Ultrasound‐mediated synthesis of camphoric acid‐based chiral salens for the enantioselective trimethylsilylcyanation of aldehydes

New chiral salen ligands were prepared by the ultrasound‐irradiated condensation of optically active (1R, 3S)‐1,2,2‐trimethyl‐1,3‐diaminocyclopentane with aromatic 1‐hydroxyaldehydes. The ultrasound‐mediated process is more convenient due to shorter reaction times, energy economy, and easier isolation of the products. The in situ formed Ti(IV)(salen) complexes, evaluated as catalysts in the enantioselective trimethylsilylcyanation of benzaldehyde, were found to be efficient for this process, originating the corresponding product in high yields (72–99%) and selectivities of up to 79%. The lowest energy transition states were determined by computational studies. These results were in qualitative agreement with the experimentally observed ones. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Superoxide dismutases—a review of the metal-associated mechanistic variations

Superoxide dismutases are enzymes that function to catalytically convert superoxide radical to oxygen and hydrogen peroxide. These enzymes carry out catalysis at near diffusion controlled rate constants via a general mechanism that involves the sequential reduction and oxidation of the metal center, with the concomitant oxidation and reduction of superoxide radicals. That the catalytically active metal can be copper, iron, manganese or, recently, nickel is one of the fascinating features of this class of enzymes. In this review, we describe these enzymes in terms of the details of their catalytic properties, with an emphasis on the mechanistic differences between the enzymes. The focus here will be concentrated mainly on two of these enzymes, copper, zinc superoxide dismutase and manganese superoxide dismutase, and some relatively subtle variations in the mechanisms by which they function.     

Moderately Thermophilic Magnetotactic Bacteria from Hot Springs in Nevada

Populations of a moderately thermophilic magnetotactic bacterium were discovered in Great Boiling Springs, Nevada, ranging from 32 to 63°C. Cells were small, Gram-negative, vibrioid to helicoid in morphology, and biomineralized a chain of bullet-shaped magnetite magnetosomes. Phylogenetically, based on 16S rRNA gene sequencing, the organism belongs to the phylum Nitrospirae.Magnetotactic bacteria are a metabolically, morphologically, and phylogenetically heterogeneous group of prokaryotes that passively align and actively swim along magnetic field lines (3). This behavior, called magnetotaxis, is due to the presence of intracellular, membrane-bounded, single-magnetic-domain crystals of magnetite (Fe₃O₄) and/or greigite (Fe₃S₄) (3).Most known cultured magnetotactic bacteria are mesophilic and do not grow much above 30°C (e.g., Magnetospirillum species and Desulfovibrio magneticus strains MV-1 and MC-1 [D. A. Bazylinski, unpublished data]). Uncultured magnetotactic bacteria have been observed in numerous habitats that were mostly at 30°C and below. There is only one report describing thermophilic magnetotactic bacteria despite a number of efforts to look for them (e.g., in hydrothermal vents [D. A. Bazylinski, unpublished data]). Nash (12) reported the presence of thermophilic magnetotactic bacteria in microbial mats at about 45 to 55°C adjacent to the main flow in Little Hot Creek (but not in other springs in the same area at 40 to 80°C) and in microbial mats of other springs in central California at up to 58°C, all on the east side of the Sierra Nevada mountains. Cells biomineralized bullet-shaped crystals of magnetite and were phylogenetically affiliated with the phylum Nitrospirae (12). Few additional details were provided regarding the organisms and their habitat.In this study, water and surface sediment samples were taken from the Great Boiling Springs (GBS) geothermal field in Gerlach, NV. GBS is a series of hot springs that range from ambient temperature to ∼96°C (2, 5). The geology, chemistry, and microbial ecology of the springs have been described in some detail (2, 5). The pHs of the samples ranged from 6.4 to 7.5, while the salinities were about 4 to 5 ppt, as determined with a handheld Palm Abbe PA203 digital refractometer (MISCO Refractometer, Cleveland, OH). Samples were examined for the presence of magnetotactic bacteria using the hanging drop technique on-site and in the laboratory at room temperature with and without magnetic enrichment of the sample (15). Some samples taken back to the laboratory were kept at an elevated temperature (∼62°C), while others were kept at ambient temperature. There did not appear to be a significant difference in the number of magnetotactic cells in samples taken back to the laboratory and kept at these two temperatures. Only one morphotype of magnetotactic bacteria was found in samples from nine springs whose temperatures ranged from 32 to 63°C, and we estimate their numbers to be between 10³ to 10⁵ cells ml⁻¹ in surface sediments in sample bottles. We did not observe magnetotactic cells of this type in a large number of springs or pools that were at <32°C. Only one spring positive for the presence of these magnetotactic bacteria had sediment that was partially covered with a microbial mat, while sediment at most of the springs was dark gray in color. Cells were small (1.8 ± 0.4 by 0.4 ± 0.1 μm; n = 59), Gram negative, vibrioid to helicoid in morphology, and possessed a single polar flagellum (Fig. ​(Fig.1A).1A). Magnetotactic bacteria were not observed in springs that were at 67°C and above, suggesting the maximum survival and perhaps growth temperature for the organism is about 63°C. In the lab, cells remained viable and motile in samples kept at 25 to 62°C for several months. We refer to this organism as strain HSMV-1.Open in a separate windowFIG. 1.Transmission electron microscope (TEM) images of cells and magnetosomes of strain HSMV-1. (A) TEM image of unstained cell of HSMV-1 showing a single polar flagellum and a single chain of bullet-shaped magnetosomes. The electron-dense structures at the poles were found to be phosphorus-rich based on energy-dispersive X-ray analysis (data not shown) and therefore likely represent polyphosphate granules. (B) Higher-magnification TEM image of the magnetosome chain. (C) High-magnification TEM image of magnetosomes from which a selected area electron diffraction (SAED) pattern was obtained (inset of B). The SAED pattern corresponds to the [1 0−1] zone of magnetite, Fe₃O₄: reflection o, (0 0 0); reflection a, (1 −1 1) (0.48 nm); reflection b, (1 1 1) (0.48 nm); reflection c, (2 0 2) (0.30 nm); angle a-o-b, 70.5°. (D) Iron, sulfur, and oxygen elemental maps, derived from energy-filtering transmission electron microscopy (EFTEM), showing that the positions of the magnetosome crystals correlate with increased concentrations of Fe and O, but not S, consistent with the iron oxide magnetite (Fe₃O₄).Cells of HSMV-1 biomineralized a single chain of magnetosomes that traversed the cells along their long axis (Fig. 1A to C). Selected area electron diffraction (SAED) and energy-filtering transmission electron microscopy (EFTEM) elemental maps were determined on magnetosome crystals using a Tecnai model G2 F30 Super-Twin transmission electron microscope (FEI Company, Hillsboro OR). SAED patterns of HSMV-1 magnetosome crystals (Fig. ​(Fig.1B,1B, inset) indicated that they consisted of magnetite, while EFTEM elemental maps (Fe, O and S) (Fig. ​(Fig.1D)1D) clearly showed that the crystals consisted of an iron oxide and not an iron sulfide, again consistent with the mineral magnetite. Cells contained an average of 12 ± 6 magnetosome crystals per cell (n = 15 cells) that averaged 113 ± 34 by 40 ± 5 nm in size (n = 179). A plot of the length of the crystals as a function of the shape factor (width/length ratio) is provided in Figure S1 in the supplemental material and shows that the crystals fit in the theoretical single-magnetic-domain size range (4), along with all known mature magnetosome magnetite crystals from magnetotactic bacteria (3).Whole-cell PCR amplification of the 16S rRNA gene was performed by first magnetically purifying cells of HSMV-1 using the “capillary racetrack” described by Wolfe et al. (18). Purity of the collected cells was determined by microscopic examination, and contaminating cells were never observed. The 16S rRNA gene was amplified using bacteria-specific primers 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5′-TACGGHTACCTTGTTACGACTT-3′ (11). PCR products were cloned into pGEM-T Easy vector (Promega Corporation, Madison, WI) and sequenced (Functional Biosciences, Inc., Madison, WI). Six of eight clones sequenced had identical inserts.Alignment of 16S rRNA gene sequences was performed using the CLUSTAL W multiple alignment accessory application in the BioEdit sequence alignment editor (7). Phylogenetic trees were constructed using MEGA version 4 (17) by applying the neighbor-joining method (14). Bootstrap values were calculated with 1,000 replicates. The 16S rRNA gene sequence of strain HSMV-1 places the organism in the phylum Nitrospirae (Fig. ​(Fig.2),2), with its closest relative in culture being Thermodesulfovibrio hydrogeniphilus (87.2% identity) (8). Two other uncultured magnetotactic bacteria are phylogenetically affiliated with the phylum Nitrospirae, including the unnamed rod-shaped bacterium strain MHB-1 (86.5% identity) (6) and the very large Candidatus Magnetobacterium bavaricum (86.4% identity) (16). Interestingly, all the magnetotactic bacteria associated with the phylum Nitrospirae thus far (e.g., Candidatus Magnetobacterium bavaricum) contain bullet-shaped magnetite crystals in their magnetosomes.Open in a separate windowFIG. 2.Phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic position of strain HSMV-1 in the phylum Nitrospirae. Bootstrap values at nodes are percentages of 1,000 replicates. The magnetotactic bacteria Desulfovibrio magneticus and Candidatus Magnetoglobus multicellularis (outgroup; deltaproteobacteria) were used to root the tree. GenBank accession numbers are given in parentheses. Bar represents 2% sequence divergence.Fluorescent in situ hybridization (FISH) was used to authenticate the 16S rRNA gene sequence. A specific Alexa594-labeled probe for HSMV-1 was designed (HSMVp, 5′-CCTTCGCCACAGGCCTTCTA-3′, complementary to nucleotides 690 to 709 of the 16S rRNA molecule) based on the alignment of 10 of the most similar 16S rRNA gene sequences found in GenBank after BLAST analysis (1) and on cultivated members of the phylum Nitrospirae. FISH with the Alexa594-labeled probe was carried out after fixation of magnetically concentrated cells directly on the wells of gelatin-coated hydrophobic microscope slides with 4% paraformaldehyde. FISH was performed according to the work of Pernthaler et al. (13). The hybridization solution contained 10 ng/ml of the probe, 20% formamide, 0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM Na₂EDTA, and 0.01% sodium dodecyl sulfate (SDS). Cells of HSMV-1 hybridized to the HSMVp probe, while other cells in the sample did not (Fig. ​(Fig.3),3), indicating that the 16S rRNA gene sequence we obtained is from the magnetotactic bacterium under study. Strain HSMV-1 clearly represents a new genus (Fig. ​(Fig.2),2), and based on the phylogeny and what we currently know phenotypically about strain HSMV-1, we propose the name Candidatus Thermomagnetovibrio paiutensis (the GBS site was originally occupied by the Paiute Indian Tribe).Open in a separate windowFIG. 3.Fluorescent in situ hybridization (FISH) of cells of strain HSMV-1 using an HSMV-1-specific oligonucleotide rRNA probe (HSMVp). Cells used for FISH were magnetically concentrated by placing a magnet next to the side of the sample bottle for 30 min and then removed with a Pasteur pipette. This technique was used rather than the magnetic racetrack method in order to have many HSMV-1 cells as well as some other cells that could be used as a negative control. (A) Differential interference contrast (DIC) image of HSMV-1 cells (filled arrows) and other cells (negative control; empty arrows) from hot spring samples; (B) cells stained with 4′,6-diamidino-2-phenylindole (DAPI); (C) cells hybridized with the specific probe HSMVp.Nash (12) first reported thermophilic magnetotactic bacteria phylogenetically affiliated with the Nitrospirae phylum in hot springs, and it would be interesting and important to compare these organisms and their habitats. However, little can be compared at this time due to lack of information. Nash (12) reported that the one spring at Little Hot Creek was freshwater and that microbial mats were present at all springs where thermophilic magnetotactic bacteria were found. The water at our sampling sites was brackish, not freshwater, and microbial mats were not an important feature of our springs. Thus, it is difficult to determine without knowing the relationship between the organisms found by Nash (12) and strain HSMV-1 what environmental parameters are important to the growth and survival of these bacteria.It is also difficult to determine the temperature ranges for the survival and growth for strain HSMV-1 without having a pure culture. Data presented here suggest that the temperature range for both is quite wide, and this would be important for the continued presence of HSMV-1 at GBS, as temperatures in the hot springs are known to fluctuate greatly (2). Even if the maximum growth temperature of HSMV-1 is slightly lower than the maximum survival temperature (a conservative estimate) that we know of (63°C), it would still be considered a moderately thermophilic bacterium.The results presented here clearly show that some magnetotactic bacteria can be considered at least moderately thermophilic. They extend the upper temperature limit for environments where magnetotactic bacteria exist and likely grow (∼63°C) and where magnetosome magnetite is deposited, a finding that may prove significant in the study and interpretation of magnetofossils (9, 10).     

New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase,Emerging from Nonconventional Codon Usage and Directed Mutation

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k_cat/K_m) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes'' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.β-Fructofuranosidases (EC 3.2.1.26) are enzymes of biotechnological interest that catalyze the release of β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. In general, they exhibit a high degree of sequence homology, and based on their amino acid sequences, they fall into family 32 of the glycosyl-hydrolases (GH), along with invertases, inulinases, and fructosyltransferases (http://www.cazy.org). The GH32 family has been studied intensely, and some three-dimensional structures are now available, such as that of inulinase from Aspergillus awamorii (26), fructan-exohydrolase from Cichorium intybus (CiFEH) (34, 36), or invertase from Thermotoga maritima (2, 3) and Arabidopsis thaliana (35). These proteins contain a five-blade β-propeller N-terminal catalytic module and a C-terminal β-sandwich domain (19). Multiple-sequence alignment of GH32 proteins, which are included in the GH-J clan together with the GH68 proteins of the inulosucrase family, reveals the presence of three conserved motifs, each containing a key acidic residue (in boldface) implicated in substrate binding and hydrolysis: Asn-Asp-Pro-Asn-Gly (NDPNG), Arg-Asp-Pro (RDP), and Glu-Cys (EC) (28). These conserved residues are implicated in a double-displacement reaction in which a covalent glycosyl-enzyme intermediate is formed. Thus, the catalytic mechanism proposed for the Saccharomyces cerevisiae invertase implies that Asp23 (NDPNG) acts as a nucleophile and Glu204 (EC) acts as the acid/base catalyst (29), whereas Asp309 (RDP) of Acetobacter diazotropicus levansucrase influences the efficiency of sucrose hydrolysis (7) and Arg188 and Asp189 of the latter motif define the substrate binding and specificity of exoinulinase from A. awamorii toward fructopyranosyl residues (26).As well as hydrolyzing sucrose, β-fructofuranosidases may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose skeleton by different glycosidic bonds, depending on the source of the enzyme (12, 21, 31). FOS act as prebiotics, and they exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, and osteoporosis (16). Currently, FOS are mainly produced by Aspergillus fructosyltransferase in industry (10, 31), providing a mixture of FOS with an inulin-type structure that contains β-(2→1)-linked fructose oligomers (¹F-FOS: 1-kestose or nystose). Curiously, when the link between two fructose units (⁶F-FOS: 6-kestose) or between fructose and the glucosyl moiety (⁶G-FOS: neokestose) involves a β-(2→6) link, the prebiotic properties of the FOS may be enhanced beyond that of commercial FOS (23).The yeast Schwanniomyces occidentalis (also called Debaryomyces occidentalis) produces a number of extracellular enzymes that make it of interest in biotechnology. Several of its amylolytic enzymes have been characterized, including amylases and glucoamylase (1, 9), as well as an invertase (17). In addition, we also characterized an extracellular β-fructofuranosidase (Ffase) from this yeast that hydrolyzes sucrose, 1-kestose, and nystose (5). This enzyme exhibited a transfructosylating activity that efficiently produces the trisaccharides 6-kestose and 1-kestose in the ratio 3:1, generating the highest 6-kestose yield yet reported, as far as we know. The Ffase three-dimensional structure has recently been solved (6) and represented as a homodimer, each modular subunit arranged like other GH32 enzymes. The Asp50 (NDPNG) and Glu230 (EC) located at the center of the propeller are the catalytic residues implicated in substrate binding and hydrolysis, whereas Arg178 and Asp179 form the RDP motif (6).The genetic codes of some yeasts incorporate certain variations. For example, while CUG was believed to be a universal codon for leucine, in the cytoplasm of certain species of the genus Candida (15) it encodes a serine, as in Pichia farinosa (33). The reassignment of this codon is mediated by a novel serine-tRNA that acquired a leucine 5′-CAG-3′ anticodon (25).Here, we show that deviation from the standard use of the CUG leucine codon to encode serine was correlated with the transferase capacity and specificity of the Ffase enzyme. Indeed, the S196L substitution enhanced the transferase activity of the enzyme 3-fold. Several site-directed mutants were generated and characterized to study their transferase capacities. These results are considered on the basis of the enzymes'' three-dimensional structure, which enables a novel putative binding site of sucrose that serves as a water substitute donor in the hydrolytic reaction yielding the tranglycosylation product 6-kestose to be identified.     

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH₂ (RGE) and IVYYPDRGETGL-NH₂ (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED₅₀ 0.16% and LD₅₀ 0.09%), this being even more effective than the native protein.     

Mangrove dynamics in the Cispata lagoon system (Colombian Caribbean) during last 900 years

The lagoon complex of Cispatá (old Sinú river delta) located at the Northwestern coast of the Colombian Caribbean, encloses one of the biggest mangrove areas in this region. This area has changed during the last 330 years because of several environmental and climatic causes, mainly changes in the position of the delta (Sinú River), which is the main freshwater source in this area, and sea level rise. We hypothesized that the climatic and geomorphologic dynamics has caused changes in the extension and composition of mangrove vegetation, especially during last 150 years. The dynamics of mangroves during the last 900 years was reconstructed based on the changes in the stratigraphy, pollen record, calcite concentrations (CaCO3) and C/N ratio, along two sediment cores from La Flotante and Navio lagoons, located in Cispatá complex. The age model was built based on lineal interpolation of 210Pb ages and changes in granulometry. Establishment and expansion of mangrove forests during the last 900 years were related to fluviomarine dynamics in the area and the lagoon formation. During the period encompassed between 1064 and 1762 A.D., the Mestizos spit was formed when marine conditions predominated in the surroundings of La Flotante Lagoon. At the site of Navío, a river dominated lagoon, terrigenous conditions dominated since 1830. Although the colonization of herbaceous pioneer vegetation started between 1142 and 1331 A.D., mangrove colonization only took place since 1717 A.D. Mangrove colonization was a result of the delta progradation. In 1849 A.D. the Sinú river delta migrated to the Cispatá bay. The eustatic sea level rise, the increase in river discharges and sedimentation rates produced the establishment of mangrove forests dominated by Rhizophora since 1849. Since 1900 a marine intrusion was recorded in both lagoons. In 1938, the migration of the delta toward its actual location in Tinajones gave place to the formation of the present lagoon system and to the expansion of mangrove forests, which reflects the balance between the high alluvial sediment input and the current sea level rise as has been recorded in similar ecosystems.     

Molecular markers show a complex mosaic pattern of wheat-Thinopyrum intermedium translocations carrying resistance to YDV

Thinopyrum intermedium translocations derived from the wheat (Triticum aestivum L.) substitution line P-29 were previously characterized by RFLP. We have further analyzed these lines and additional related germplasm with publicly available STS and SSRs. Primers which showed a polymorphism between wheat and P-29, were tested in all recombinant and nulli-tetrasomic lines confirming their position on chromosome 7D. The resulting 7D/7E chromosome maps appeared as a mosaic of wheat and Th. intermedium chromatin sections. To verify the composition of the translocation lines suggested by the RFLP-PCR map, F₂ progeny of two crosses (CS/216-1 and CS/260-1) were analyzed with molecular markers. Both populations gave an unexpectedly diverse number of recombinant individuals, suggesting that interstitial translocations occur more frequently than previously thought. This analysis also showed that there is a wide range in the number and position of the interstitial translocations within a given line such as the mosaic chromosome in recombinant line 260-1/CS-26, which has four Th. intermedium chromosome segments. Phenotypic data of the two populations suggested the presence of one gene which we have called Bdv3 to differentiate it from the previously reported orthologous gene Bdv2. Using the PCR-based molecular markers identified in this study, 5 out of 12 elite lines that showed good yields and no YDV symptoms contained Th. intermedium chromatin. Due to the multiple components involved in the YDV disease complex, combining selection for YDV resistance with the molecular markers and maps identified in this study will increase the efficiency of introgressing Th. intermedium chromatin containing YDV resistance or other beneficial traits into elite wheat germplasm.     

Interplay of cysteinyl leukotrienes and TGF-β in the activation of hepatic stellate cells from Schistosoma mansoni granulomas

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC₄-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB_4, into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.