Intracellular β-Carbonic Anhydrase of the Unicellular Green Alga Coccomyxa : Cloning of the cDNA and Characterization of the Functional Enzyme Overexpressed in Escherichia coli

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO₂, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO₂-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.     

Mutational analysis of basic residues in the N-terminus of the rRNA:m<Superscript>6</Superscript>A methyltransferase ErmC′

Erm methyltransferases mediate the resistance to the macrolide-lincosamide-streptogramin B antibiotics via dimethylation of a specific adenine residue in 23S rRNA. The role of positively charged N-terminal residues of the ErmC' methyltransferase in RNA binding and/or catalysis was determined. Mutational analysis of amino acids K4 and K7 was performed and the mutants were characterized in in vivo and in vitro experiments. The K4 and K7 residues were suggested not to be essential for the enzyme activity but to provide a considerable support for the catalytic step of the reaction, probably by maintaining the optimum conformation of the transition state through interactions with the phosphate backbone of RNA.     

Review: Transport of tRNA out of the Nucleus—Direct Channeling to the Ribosome?

Although tRNA was the first substrate whose export from the nuclei of eukaryotic cells had been shown to be carrier-mediated and active, it has only been in the last 2 years that the first mechanistic details of this nucleocytoplasmic transport pathway have begun to emerge. A member of the importin/karyopherin β superfamily, Los1p in yeast and Xpo-t in vertebrates, has been shown to export tRNA in cooperation with the small GTPase Ran (Gsp1p) from the nucleus into the cytoplasm, where tRNA becomes available for translation. However, Los1p is not essential for viability in yeast cells, suggesting that alternative tRNA export pathways exist. Recent results show that aminoacylation and a translation factor are also required for efficient nuclear tRNA export. Thus, protein translation and nuclear export of tRNA appear to be coupled processes.     

Integral role of the I′-helix in the function of the “inactive” C-terminal domain of catalase–peroxidase (KatG)

Catalase–peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I′-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I′-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatG^C). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed substantial increases in hexa-coordinate low-spin heme and diminished enzyme activity, similar to that observed for the N-terminal domain on its own (KatG^N). The analogous variants of KatG^C showed a much more profound loss of function as evaluated by their ability to return KatG^N to its active conformation. All of the single variants showed a substantial decrease in the rate and extent of KatG^N reactivation, but with two substitutions, KatG^C completely lost its capacity for the reactivation of KatG^N. These results suggest that the I′-helix is central to direct structural adjustments in the adjacent N-terminal domain and supports the hypothesis that the C-terminal domain serves as a platform to direct N-terminal domain conformation and bifunctionality.     

Assessing the Efficiency of the Ability to Intentionally Variate the Power of β<Subscript>2</Subscript> Frequencies in the Frontal Lobes of the Cerebral Cortex

We performed longitudinal examinations by neurofeedback in 17 subjects. The subjects were trained for 12 training seßsions (three weeks) to voluntarily increase the intensity of the ß₂ frequencies in the frontal EEG electrodes of the right (the D scenario) and the left (the S scenario) hemispheres. All the subjects were divided into three groups depending on the training efficacy: a group of subjects that successfully controlled the ß activity in the frontal electrodes of both hemispheres (nine subjects), a group of subjects that successfully controlled this activity only in the right hemisphere (four subjects), and a group of subjects that failed to train during the specified period (four subjects). Analysis of the obtained data showed that the training efficacy depended on the cognitive activity that was focused on achieving the corresponding EEG effects and on the individual personality characteristics.     

Effect of dehydroleucodine on intestinal transit: structural basis of the interaction with the α<Subscript>2</Subscript>-adrenergic receptor

The activity of dehydroleucodine, a sesquiterpene lactone obtained from Artemisia douglasiana, was studied in mice small intestinal transit. Its mechanism was evaluated in the presence of several adrenergic and cholinergic antagonist drugs and one opioid antagonist. Docking of dehydroleucodine into the homology model of the α2-adrenergic receptor allowed us to analyze the structural basis of their interactions. The experiments showed that dehydroleucodine delayed intestinal transit. The docking of dehydroleucodine showed a unique binding site, equivalent to the binding site of carozolol in the β-adrenergic receptor. The results suggested that dehydroleucodine produced an inhibitory effect on intestinal transit. Its action could be mediated, at least in part, through the α2-adrenergic receptor.     

Phylogeny of γ‐proteobacteria: resolution of one branch of the universal tree?

The reconstruction of bacterial evolutionary relationships has proven to be a daunting task because variable mutation rates and horizontal gene transfer (HGT) among species can cause grave incongruities between phylogenetic trees based on single genes. Recently, a highly robust phylogenetic tree was constructed for 13 gamma-proteobacteria using the combined alignments of 205 conserved orthologous proteins.1 Only two proteins had incongruent tree topologies, which were attributed to HGT between Pseudomonas species and Vibrio cholerae or enterics. While the evolutionary relationships among these species appears to be resolved, further analysis suggests that HGT events with other bacterial partners likely occurred; this alters the implicit assumption of gamma-proteobacteria monophyly. Thus, any thorough reconstruction of bacterial evolution must not only choose a suitable set of molecular markers but also strive to reduce potential bias in the selection of species.     

Accuracy of the StressEraser<Superscript>®</Superscript> in the Detection of Cardiac Rhythms

StressEraser is a commercially marketed biofeedback device designed to enhance heart rate variability. StressEraser makes its internal calculations on beat-to-beat measures of finger pulse intervals. However, the accuracy and precision of StressEraser in quantifying interbeat intervals using finger pulse intervals has not been evaluated against standard laboratory equipment using R-R intervals. Accuracy was assessed by simultaneously recording interbeat intervals using StressEraser and a standard laboratory ECG system. The interbeat intervals were highly correlated between the systems. The average deviation in interbeat interval recordings between the systems was approximately 6 ms. Moreover, correlations approached unity between the systems on estimates of heart period, heart rate, and heart rate variability. Feedback from StressEraser is based on an interbeat time series that provides sufficient information to provide an excellent estimate of the dynamic changes in heart rate and heart rate variability. The slight variations between StressEraser and the laboratory equipment in quantifying heart rate and heart rate variability are due to features related to monitoring heart rate with finger pulse: (1) a lack in precision in the peak of the finger pulse relative to the clearly defined inflection point in the R-wave, and (2) contribution of variations in pulse transit time.     

Study of the Mechanism of Interaction of Oligonucleotides with the 3′-Terminal Region of tRNA<Superscript>Phe</Superscript> by Computer Modeling

Three-dimensional atomic models of complexes between yeast tRNA^Phe and 10- or 15-mer oligonucleotides complementary to the 3′-terminal tRNA sequence have been constructed using computer modeling. It has been found that rapidly formed primary complexes appear when an oligonucleotide binds to the coaxial acceptor and T stems of the tRNA^Phe along the major groove, which results in the formation of a triplex. Long stems allow the formation of a sufficiently strong complex with the oligonucleotide, which delivers its 3′-terminal nucleotides to the vicinity of the T loop adjoining the stem. These nucleotides destabilize the loop structure and initiate conformational rearrangements involving local tRNA^Phe destruction and formation of the final tRNA^Phe-oligonucleotide complementary complex. The primary complex formation and the following tRNA^Phe destruction constitute the “molecular wedge” mechanism. An effective antisence oligonucleotide should consist of three segments—(1) complex initiator, (2) primary complex stabilizer, and (3) loop destructor—and be complementary to the (free end)/loop-stem-loop tRNA structural element.     

The < folding problem >: can one predict the structure of proteins?

A protein's three-dimensional structure is encoded in its amino acid sequence. The < folding problem > consists in predicting one based on the other. This classic problem of molecular biology has seen important steps forward in recent years. The raw power of today's computers, along with the mobilization of thousands of internauts, have allowed several small proteins to be literally folded up in a computer, through simulations. Moreover, international programs for structural genomics aim to determine the experimental structures of hundreds of proteins in several organisms, and to model the others by homology to known structures. This will lead to a nearly-complete map of the protein structure universe, shedding light on the past evolution and current functions of today's proteins, and suggesting new targets for therapeutic strategies.     

&quot;Who owns your poop?&quot;: insights regarding the intersection of human microbiome research and the ELSI aspects of biobanking and related studies

Background

While the social, ethical, and legal implications of biobanking and large scale data sharing are already complicated enough, they may be further compounded by research on the human microbiome.

Discussion

The human microbiome is the entire complement of microorganisms that exists in and on every human body. Currently most biobanks focus primarily on human tissues and/or associated data (e.g. health records). Accordingly, most discussions in the social sciences and humanities on these issues are focused (appropriately so) on the implications of biobanks and sharing data derived from human tissues. However, rapid advances in human microbiome research involve collecting large amounts of data on microorganisms that exist in symbiotic relationships with the human body. Currently it is not clear whether these microorganisms should be considered part of or separate from the human body. Arguments can be made for both, but ultimately it seems that the dichotomy of human versus non-human and self versus non-self inevitably breaks down in this context. This situation has the potential to add further complications to debates on biobanking.

Summary

In this paper, we revisit some of the core problem areas of privacy, consent, ownership, return of results, governance, and benefit sharing, and consider how they might be impacted upon by human microbiome research. Some of the issues discussed also have relevance to other forms of microbial research. Discussion of these themes is guided by conceptual analysis of microbiome research and interviews with leading Canadian scientists in the field.

     

On the species of the genus <Emphasis Type="Italic">Diplocheila</Emphasis> Brullé (Coleoptera: Carabidae) of the Far East of Russia,with a brief review of the East Asian species

A taxonomic review of seven species of Diplocheila of the Far East of Russia and adjacent lands is given. Three species are known from the Russian Far East, two of which, D. zeelandica Redt. and D. minima Jedl., are recorded from Russia for the first time. A key to the East Asian species is presented. Composition and diagnoses of subgenera are discussed. The subgenus Submera Habu is restored.     

Indigenous Fire Management in the <Emphasis Type="Italic">cerrado</Emphasis> of Brazil: The Case of the Krahô of Tocantíns

Indigenous peoples have been using fire in the cerrado (savannas) of Brazil as a form of management for thousands of years, yet we have little information on why, when and how these fire practices take place. The aim of this paper was to explore the traditional use of fire as a management tool by the Krahô indigenous group living in the north-eastern region of Tocantíns state, Brazil. The results indicate that the Krahô burn for a variety of reasons throughout the dry season, thereby producing a mosaic of burned and unburned patches in the landscape. The paper discusses this burning regime in the context of contemporary issues regarding fire management, and in the face of changing perceptions to fire by the Krahô themselves.     

Biosynthesis of the respiratory formate dehydrogenases from <Emphasis Type="Italic">Escherichia coli</Emphasis>: characterization of the FdhE protein

Escherichia coli can perform two modes of formate metabolism. Under respiratory conditions, two periplasmically-located formate dehydrogenase isoenzymes couple formate oxidation to the generation of a transmembrane electrochemical gradient; and under fermentative conditions a third cytoplasmic isoenzyme is involved in the disproportionation of formate to CO₂ and H₂. The respiratory formate dehydrogenases are redox enzymes that comprise three subunits: a molybdenum cofactor- and FeS cluster-containing catalytic subunit; an electron-transferring ferredoxin; and a membrane-integral cytochrome b. The catalytic subunit and its ferredoxin partner are targeted to the periplasm as a complex by the twin-arginine transport (Tat) pathway. Biosynthesis of these enzymes is under control of an accessory protein termed FdhE. In this study, it is shown that E. coli FdhE interacts with the catalytic subunits of the respiratory formate dehydrogenases. Purification of recombinant FdhE demonstrates the protein is an iron-binding rubredoxin that can adopt monomeric and homodimeric forms. Bacterial two-hybrid analysis suggests the homodimer form of FdhE is stabilized by anaerobiosis. Site-directed mutagenesis shows that conserved cysteine motifs are essential for the physiological activity of the FdhE protein and are also involved in iron ligation.