Characterization of Bacillus probiotics available for human use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

A complete library of amino acid alterations at N304 in Streptomyces clavuligerus deacetoxycephalosporin C synthase elucidates the basis for enhanced penicillin analogue conversion

N304 of Streptomyces clavuligerus deacetoxycephalosporin C synthase was mutagenized to alter its catalytic ability. Given that N304A, N304K, N304L, and N304R mutant enzymes exhibited significant improvements in penicillin analogue conversions, we advocate that replacement of N304 with residues with aliphatic or basic side chains is preferable for engineering of a hypercatalytic enzyme.     

Solid-Phase Artificial Chaperone-Assisted Refolding using Insoluble β-Cyclodextrin–Acrylamide Copolymer Beads

Solid-phase refolding methods are advantageous since they facilitate both separation of solid additives from the refolded protein and recycling of the additives. -Cyclodextrin–acrylamide copolymer hydrogel beads were used as a matrix for detergents in solid-phase artificial chaperone-assisted refolding and improved the yield of lysozyme (up to 65%) and carbonic anhydrase B (up to 80%), compared with conventional solid host matrices.Revisions received 29 September 2004     

Purification of a fibrinolytic enzyme (myulchikinase) from pickled anchovy and its cytotoxicity to the tumor cell lines

A fibrinolytic enzyme, myulchikinase, from a Korean seasoning ingredient, myul-chi-jeot-gal, has been purified to electrophoretic homogeneity. The molecular mass of the myulchikinase was estimated to about 28 kDa by SDS-PAGE and gel filtration. Amino acid sequence of the NH2-terminal of myulchikinase showed significant homology with other fibrinolytic enzymes including trypsin from starfish, katsuwokinase, and rat pancreatic elastase II. The purified myulchikinase hydrolyzed various synthetic substrates with different substrate specificity and cytotoxic to the tumor cell lines.     

Selective extraction of acetic acid from the fermentation broth produced by Mannheimia succiniciproducens

Acetic acid is by-product from fermentation processes for producing succinic acid using Mannheimia succiniciproducens . To obtain pure succinic acid from the final fermentation broth, acetic acid was selectively removed based on the different extractability of succinic acid and acetic acid with pH using tri-n-octylamine (TOA) as extractant. When successive batch extractions were performed using 0.25 mol TOA kg(-1) dissolved in 1-octanol at pH 5, the mol ratio of succinic acid to acetic acid before extraction was 4.9 and the final ratio after the fourth batch was 9.4.     

Effect of Eu3+ on characterization of photosystem II particles from spinach by spectroscopy

Photosystem II (PSII) particles were purified from Eu³⁺-treated spinach and studied by spectroscopy. The results showed that electron transport rate of PS II was accelerated by Eu³⁺ treating, that violet shift of the PSII Soret band or Q-band was 6 nm or 2 nm for the ultraviolet-visible (UV-Vis) spectrum, that the violet shift of the PSII fluorescence emission peak was 9 nm for fluorescence emission spectrum, that the PSII Signal II’s of low-temperature electron paramagnetic resonance (EPR) spectrum was intensified under light, and that the PSII CD spectrum was similar to that of control. It is suggested that Eu³⁺ might bind to the PSII reaction center complex and enhance the electron transport rate of PSII CD; however, Eu³⁺ treatment does not change the configuration of the PSII reaction center complex.     

Members of a New Group of Chitinase-Like Genes are Expressed Preferentially in Cotton Cells with Secondary Walls

Two homologous cotton (Gossypium hirsutum L.) genes, GhCTL1 and GhCTL2, encode members of a new group of chitinase-like proteins (called the GhCTL group) that includes other proteins from two cotton species, Arabidopsis, rice, and pea. Members of the GhCTL group are assigned to family GH19 glycoside hydrolases along with numerous authentic chitinases (http://afmb.cnrs-mrs.fr/CAZY/index.html), but the proteins have novel consensus sequences in two regions that are essential for chitinase activity and that were previously thought to be conserved. Maximum parsimony phylogenetic analyses, as well as Neighbor-Joining distance analyses, of numerous chitinases confirmed that the GhCTL group is distinct. A molecular model of GhCTL2 (based on the three-dimensional structure of a barley chitinase) had changes in the catalytic site that are likely to abolish catalytic activity while retaining potential to bind chitin oligosaccharides. RNA blot analysis showed that members of the GhCTL group had preferential expression during secondary wall deposition in cotton lint fiber. Cotton transformed with a fusion of the GhCTL2 promoter to the beta -d-glucuronidase gene showed preferential reporter gene activity in numerous cells during secondary wall deposition. Together with evidence from other researchers that mutants in an Arabidopsis gene within the GhCTL group are cellulose-deficient with phenotypes indicative of altered primary cell walls, these data suggest that members of the GhCTL group of chitinase-like proteins are essential for cellulose synthesis in primary and secondary cell walls. However, the mechanism by which they act is more likely to involve binding of chitin oligosaccharides than catalysis.     

Characterization of <Emphasis Type="Italic">HoMADS 1</Emphasis> and its induction by plant hormones during <Emphasis Type="Italic">in vitro</Emphasis> ovule development in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

To understand the molecular mechanism of ovule development, a MADS box gene,HoMADS 1, has been isolated from the ovule tissues of Hyacinthus. Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated.