Ribosome specificity of archaebacterial elongation factor 2. Studies with hybrid polyphenylalanine synthesis systems

Polyphenylalanine synthesis with ribosomes and two separated, partially purified elongation factors (EF) was measured in cell-free systems from the archaebacteria Thermoplasma acidophilum and Methanococcus vannielii, in an eukaryotic system from rat liver and an eubacterial one with Escherichia coli ribosomes and factors from Thermus thermophilus. By substitution of heterologous EF-2 or EF-G, respectively, for the homologous factors, ribosome specificity was shown to be restricted to factors from the same kingdom. In contrast, EF-1 from T. thermophilus significantly cooperated with ribosomes from T. acidophilum.     

ROS generation and multiple forms of mammalian mitochondrial glycerol-3-phosphate dehydrogenase

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.     

Characterization and kinetic analysis of enzyme–substrate recognition by three recombinant lactococcal tripeptidases

Tripeptidases from Lactococcus lactis subsp. lactis (L9PepTR), L. lactis subsp. cremoris (L6PepTR), and L. lactis subsp. hordniae (hTPepTR) were cloned, overexpressed, purified, and characterized. Although these enzymes contained three to seven naturally occurring amino acid differences, both metal-binding and catalytic sites were highly conserved. The k_cat values of hTPepTR were approximately 1.5- to 2-fold higher than those of L9PepTR, while, for L6PepTR, they were approximately 0.8- to 1.4-times the L9PepTR values. The K_m of tripeptidase from subsp. lactis (L9PepTR) was considerably larger when glycine was the amino acid located at both the N- and C-terminus of the peptide substrate. In addition, the K_m values of L9PepTR increased in the following order for YGG, LGG, FGG, SGG, and α-aminoisobutyrylglycylglycine, while the k_cat/K_m decreased in the same order. These results suggest that the dipole moment and steric hindrance of the N-terminal amino acid side chain may be the most important factors controlling substrate specificity.     

Voracity and feeding preferences of two aphidophagous coccinellids on Aphis citricola and Tetranychus urticae

Voracity and feeding preferences of adult Coccinella septempunctata L. and Harmonia axyridis Pallas (Coleoptera: Coccinellidae, tribe Coccinellini) were evaluated in the laboratory on a common prey, the spirea aphid, Aphis citricola van der Goot (Homoptera: Aphididae), and on the twospotted spider mite, Tetranychus urticae Koch (Acarina: Tetranychidae), a prey previously unrecorded for these two predators. The experiments were carried out in the laboratory on apple saplings (Malus domestica Borkhausen). Adult males and females of H. axyridis consumed significantly more mites than adults of C. septempunctata. For H. axyridis, males consumed 41.3 spider mites in 24 h and females 48.4, whereas for C. septempunctata males consumed 14.1 prey and females 15.2. The consumption of spirea aphids by the males was similar for the two species. Consumption by the females was significantly greater for H. axyridis (46.5) than for C. septempunctata (22.2). The two coccinellids showed a significant preference for A. citricola in the presence of T. urticae. This preference was more pronounced for C. septempunctata. The total number of prey consumed and the percentage of exploited biomass decreased significantly for C. septempunctata and stayed relatively constant for H. axyridis as the number of mites increased in the prey ratio. Our results suggest that T. urticae is only an alternative prey for both predators, and that H. axyridis should be more efficient than C. septempunctata in a prey assemblage with aphids and mites.     

Dual Role of Plasma Membrane Electron Transport Systems in Defense

Bcause oxidative stress is one of the main sources of severe cellular damage, cells have different defense weapons against reactive oxygen species. Ubiquitous plasma membrane redox systems play a role in defense against oxidative stress damage. On the other hand, a tightly controlled and localized production of reactive oxygen species by a plasma membrane NADPH oxidase can be used as a potent microbicidal weapon. This dual, prooxidant and antioxidant role of plasma membrane electron transport systems in defense is studied and discussed.     

Tailoring the Peptide-Binding Specificity Of An RNA by Combinations of Specificity-Altering Mutations

In this study, the ability to tailor the peptide-binding specificity of an RNA was investigated. First, variants of the Rev-response element (RRE) RNA with different specificities toward the natural binding partner, Rev, and two RRE-binding aptamers, the RSG-1.2 and the Kl peptides, were identified. Next, hybrid RRE mutants with combinations of two sets of specificity-altering substitutions were tested for peptide-binding specificity. It was shown that in most cases the results of the combination of individual mutations were of an additive nature, therefore providing a way to manipulate the peptide-binding specificity of an RNA in a predictable manner.     

Prevalence of antibodies against Simkania negevensis in a healthy Japanese population determined by the microimmunofluorescence test

Simkania negevensis has been associated with bronchiolitis in infants and community-acquired pneumonia in adults. Reports of exposure to this microorganism are only available from Israel, North America and Western Europe. Currently, no standard method for diagnosis of S. negevensis infection has been established nor have prevalence rates been shown in Japan. For the first time we demonstrated the ability of the microimmunofluorescence (MIF) test to detect S. negevensis-specific immunoglobulin G and exposure to S. negevensis in Japan. The positive rate in healthy volunteers was 4.3% (25/588), with rates increasing with age. Results indicate the usefulness of the MIF test as a serological method for detecting S. negevensis-specific antibodies. A standard serological test for infection with S. negevensis is needed.     

Plant-bacteria interactions with special emphasis on the kallar grass association

Kallar grass is a highly salt-tolerant grass grown as a pioneer plant on alkaline, salt-affected soils in Pakistan. Nitrogen-fixing bacteria and kallar grass were found to be in close association, which was even root-zone specific: rhizoplane and endorhizosphere were colonized by two different populations. Among theAzospirillum isolates originating from the root surface, some were of a new species, now namedA. halopraeferens. To study plant-bacterium interactions, this natural kallar grass association was chosen. The possible role of bacterial chemotaxis and oxygen tolerance are discussed.     

Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

Nucleotide sequence and style-specific expression of a novel proline-rich protein gene from Nicotiana alata

cDNA clones encoding a novel proline-rich protein (NaPRP4) have been isolated from a Nicotiana alata stylar cDNA library. The N-terminal part of the derived protein is highly rich in proline (32.2%) and contains several repeats such as Lys-Pro-Pro (7 times) and Pro-Thr-Lys-Pro-Pro-Thr-Tyr-Ser-Pro-Ser-Lys-Pro-Pro (twice); the C-terminal part, on the other hand, has a lower proline content (9.9%) and contains two potential N-glycosylation sites and all the six cysteine residues. Northern blot and in situ hybridisation analyses indicate that expression of the NaPRP4 gene is restricted to cells of the transmitting tract of the style.