Identification of C-terminal neighbours of amino acid residues without an aliphatic <Superscript>13</Superscript>C<Superscript>γ</Superscript> as an aid to NMR assignments in proteins

We propose a methodology that uses GFT (3,2)D CB(CACO)NNH experiment to rapidly collect the data and readily identify six amino acid residue types (Ala, Asn, Asp, Cys, Gly and Ser) in any given protein. Further, the experiment can distinguish the redox state of Cys residues. The proposed experiment in its two forms will have wide range of applications in resonance assignment strategies and structure determination of proteins.     

Screening and identification of an <Emphasis Type="Italic">Enterobacter ludwigii</Emphasis> strain expressing an active <Emphasis Type="Italic">β</Emphasis>-xylosidase

Researchers have expressed increasing interest in the xylanolytic enzymes used in hemicellulose hydrolysis that convert wood and agricultural residues to second-generation biofuels. In our study, 32 isolates showed clear hydrolysis zones on agar plates containing xylan after Congo red staining. Among these isolates, strain LY-62 exhibited the highest β-xylosidase activity (1.29?±?0.05 U/mL). According to the phylogenetic analysis of the 16S rDNA, strain LY-62 belongs to the Enterobacter genus. Using a combination of electron microscopy, Gram-staining, and conventional physiological and biochemical examinations, the strain LY-62 was identified as Enterobacter ludwigii. The β-xylosidase gene from Enterobacter ludwigii LY-62 was cloned, and the full-length protein was expressed in Escherichia coli as an N-terminal or C-terminal His-tagged fusions protein. Optimal β-xylosidase activity was achieved at pH 7.0 and 40 °C. The Michaelis constant K_M values for His-Xyl62 and Xyl62-His were 1.55 and 2.8 mmol/L, respectively. The k_cat values for His-Xyl62 and Xyl62-His were 8.51 and 6.94 s^?1, respectively. The catalytic efficiencies of His-Xyl62 and Xyl62-His were 5.49 and 2.48 s^?1?×?mM^?1, respectively. Thus, Xyl62 is a functional β-xylosidase, and our study represents the first report of a β-xylosidase from Enterobacter ludwigii.     

Primary cilium—is it an osteocyte's strain‐sensing flowmeter?

With few exceptions, the non-cycling cells in a vast range of animals including humans have a non-motile primary cilium that extends from the mother centriole of the pair of centrioles in their centrosomes located between their Golgi apparatuses and nuclei. It has very recently been shown that the primary cilium of a dog or a mouse embryonic kidney cell is a fluid flowmeter studded with heterodimeric complexes of mechanoreceptors linked to Ca(2+)-permeable cation channels that when the cilium is bent can send Ca(2+) signals into the cell and beyond to neighboring cells through gap junctions. More than 30 years ago, osteocytes were reported also to have primary cilia, but this was promptly ignored or forgotten. Osteocytes are the bones' strain sensors, which measure skeletal activity from the effects of currents of extracellular fluid caused by their bones being bent and squeezed during various activities such as walking and running. Since bending a kidney cell's primary cilium can send a Ca(2+) wave surging through itself and its neighbors, the bending of an osteocyte's primary cilium by sloshing extracellular fluid is likely to do the same thing and thus be involved in measuring and responding to bone strain.     

Does gabapentin act as an agonist at native GABA<Subscript>B</Subscript> receptors?

Gabapentin, a novel anticonvulsant and analgesic, is a -aminobutyric acid (GABA) analogue but was shown initially to have little affinity at GABA_A or GABA_B receptors. It was recently reported to be a selective agonist at GABA_B receptors containing GABA_B1a-GABA_B2 heterodimers, although several subsequent studies disproved that conclusion. In the present study, we examined whether gabapentin is an agonist at native GABA_B receptors using a rat model of postoperative pain in vivo and periaqueductal gray (PAG) slices in vitro; PAG contains GABA_B receptors, and their activation results in antinociception. An intrathecal injection of gabapentin or baclofen, a GABA_B receptor agonist, induced antiallodynia in this postoperative pain model. Intrathecal injection of GABA_B receptor antagonists CGP 35348 and CGP 55845 antagonized baclofen- but not gabapentin-induced antiallodynia. In ventrolateral PAG neurons, baclofen activated G-protein-coupled inwardly rectifying K⁺ (GIRK) channels in a manner blocked by CGP 35348 or CGP 55845. However, gabapentin displayed no effect on the membrane current. In neurons unaffected by gabapentin, baclofen activated GIRK channels through GABA_B receptors. It is concluded that gabapentin is not an agonist at GABA_B receptors that are functional in baclofeninduced antiallodynia in the postoperative pain model in vivo and in GIRK channel activation in ventrolateral PAG neurons in vitro.     

The introduced <Emphasis Type="Italic">Micropterus salmoides</Emphasis> in an equatorial lake: a paradoxical loser in an invasion meltdown scenario?

Micropterus salmoides is a North American piscivorous fish on the IUCN list of 100 of the world’s worst invasive alien species. Introduced into Lake Naivasha (Kenya) in 1929, their current population abundance is significantly depressed in a lake that has recently become dominated by fishes of the Cyprinidae family; the introduced cyprinid Cyprinus carpio now dominates catches in the commercial fishery and Barbus paludinous is now numerically dominant in the fish community. Long-term diet studies of M. salmoides based on gut contents analysis (GCA) have defined their diet spectrum, feeding preferences and ontogenetic dietary shifts. Between 1987 and 1991, diet was size-structured; fish <260 mm were mainly insectivorous and fish >260 mm fed mainly on invasive crayfish Procambarus clarkia with B. paludinosus rarely taken. More recent GCA data revealed that up to 2003, these size-structured trophic relationships were still evident, but there has been a subsequent shift to their feeding almost exclusively on small (<100 mm) B. paludinosus, coincident with a size-related functional switch whereby M. salmoides >120 mm were now piscivorous. However, a Bayesian stable isotope mixing model (SIAR) suggested M. salmoides diet actually remained relatively varied in 2006 and 2007; it indicated P. clarkii were still contributing more to their diet than B. paludinosus in fish <260 mm and provided only partial support for the functional shift. The consequence of the M. salmoides depressed population abundance is their predation pressure on prey fishes is limited and preventing top-down effects. This is in contrast to their invasive populations elsewhere in the world and the likely result of invasion meltdown processes in Naivasha involving the introduced C. carpio and P. clarkii that have produced sub-optimal foraging conditions for M. salmoides.     

Spezifität beim „Attachment” von Agrobacterium an Wundrandzellen von Kalanchoe?

Specificity of the Attachment of Agrobacterium to Wound Cells of Kalanchoe? Tumor induction by A. tumefaciens on leaves of Kalanchoe is severely inhibited by cell wall preparations from young and mature tissucs of monocotyledons and dicoryledons and from tumor cells and also by filter paper homogenates and living or dead Pseudomonas cells. The inhibition a demonstrable if the cell wall preparations or Pseudomonas cells are inoculated into the plant wounds at the same time with A tumefaciens or before A. tumefaciens a. Postinoculation does affect tumor These results demonstrate site binding as an important step of tumor induction without any doubt but at the same time they question the specificity of this process.     

The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’?

Hydrogen sulfide (H2S) is a well-known cytotoxic gas. Recently it has been shown to stimulate N-methyl-D-aspartate (NMDA) receptors to enhance long-term potentiation suggesting a novel neuromodulatory role in vivo. Endogenous levels of H2S in the brain are reported to range between 10 and 160 microm. Considerably lower H2S levels are reported in the brains of Alzheimer's disease (AD) patients, where levels of brain protein nitration (probably mediated by peroxynitrite) are markedly increased. Activation of NMDA receptors leads to intracellular tyrosine nitration by peroxynitrite. Because H2S and peroxynitrite are important mediators in brain function and disease, we investigated the effects of the H2S 'donor', sodium hydrogen sulfide (NaSH) on peroxynitrite-mediated damage to biomolecules and to cultured human SH-SY5Y cells. H2S significantly inhibited peroxynitrite-mediated tyrosine nitration and inactivation of alpha1-antiproteinase to a similar extent to reduced glutathione at each concentration tested (30-250 microm). H2S also inhibited peroxynitrite-induced cytotoxicity, intracellular protein nitration and protein oxidation in human neuroblastoma SH-SY5Y cells. These data suggest that H2S has the potential to act as an inhibitor of peroxynitrite-mediated processes in vivo and that the potential antioxidant action of H2S deserves further study, given that extracellular GSH levels in the brain are very low.     

Are <Emphasis Type="Italic">Drosophila</Emphasis> telomeres an exception or the rule?

At the ends of eukaryotic chromosomes are telomeres, specialized structures with unusual properties. The repetitive structure of telomere regions makes them difficult to deal with in general genome-sequencing projects. Specific efforts to compare sequences and properties of telomeres across species can reveal the generalities of telomere properties.     

Herbivore population suppression by an intermediate predator, <Emphasis Type="Italic">Phytoseiulus macropilis</Emphasis>, is insensitive to the presence of an intraguild predator: an advantage of small body size?

Recent work in terrestrial communities has highlighted a new question: what makes a predator act as a consumer of herbivores versus acting as a consumer of other predators? Here we test three predictions from a model (Rosenheim and Corbett in Ecology 84:2538–2548) that links predator foraging behavior with predator ecology: (1) widely foraging predators have the potential to suppress populations of sedentary herbivores; (2) sit and wait predators are unlikely to suppress populations of sedentary herbivores; and (3) sit and wait predators may act as top predators, suppressing populations of widely foraging intermediate predators and thereby releasing sedentary herbivore populations from control. Manipulative field experiments conducted with the arthropod community found on papaya, Carica papaya, provided support for the first two predictions: (1) the widely foraging predatory mite Phytoseiulus macropilis strongly suppressed populations of a sedentary herbivore, the spider mite Tetranychus cinnabarinus, whereas (2) the tangle-web spider Nesticodes rufipes, a classic sit and wait predator, failed to suppress Tetranychus population growth rates. However, our experiments provided no support for the third hypothesis; the sit and wait predator Nesticodes did not disrupt the suppression of Tetranychus populations by Phytoseiulus. This contrasts with an earlier study that demonstrated that Nesticodes can disrupt control of Tetranychus generated by another widely foraging predator, Stethorus siphonulus. Behavioral observations suggested a simple explanation for the differing sensitivity of Phytoseiulus and Stethorus to Nesticodes predation. Phytoseiulus is a much smaller predator than Stethorus, has a lower rate of prey consumption, and thus has a much smaller requirement to forage across the leaf surface for prey, thereby reducing its probability of encountering Nesticodes webs. Small body size may be a general means by which widely foraging intermediate predators can ameliorate their risk of predation by sit and wait top predators. This effect may partially or fully offset the general expectation from size-structured trophic interactions that smaller predators are subject to more intense intraguild predation.     

In vitro induction and characterization of hexaploid Pennisetum × advena,an ornamental grass

Plant Cell, Tissue and Organ Culture (PCTOC) - Pennisetum&nbsp;×&nbsp;advena (purple fountain grass) is an ornamental grass with considerable commercial value. In vitro induction of...     

Transition-metal-mediated uncaging in living human cells — an emerging alternative to photolabile protecting groups

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Molecular cloning and expression of an extracellular α-amylase gene from an Antarctic deep sea psychrotolerant <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain 7193

Psychrotolerant Pseudomonas stutzeri strain 7193 capable of producing an extracellular α-amylase was isolated from deep sea sediments of Prydz Bay, Antarctic. The 59678-Da protein (AmyP) was encoded by 1665-bp gene (amyP). The deduced amino acid sequence was identified with four regions, which are conserved in amylolytic enzymes and form a catalytic domain, and was predicted to be maltotetraose forming extracellular amylase by using the I-TASSER online server. Purification of AmyP amylases from both the recombinant of Escherichia coli Top 10 F′ and strain 7193 was conducted. Biochemical characterization revealed that the optimal amylase activity was observed at pH 9.0 and temperature 40°C. The enzymes were unstable at temperatures above 30°C, and only retain half of their highest activity after incubation at 60°C for 5 min. Thin-layer chromatography analysis of the products of the amylolytic reaction showed the presence of maltotetraose, maltotriose, maltose and glucose in the starch hydrolysate.     

Secretion of acid phosphatase in<Emphasis Type="Italic">Claviceps purpurea</Emphasis> — an ultracytochemical study

The lead phosphate precipitation method showed the reaction product of acid phosphatase (which reflects the presence of the enzyme glycoprotein) in peripheral cytoplasmic vesicles in the ascomycetous fungus Claviceps purpurea. The product appeared to diffuse from these vesicles (diameter 100-200 nm) towards the cell wall, usually to its sites covered by the capsular fibres exhibiting also acid phosphatase activity. This observation of diffusion of secretory glycoprotein in the cytoplasmic matrix and its orientation to the plasmalemma and capsular fibrils suggests an alternative to the well-described secretory mechanism of transport and exocytosis of glycoproteins via membrane-bound transport conveyors fusing with the cell membrane. It confirms and enlarges our previous finding of the reaction product of acid phosphatase performed by ultrastructural cytochemistry in vacuoles (lysosomes), in the growing cell septum, in cytoplasmic vesicles and in the fibres of the external capsule.     

The <Emphasis Type="Italic">pydA</Emphasis>–<Emphasis Type="Italic">pydB</Emphasis> fusion gene produces an active dioxygenase–hydrolase that degrades 3-hydroxy-4-pyridone,an intermediate of mimosine metabolism

The objective of this research was to construct a pydA–pydB hybrid gene that encodes a functional dioxygenase–hydrolase (PydA–PydB) fusion protein for degradation of 3-hydroxy-4-pyridone (HP). HP is an intermediate in both synthesis and degradation of mimosine, a toxic amino acid produced by the tree legume Leucaena leucocephala. Computer-generated models of the fusion proteins suggested that joining of PydA and PydB with 0, 3, or 7 glycine residues as a linker should produce a functional PydA–PydB fusion protein. Accordingly, three hybrid genes, G0, G3, and G7, were constructed in which pydA and pydB were connected with 0, 9, and 21 nucleotides, respectively, encoding the glycine residues of the linker region. When these hybrid genes were expressed in Rhizobium and Escherichia coli, only one of them, G3, produced a functional PydA–PydB fusion protein, having both the dioxygenase and hydrolase activities. The G3 hybrid gene could complement both pydA and pydB mutants of Rhizobium, and E. coli lysate containing the overexpressed G3 protein was able to degrade HP. This hybrid gene may be useful for developing mimosine-free L. leucocephala plants in the future.     

Metabolomics – an overview. From basic principles to potential biomarkers (part 2)

The metabolome, which represents the complete set of molecules (metabolites) of a biological sample (cell, tissue, organ, organism), is the final downstream product of the metabolic cell process that involves the genome and exogenous sources. The metabolome is characterized by a large number of small molecules with a huge diversity of chemical structures and abundances. Exploring the metabolome requires complementary analytical platforms to reach its extensive coverage. The metabolome is continually evolving, reflecting the continuous flux of metabolic and signaling pathways. Metabolomic research aims to study the biochemical processes by detecting and quantifying metabolites to obtain a metabolic picture able to give a functional readout of the physiological state. Recent advances in mass spectrometry (one of the mostly used technologies for metabolomics studies) have given the opportunity to determine the spatial distribution of metabolites in tissues. In a two-part article, we describe the usual metabolomics technologies, workflows and strategies leading to the implementation of new clinical biomarkers. In this second part, we first develop the steps of a metabolomic analysis from sample collection to biomarker validation. Then with two examples, autism spectrum disorders and Alzheimer's disease, we illustrate the contributions of metabolomics to clinical practice. Finally, we discuss the complementarity of in vivo (positron emission tomography) and in vitro (metabolomics) molecular explorations for biomarker research.     

<Emphasis Type="Italic">Ganoderma </Emphasis>diseases of perennial crops in India – an overview

The species of Ganoderma recorded from India as causing diseases of perennial crops are listed, and their host range and taxonomy discussed. Four new hosts of G. lucidum are also reported. A decline in productivity and the death of trees are the main economic impacts due to Ganoderma diseases, and the fungus is identified as a serious pathogen of cash crops, forest plantations and trees in natural forests in the country. Ganoderma diseases have been recorded on 144 hosts in India, the major pathogens being G. lucidum and G. applanatum. G. lucidum has been recorded on 91 hosts, and appears to cause the most widespread diseases. Identification has largely been made from morphological and cultural characters, and the names currently in use should therefore be treated with caution. Cultural methods of disease control are largely inefficient in minimising inoculum pressure and in reducing the disease incidence. Chemical methods in combination with soil amendments form short-term solutions for managing the disease and improving productivity. The immediate priorities for developing an efficient management system for Ganoderma diseases in India are: (1) a thorough understanding of the etiology and epidemiology of the diseases on different hosts, (2) clarifying current ambiguity in species names, (3) assessing the inter-relationships between populations of Ganoderma on different hosts and (4) developing tools for early detection of diseases in important crops.