Enzymatic synthesis of amino acid-sugar alcohol conjugates in organic media

Amino acid-sugar alcohol conjugates were synthesized by a commercial serine protease, Optimase M-440, in organic media. Optimase M-440 showed broad substrate specificity towards N-t-Boc-protected l-amino acids as acyl donors and sugar alcohols as nucleophiles. Among various solvents tested Optimase M-440 showed the highest activity in pyridine. The regioselective acylation of the primary –OH groups of sugar alcohols gave the amino acid conjugates in good yields without byproducts.     

Concentration of 6-aminopenicillanic acid from penicillin bioconversion solution and its mother liquor by nanofiltration membrane

In this study, nanofiltration was applied to the concentration of the 6-aminopenicillinic acid (6-APA) from bioconverted penicillin solution and also to its mother liquor. The 6-APA in the solution was concentrated from 0.211 mol/L to 0.746 mol/L by nanofiltration. The final maximum concentration was 3.6 times higher than the initial concentration and the recovery yield was 97% to 99% of the original 6-APA. The concentrated solution was crystallized with the yields of 88.9–90.2% and the purity of the crystallized product was about 98%. The concentration of 6-APA in the mother liquor after crystallization was 0.014 mol/L and thus was concentrated 20–30 fold by nanofiltration and crystallization. The recovery of 6-APA was over 98%. The salts contained in the mother liquor, such as NH₄Cl and KCl, could be removed by allowing them to permeate through the membrane.     

Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases

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Effects of Atmospheric O3 on Azolla-Anabaena Symbiosis

Cultures of water fern Azolla pinnata R. Br. exposed for 1 weekto either 30, 50 or 80 nl l^-1 O₃ showed significant reductionsin rates of growth and N₂ fixation, and had fewer heterocysts.Although the levels of glutamine synthetase (GS) and glutamatedehydrogenase (GDH) activity were decreased by low concentrationsof O₃ exposures (30 or 50 nl l^-1), significant increases inlevels of the same enzymes were caused by higher concentrationsof O₃ (80 nl l^-1). Increased levels of total protein, polyamines(putrescine and spermidine), and the xanthophyll-cycle precursorof abscisic acid (ABA), violaxanthin, were also found with higherlevels of O₃ (80 nl l^-1). Levels of ABA itself were significantlyincreased by low level O₃ fumigation (30 nl l^-1) but significantlydecreased by exposure to 80 nl l^-1 O₃. This may indicate thathigher levels of atmospheric O₃ inhibit the final stages ofABA biosynthesis from violaxanthin.Copyright 1994, 1999 AcademicPress Abscisic acid, nitrogen assimilation, nitrogen fixation, ozone pollution, polyamines, violaxanthin     

Label-Free Enrichment of Adrenal Cortical Progenitor Cells Using Inertial Microfluidics

Passive and label-free isolation of viable target cells based on intrinsic biophysical cellular properties would allow for cost savings in applications where molecular biomarkers are known as well as potentially enable the separation of cells with little-to-no known molecular biomarkers. We have demonstrated the purification of adrenal cortical progenitor cells from digestions of murine adrenal glands utilizing hydrodynamic inertial lift forces that single cells and multicellular clusters differentially experience as they flow through a microchannel. Fluorescence staining, along with gene expression measurements, confirmed that populations of cells collected in different outlets were distinct from one another. Furthermore, primary murine cells processed through the device remained highly viable and could be cultured for 10 days in vitro. The proposed target cell isolation technique can provide a practical means to collect significant quantities of viable intact cells required to translate stem cell biology to regenerative medicine in a simple label-free manner.     

Hepatitis C virus (HCV) genotyping by annealing reverse transcription-PCR products with genotype-specific capture probes

The genotype of the hepatitis C virus (HCV) strain infecting a given patient is an important predictive factor for the clinical outcome of chronic liver disease and its response to anti-viral therapeutic agents. We herein sought to develop a new easy, sensitive and accurate HCV genotyping method using annealing genotype-specific capture probes (AGSCP) in an automation-friendly 96-well plate format. The validation of our new AGSCP was performed using the Standard HCV Genotype Panel. We then used both our AGSCP and the commercially available INNO-LiPA assay to analyze the HCV genotypes from 111 Korean patients. Discordant results were analyzed by direct sequencing. AGSCP successfully genotyped the standard panel. The genotypes of 111 patient samples were also obtained successfully by AGSCP and INNO-LiPA. We observed a high concordance rate (93 matched samples, 83.8%) between the two assays. Sequencing analysis of the 18 discordant results revealed that the AGSCP had correctly identified 12 samples, whereas the INNO-LiPA had correctly identified only 6. These results collectively indicate that AGSCP assay is a convenient and sensitive method for large-scale genotyping, and it may be a promising tool for the determination of HCV and other genotypes in clinical settings.     

The resolution of five racemic alpha-lactams by HPLC

The resolution of five racemic alpha-lactams (1a-d,g) using HPLC is reported. Five different Pirkle-type stationary phases were tested. The enantiomers of alpha-lactams containing the trityl group (1a-d) were separated (selectivity factors ranging from 1.08 to 1.20) using a mobile phase of hexane/2-propanol:98/2 and a stationary phase consisting of the 3,5-dinitroaniline derivative of (S)-valine with a urea linkage. Among the dialkyl-substituted alpha-lactams (1e-g), only 1,3-di-tert-butylaziridinone (1g) could be resolved, but only partially (selectivity factor = 1.07), with a mobile phase of hexane/1,2-dichloroethane:95/5 and the stationary phase consisting of the 3,5-dinitrobenzoic acid derivative of (R)-1-naphthylglycine.     

Degenerate tetraploidy was established before bdelloid rotifer families diverged

Rotifers of Class Bdelloidea are abundant freshwater invertebrates known for their remarkable ability to survive desiccation and their lack of males and meiosis. Sequencing and annotation of approximately 50-kb regions containing the four hsp82 heat shock genes of the bdelloid Philodina roseola, each located on a separate chromosome, have suggested that its genome is that of a degenerate tetraploid. In order to determine whether a similar structure exists in a bdelloid distantly related to P. roseola and if degenerate tetraploidy was established before the two species separated, we sequenced regions containing the hsp82 genes of a bdelloid belonging to a different family, Adineta vaga, and the histone gene clusters of P. roseola and A. vaga. Our findings are entirely consistent with degenerate tetraploidy and show that it was established before the two bdelloid families diverged and therefore probably before the bdelloid radiation.     

Biogenic Formation of As-S Nanotubes by Diverse Shewanella Strains

Shewanella sp. strain HN-41 was previously shown to produce novel, photoactive, As-S nanotubes via the reduction of As(V) and S₂O₃²⁻ under anaerobic conditions. To determine if this ability was unique to this bacterium, 10 different Shewanella strains, including Shewanella sp. strain HN-41, Shewanella sp. strain PV-4, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were examined for production of As-S nanotubes under standardized conditions. Of the 10 strains examined, three formed As-S nanotubes like those of strain HN-41. While Shewanella sp. strain HN-41 and S. putrefaciens CN-32 rapidly formed As-S precipitates in 7 days, strains S. alga BrY and S. oneidensis MR-1 reduced As(V) at a much lower rate and formed yellow As-S after 30 days. Electron microscopy, energy-dispersive X-ray spectroscopy, and extended X-ray absorption fine-structure spectroscopy analyses showed that the morphological and chemical properties of As-S formed by strains S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 were similar to those previously determined for Shewanella sp. strain HN-41 As-S nanotubes. These studies indicated that the formation of As-S nanotubes is widespread among Shewanella strains and is closely related to bacterial growth and the reduction rate of As(V) and thiosulfate.A number of bacterial strains have been shown to contribute to the formation of diverse arsenic minerals (4). If sulfide is present as a ligand for immobilization of arsenic, As-S precipitates often form. Desulfosporosinus auripigmentum, which can be isolated from lake sediments, reduces As(V) to As(III) and S(VI) to S(−II) during anaerobic respiration and forms a yellow arsenic sulfide precipitate (7). While Desulfovibrio strain Ben-RB also produces precipitated arsenic sulfide in culture media, As reduction was not correlated with energy conservation (6). Other taxonomically divergent microorganisms isolated from various arsenic-rich sites have also been shown to reduce As(V) to As(III) and form arsenic sulfide precipitates (1, 2).We previously reported that Shewanella sp. strain HN-41 produces an extensive extracellular network of filamentous arsenic-sulfide (As-S) nanotubes via its dissimilatory metal-reducing activity (4). The As-S nanotubes, which formed via the reduction of As(V) and S₂O₃²⁻, were initially amorphous As₂S₃ but evolved with increasing incubation time toward polycrystalline phases of the chalcogenide minerals realgar (AsS) and duranusite (As₄S). Because the Shewanella As-S nanotubes behaved both as metals and as semiconductors, in terms of their electrical and photoconductive properties, respectively, it was postulated that they may provide useful materials for novel nano- and optoelectronic devices (4).While several bacterial species have been shown to produce amorphous and particulate As-S precipitates (1, 2, 4, 7), the formation of the As-S nanotubes by other bacteria has not yet been described, suggesting that this may be a unique property of Shewanella strains. To test this hypothesis, 10 different Shewanella strains, including Shewanella sp. strains PV-4 and HN-41, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were inoculated into HEPES-buffered basal medium (3, 5) containing 10 mM sodium dl-lactate as the electron donor and 5 mM arsenate (Na₂HAsO₄·7H₂O) and 5 mM thiosulfate (Na₂S₂O₃·5H₂O) as the electron acceptors. All chemicals and methods for sample preparation and characterization used in this study were previously described (4).Of the 10 different Shewanella strains examined, only four strains, Shewanella sp. strain HN-41, S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1, produced As-S yellow precipitates in culture medium following incubation in the presence of arsenate and thiosulfate. Shewanella sp. strain HN-41 and S. putrefaciens CN-32 produced yellow precipitates of As-S after 7 days of incubation, whereas S. alga BrY and S. oneidensis MR-1 produced only a small amount of visible precipitate after 30 days of incubation. The remainder of the tested Shewanella strains failed to produce yellow precipitates, regardless of incubation time.The culture medium of the strains tested was periodically sampled during the bacterial incubation period to determine the concentrations of lactate, acetate, arsenic, and sulfide in the aqueous solution. Among the 10 strains examined, Shewanella strain HN-41, S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 metabolized lactate in growth medium containing arsenate and thiosulfate (Table ​(Table1).1). Shewanella sp. strain HN-41 and S. putrefaciens CN-32 rapidly consumed lactate both as an electron donor and as a carbon source (see Fig. S1 in the supplemental material). Cultures of S. alga BrY and S. oneidensis MR-1 consumed ∼1.4 mM lactate after 7 days, while Shewanella sp. strain HN-41 and S. putrefaciens CN-32 consumed 1.7 mM and 2.3 mM lactate, respectively. Although S. putrefaciens CN-32 reduced As(V) in the culture medium supplemented with 5 mM As(V) as the sole electron acceptor, Shewanella sp. strain HN-41, S. alga BrY, and S. oneidensis MR-1 did not reduce As(V) and did not oxidize lactate to acetate (data not shown). Consequently, the latter three strains could not utilize As(V) as an electron acceptor for respiratory metabolism.

TABLE 1.

Influence of thiosulfate on the consumption of lactate, reduction of As(V), and formation of As-S nanotubes by Shewanella strains in medium containing lactate and 5 mM As(V)