Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, alpha-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM alpha-ketobutyrate, and 100 mM L-glutamate with a molar yield to alpha-ketobutyrate of 4.3% in 50 mM Tris-HCl buffer (pH 7.5) containing 2 mM MnCl(2) x 4H(2)O at 28 degrees C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and alpha-ketobutyrate was purified from A. simplex AKU 626. Mn(2+) and pyridoxal 5'-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.     

Interactions of two monoclonal antibodies with BNP: high resolution epitope mapping using fluorescence correlation spectroscopy

Structure-function studies of antibody-antigen systems include the identification of amino acid residues in the antigen that interact with an antibody and elucidation of their individual contributions to binding affinity. We used fluorescence correlation spectroscopy (FCS) and alanine-scanning mutagenesis to characterize the interactions of brain natriuretic peptide (BNP) with two monoclonal antibodies. Human BNP is a 32 amino acid residue long cyclic polypeptide with the ring structure confined between cysteines in positions 10 and 26. It is an important cardiovascular hormone and a valuable diagnostic cardiac marker. We compare the binding strength of the N-terminus Alexa488-labeled BNP, native cyclic BNP, BNP alanine-substituted mutants, linear BNP, and its short fragments to determine the individual contributions of amino acid residues included in the continuous antigenic epitopes that are recognized by two different monoclonal antibodies raised toward BNP. Implementation of FCS for these studies offers all of the advantages of solution phase measurements, including high sensitivity, simplicity of manipulation with reagents, and elimination of solid phase interferences or separation steps. Significant differences in the molecular masses of the free and antibody bound BNP results in a substantial ( approximately 2.5-times) increase in the diffusion rates. Determination of the binding constants and inhibition effects by measuring the diffusion rates of the ligand at the single molecule level introduces the ultimate opportunity for researching systems where the fluorescence intensity and/or fluorescence anisotropy do not change upon interaction of the ligand with the protein. Monoclonal antibodies 106.3 and BC203 demonstrate high affinities to BNP and bind two distant epitopes forming robust antibody sandwiches. Both antibodies are used in Abbott diagnostic assays on AxSYM, IMx, and Architect platforms.     

Fungal fragments as indoor air biocontaminants

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.     

Reactive oxygen species and induction of lignin peroxidase in Phanerochaete chrysosporium

We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O(2)) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O(2) gas. The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O(2) (oxygenated cultures) than in cultures grown with atmospheric air. Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O(2) concentrations and in cultures grown with atmospheric air. These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O(2) is at least partially mediated by the intracellular ROS.     

Effect of Dipyridamole on Membrane Energization and Energy Transfer in Chromatophores of Rba. sphaeroides

Biochemistry (Moscow) - Effect of dipyridamole (DIP) at concentrations up to 1 mM on fluorescent characteristics of light-harvesting complexes LH2 and LH1, as well as on conditions of...     

Review of Indo-Pacific dwarf lionfishes (Scorpaenidae: Pteroinae) in the <Emphasis Type="Italic">Dendrochirus brachypterus</Emphasis> complex,with description of a new species from the western Indian Ocean

A taxonomic review of the Dendrochirus brachypterus complex resulted in the recognition of five species, including Dendrochirus barberi (Steindachner 1900), Dendrochirus bellus (Jordan and Hubbs 1925), Dendrochirus brachypterus (Cuvier in Cuvier and Valenciennes 1829), Dendrochirus hemprichi sp. nov. and Dendrochirus tuamotuensis Matsunuma and Motomura 2013. The complex is defined as having usually 9 dorsal-fin soft rays, usually 5 anal-fin soft rays, 17–20 (rarely 20) pectoral-fin rays, no ocellated spots on the soft-rayed portion of the dorsal fin and usually 2 (sometimes none) barbels on the snout tip. Dendrochirus barberi, known from the Hawaiian Islands and Johnston Atoll, is characterized by usually 18 pectoral-fin rays, a relatively high number of scale rows in the longitudinal series (modally 51 vs. 39–49 in other species) and mottled markings on the pectoral fin in large specimens. Dendrochirus bellus, restricted to the northwestern Pacific Ocean from the South China Sea north to southern Japan, is characterized by usually 17 pectoral-fin rays, a relatively low number of scale rows in the longitudinal series (modally 38 vs. 44–51 in other species), and the absence of skin flaps on the orbit surface and uppermost preopercular spine base. Dendrochirus tuamotuensis, recorded only from the Tuamotu Archipelago, is characterized by 19 pectoral-fin rays, the posterior margin of the pectoral fin strongly notched, and a relatively shallow and narrow head and body. Dendrochirus hemprichi sp. nov. is distributed in the western Indian Ocean, including the Red Sea. Although previously confused with a closely related congener (D. brachypterus, known from the northern and eastern Indian Ocean and western Pacific), D. hemprichi can be distinguished from the former by having fewer scale rows between the last dorsal-fin spine base and lateral line, and between the sixth dorsal-fin spine base and lateral line [4–7 (5) in D. hemprichi vs. 5–7 (6) in D. brachypterus, in both cases], a slightly greater interorbital width at the mid-orbit [5.5–10.7 (mean 7.8) % SL vs. 4.5–8.9 (6.8) % of SL] and at preocular spine base [4.4–9.1 (6.6) % SL vs. 3.5–7.8 (5.7) % of SL], and slightly shorter posteriormost (usually 13th) dorsal-fin spine length [11.8–19.9 (15.3) % SL vs. 13.3–21.3 (17.2) % of SL]. Moreover, D. hemprichi tends to have relatively more spinous points on the head spines and ridges, compared with D. brachypterus. Notwithstanding the morphological similarity between the two species, an obvious genetic difference was observed between D. hemprichi and D. brachypterus. Dendrochirus chloreus Jenkins 1903 and Dendrochirus hudsoni Jordan and Evermann 1903 were synonymized under Pterois barberi, as in some previous studies. Scorpaena koenigii Bloch 1789 was regarded as conspecific with D. brachypterus, which it predated. However, the former name should be suppressed under Reversal of Precedence.     

Pr-SNTX,a short-chain three-finger toxin from Papuan pigmy mulga snake,is an antagonist of muscle-type nicotinic acetylcholine receptor (α2βδε)

Three-finger toxins (3FTxs) are one of the major components in snake venoms. In this study, we isolated a cDNA encoding a short-chain 3FTx, Pr-SNTX, from Pseudechis rossignolii. The amino acid sequence of Pr-SNTX is nearly identical to that of its ortholog in Pseudechis australis. Pr-SNTX protein inhibited muscle-type (α₂βδε), but not neuronal α7 nicotinic acetylcholine receptor (nAChR) activity.     

The degradation of three-ringed polycyclic aromatic hydrocarbons by wood-inhabiting fungus Pleurotus ostreatus and soil-inhabiting fungus Agaricus bisporus

The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization.A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungi's affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization.     

ELCS in ice: cryo-electron microscopy of nuclear envelope-limited chromatin sheets

Nuclear envelope-limited chromatin sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ～30 nm, containing a layer of parallel chromatin fibers. In this study, the ultrastructure of ELCS was compared by three different methods: (1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining, (2) high-pressure freezing/freeze substitution into plastic/sectioning and staining, and (3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. ELCS could be clearly visualized by all three methods and, consequently, must exist in vivo and are not fixation artifacts. The ～30-nm chromatin fibers could only be observed following aldehyde fixation; none were seen in cryo-sections. Electron microscopic tomography tangential views of aldehyde-fixed ELCS suggested an ordering of the separate chromatin fibers adjacent to the nuclear envelope. Possible mechanisms of this chromatin ordering are discussed.