Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion

The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the k_cat/K_m ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a k_cat/K_m ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.     

Bioactive new withanolides from the cultured soft coral Sinularia brassica

Continuing study of the ethyl acetate (EtOAc) extract of the cultured soft coral Sinularia brassica afforded five new withanolides, sinubrasolides H–L (1–5). The structures of the new compounds were elucidated on the basis of spectroscopic analysis. The cytotoxicities of new compounds 1–5 and a known compound sinubrasolide A (6) against the proliferation of a limited panel of cancer cell lines were assayed. The anti-inflammatory activities of compounds 1–6 were evaluated by measuring their ability to suppress N-formyl-methionyl-leucyl-phenyl-alanine/cytochalasin B (fMLP/CB)-induced superoxide anion generation and elastase release in human neutrophils.     

Anti-oxidant activity and effect of Pinus morrisonicola Hay. on the survival of leukemia cell line U937

The free radical scavenging and anti-cancer activites of Pinus morrisonicola Hay. were studied using different parts of the pine, namely, needle, bark and cone. Results showed that pine needle water extract has the highest scavenging superoxide anion activity and the lowest IC50 value in inhibiting superoxide anion formation; however, the bark water extract showed the best anti-lipid peroxidation activity. Additionally, needle water extract displayed the highest inhibition of leukemia cell line U937 growth. The results indicated that P. morrisonicola Hay. possesses potential chemopreventative and therapeutic properties.     

Differential mass spectrometry of rat plasma reveals proteins that are responsive to 17beta-estradiol and a selective estrogen receptor modulator PPT

Estrogens are a class of steroid hormones that interact with two related but distinct nuclear receptors, estrogen receptor (ER) alpha and beta. To identify potential ER biomarkers, we profiled the rat plasma glycoproteome after treatment with vehicle or 17beta-estradiol (E2) or an ERalpha-selective agonist PPT by differential mass spectrometry. Our comparative proteomic experiment identifies novel E2- and PPT-responsive proteins, such as serine protease inhibitor family members.     

Immunoglobulin G galactosylation and sialylation are associated with pregnancy-induced improvement of rheumatoid arthritis and the postpartum flare: results from a large prospective cohort study

Introduction  

Improvement of rheumatoid arthritis (RA) during pregnancy has been causatively associated with increased galactosylation of immunoglobulin G (IgG) N-glycans. Since previous studies were small, did not include the postpartum flare and did not study sialylation, these issues were addressed in the present study.     

Multispectral fingerprinting for improved in vivo cell dynamics analysis

Background  

Tracing cell dynamics in the embryo becomes tremendously difficult when cell trajectories cross in space and time and tissue density obscure individual cell borders. Here, we used the chick neural crest (NC) as a model to test multicolor cell labeling and multispectral confocal imaging strategies to overcome these roadblocks.     

Variable Recombination Efficiency in Responder Transgenes Activated by Cre Recombinase in the Vasculature

Cre recombinase has become a ubiquitous tool in transgenic strategies for regulation of transgene expression in a tissue-specific manner. We report analysis of two SM22αCre lines and their ability to mediate genomic recombination in five independent Cre-responsive transgenic lines. One of the SM22αCre lines developed was a tet-on system based on the reverse tetracycline transactivator. Our goal was to use this strategy to inhibit the Notch signaling pathway specifically in smooth muscle cells. Our responder transgenes contained a constitutively expressed marker gene (chloramphenicol acetyltransferase, CAT), flanked by loxP sites in direct orientation, upstream of Notch-related transgenes. We developed two dominant negative Notch transgenic responder lines activated by Cre-mediated DNA recombination. The first is the extracellular domain of human Jagged1, and the second is the extracellular domain of the human Notch2 receptor. Despite high expression of the marker gene in all responder lines, we found that Cre-mediated genomic recombination between these five lines was highly variable, ranging from 46 to 93% of individuals using an SM22αCre activating strain, or 8–58% of individuals using an inducible SM22αrtTACre. In all cases examined, detection of recombination by PCR correlated with expression of the transgene as determined by Western blot analysis. Our studies reflect the variability in recombination success based on the responder strain, presumably due to inaccessibility of the locus of integration of the responder allele.