether phosphorylation near the C-terminus inhibits C-terminal oligomerization of oatp1a1, required for normal transport function, can be speculated upon but is as yet unknown.     

Mechanisms of gamma-glutamylcysteine ligase regulation

The principal objective of this study was to investigate the mechanisms regulating the activity of gamma-glutamylcysteine ligase (GCL; EC 6.3.2.2), the rate limiting enzyme in glutathione biosynthesis. Two phylogenetically divergent species, mouse and the fruitfly, Drosophila melanogaster were used to test the hypothesis that reversible protein phosphorylation and pyridine dinucleotide phosphate dependent allostery regulate GCL activity. GCL was almost completely inhibited under phosphorylating conditions, involving preincubations with MgATP and endogenous protein kinases. Maximal GCL inhibitions of 94%, 77%, 85%, 87%, 83%, 95% and 89% occurred, respectively, in mouse cerebellum, hippocampus, brainstem, striatum, cortex and heart, and Drosophila. These changes in GCL activity were detected using saturating levels of substrates, suggesting that V(max) was dramatically affected, whereas K(m) values showed no differences. In vitro activation of GCL, presumably due to dephosphorylation, was blocked by inhibitors of protein phosphatases, suggesting that GCL exists in vivo as a mixture of phosphorylated and dephosphorylated forms. The reversibility of the dephosphorylation-dependent activation was indicated by the time-dependent inactivation of the in vitro activated Drosophila GCL, by preincubation with MgATP. NADPH increased maximal GCL activity by up to 93%, whereas several other nucleotide analogues did not, thereby demonstrating specificity. Kinetic analysis using Hanes-Woolf replots of initial velocity data suggested that the NADPH-dependent stimulation of GCL activity is brought about by a change in the maximal activity, V(max), rather than changes in substrate affinity. Results of this study suggest that mechanisms of modulation of eukaryotic GCL enzymes may include specific binding of ligands such as pyridine dinucleotide phosphates and reversible protein phosphorylation.     

Part of the series: from dietary antioxidants to regulators in cellular signalling and gene expression. Role of reactive oxygen species and (phyto)oestrogens in the modulation of adaptive response to stress

There is increasing evidence that reactive oxygen species (ROS) are not only toxic but play an important role in cellular signalling and in the regulation of gene expression. We, here, discuss two examples of improved adaptive response to an altered cellular redox state. First, differences in longevity between males and females may be explained by a higher expression of antioxidant enzymes in females resulting in a lower yield of mitochondrial ROS. Oestrogens are made responsible for these phenomena. Oestradiol induces glutathione peroxidase-1 and MnSOD by processes requiring the cell surface oestrogen receptor (ER) and the activation of pathways usually involved in oxidative stress response. Second, oxygen radicals produced during moderate exercise as performed during training up-regulate the expression of antioxidant enzymes in muscle cells. An increased level of these enzymes might prevent oxidative damage during exhaustive exercise and should, therefore, not be prevented by antioxidants. The relevance of these findings is discussed in the context with observations made in transgenic animals overexpressing MnSOD or catalase.     

Sonographic evaluation of thyroid morphology during different reproductive events in female Indo‐Pacific bottlenose dolphins,Tursiops aduncus

Thyroid morphology and function are likely affected by the cyclic hormonal environment during different reproductive events in females. The present study was undertaken to evaluate the variation of thyroid morphology at different reproductive events (anestrus, estrus, lactation, and pregnancy) in a captive group of Indo‐Pacific bottlenose dolphins (Tursiops aduncus) measured using sonography. Sonographic examinations of the thyroid gland and ovaries in nine sexually mature female subjects were performed weekly for 2.5 yr. A generalized linear mixed model was used to estimate the effects of the reproductive events for thyroid volume. Reproductive event was found to be a significant predictor for thyroid volume measurement and significant variation in thyroid volume was found between different reproductive events. A significantly larger thyroid volume in lactating females was observed when compared with estrous and anestrous females, possibly due to the high energy requirements and milk production during lactation. Taken together, thyroid volume variation during different reproductive events in female dolphins should be considered so as to obtain a diagnostically meaningful assessment when conducting routine examinations.     

Mitochondrial Complex II Can Generate Reactive Oxygen Species at High Rates in Both the Forward and Reverse Reactions

Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.     

The original colours of fossil beetles

Structural colours, the most intense, reflective and pure colours in nature, are generated when light is scattered by complex nanostructures. Metallic structural colours are widespread among modern insects and can be preserved in their fossil counterparts, but it is unclear whether the colours have been altered during fossilization, and whether the absence of colours is always real. To resolve these issues, we investigated fossil beetles from five Cenozoic biotas. Metallic colours in these specimens are generated by an epicuticular multi-layer reflector; the fidelity of its preservation correlates with that of other key cuticular ultrastructures. Where these other ultrastructures are well preserved in non-metallic fossil specimens, we can infer that the original cuticle lacked a multi-layer reflector; its absence in the fossil is not a preservational artefact. Reconstructions of the original colours of the fossils based on the structure of the multi-layer reflector show that the preserved colours are offset systematically to longer wavelengths; this probably reflects alteration of the refractive index of the epicuticle during fossilization. These findings will allow the former presence, and original hue, of metallic structural colours to be identified in diverse fossil insects, thus providing critical evidence of the evolution of structural colour in this group.     

Human preferences for symmetry: subjective experience, cognitive conflict and cortical brain activity

This study examines the links between human perceptions, cognitive biases and neural processing of symmetrical stimuli. While preferences for symmetry have largely been examined in the context of disorders such as obsessive-compulsive disorder and autism spectrum disorders, we examine various these phenomena in non-clinical subjects and suggest that such preferences are distributed throughout the typical population as part of our cognitive and neural architecture. In Experiment 1, 82 young adults reported on the frequency of their obsessive-compulsive spectrum behaviors. Subjects also performed an emotional Stroop or variant of an Implicit Association Task (the OC-CIT) developed to assess cognitive biases for symmetry. Data not only reveal that subjects evidence a cognitive conflict when asked to match images of positive affect with asymmetrical stimuli, and disgust with symmetry, but also that their slowed reaction times when asked to do so were predicted by reports of OC behavior, particularly checking behavior. In Experiment 2, 26 participants were administered an oddball Event-Related Potential task specifically designed to assess sensitivity to symmetry as well as the OC-CIT. These data revealed that reaction times on the OC-CIT were strongly predicted by frontal electrode sites indicating faster processing of an asymmetrical stimulus (unparallel lines) relative to a symmetrical stimulus (parallel lines). The results point to an overall cognitive bias linking disgust with asymmetry and suggest that such cognitive biases are reflected in neural responses to symmetrical/asymmetrical stimuli.