Expression and characterization of polyketide synthase module involved in the late step of cephabacin biosynthesis from Lysobacter lactamgenus

The cephabacins produced by Lysobacter lactamgenus are beta-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, beta-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of His6-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-LAla- L-3H-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/ PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with 14C-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.     

Biosynthesis of 2-amino-3-hydroxycyclopent-2-enone moiety of bafilomycin in <Emphasis Type="Italic">Kitasatospora cheerisanensis</Emphasis> KCTC2395

Bafilomycins produced by Kitasatospora cheerisanensis KCTC- 2395 belong to the 16-membered macrolactone family plecomacrolide antibiotics. Bafilomycin B₁ contains 2-amino- 3-hydroxycyclopent-2-enone (C₅N), a five membered ring, which gets condensed via an amide linkage to bafilomycin polyketide. To study the biosynthetic pathway of C₅N during bafilomycin biosynthesis in K. cheerisanensis KCTC2395, we attempted the functional analysis of two putative genes, encoding 5-aminolevulinic acid synthase (ALAS) and acyl- CoA ligase (ACL). The amplified putative genes for ALAS and ACL were cloned into the E. coli expression vector pET- 32a(+) plasmid, following which the soluble recombinant ALAS and ACL proteins were purified through nickel-affinity column chromatography. Through HPLC analysis of the enzyme reaction mixture, we confirmed the products of putative ALAS and ACL reaction as 5-aminolevulinic acid (5-ALA) and 5-ALA-CoA, respectively. The optimal pH for the putative ALAS reaction was 7.5, and for putative ACL reaction was 7.0, as confirmed by the colorimetric assay. Furthermore, pyridoxal 5'-phosphate (PLP) was found to be an essential cofactor in the putative ALAS reaction, and ATP was a cofactor for the putative ACL catalysis. Finally, we also confirmed that the simultaneous treatment of putative ACL and putative ALAS enzymes resulted in the production of C5N compound from 5-ALA.     

The expression profile and function of Satb2 in zebrafish embryonic development

The present study shows the expression profile and function of the homeobox gene, satb2 during zebrafish embryonic development. Satb2 was ubiquitously expressed from the 1 cell stage to the 10-somite stage in zebrafish embryos. Satb2 showed stage-specific expression profiles such as in the pronephric duct at 24 hpf, the branchial arches at 36 hpf, and the ganglion cell layer of the retina and fins at 48 hpf. Additionally, satb2 knockdown embryos were arrested at 50–60% epiboly, and transplantation experiments with satb2 knockdown cells showed migration defects. Interestingly, satb2 knockdown cells also exhibited down-regulation of dynamin II and VAMP4, which are involved in exocytosis and endocytosis, respectively. Furthermore, satb2 knockdown cells have a disorganized actin distribution and an underdeveloped external yolk syncytial layer, both of which are involved in epiboly. These results suggest that satb2 has a functional role in epiboly. This role may potentially be the regulation of endo-exocytic vesicle transport-dependent cell migration and/or the regulation of the development of the yolk syncytial layer.     

Metabolic Alterations in Two Cirsium Species Identified at Distinct Phenological Stages using UPLC‐QTOF/MS

Introduction

Cirsium chanroenicum and C. setidens are commonly used both in traditional folk medicine and as a food source. The quality of different species of Cirsium at different harvest times is a function of their metabolite composition, which is determined by the phenological stage.

Objective

We sought to determine the differences in the metabolite composition of two species of Cirsium during different phenological stages using ultra‐performance liquid chromatography (UPLC) quadrupole time‐of‐flight (QTOF) mass spectrometry (MS).

Methodology

Cirsium chanroenicum and C. setidens plants were collected at the floral budding and full flowering stages. Metabolic profiles of Cirsium extracts were determined using UPLC‐QTOF/MS to characterise the differences between phenological stages, and the major metabolites were quantified using UPLC‐QTOF/MS‐multiple reaction monitoring (MRM).

Results

At the full flowering stage, the levels of phenolic acids as well as components of the phenylpropanoid pathway were increased. Flavonoids predominated at the full flowering stage in both species. The levels of coumaric acid, kaempferol, and pectolinarigenin differed between the two species of Cirsium. Overall, these results suggest that components of the phenylpropanoid metabolic pathway are upregulated in the full flowering stage in Cirsium, although we did observe some variation between the species.

Conclusion

These results will help elucidate the metabolic pathways related to the different phases of the vegetative cycle, and may help determine the optimal season for the harvest of Cirsium with the highest levels of bioactive compounds. Copyright © 2017 John Wiley & Sons, Ltd.     

Subcellular localization of presenilins during mouse preimplantation development.

The genes defective in familial Alzheimer's disease encode the proteins presenilin 1 and 2 (PS1 and 2). Expression of presenilins (PSs) and their proteolytic processing are regulated during neuronal development. Even though these proteins are detected and regulated mainly in Golgi and endoplasmic reticulum, their subcellular distribution during the development is not known. The present study aimed to investigate the localization of PSs and their role during early developmental stage using mouse embryo model. At preimplantation stage, PSs were detected not only in cytoplasm, but also in the nucleus from oocyte to 2.5 dpc (day postcoitum), then disappeared in the nucleus at blastocyst stage (3.5 dpc). Antisense against PS1 and PS2 decreased the transition to blastocyst stage, whereas each antisense alone had no effect. Treatment with lactacystin (26S proteosome inhibitor), which arrest cell cycle at M phase, redistributed PSs into centrosome-kinetochore microtubule. PS2 overexpression in HEK 293 cell arrested cell cycle at S phase. These data suggest that PSs play key roles in cell division and differentiation during early development.     

Postpartum changes in body composition

Parity is associated with weight retention and has long-lasting and detrimental effects on the health of women. Previous studies have shown that increasing parity was independently associated with an increased prevalence of metabolic syndrome. Postpartum weight is made up of several components including uterine and mammary tissues, body water (intracellular (ICW) and extracellular water (ECW)), and fat. These components change in variable amounts postpartum, thereby distinctly affecting the interpretation of individual weight retention; however, it is unclear which components contribute to weight retention. The aims of this longitudinal study were to evaluate changes in body composition during the postpartum period and to investigate their effects on weight retention. This prospective study examined 41 healthy, pregnant women who gave birth at Korea University Guro Hospital. We measured body composition at 2 days, 2 weeks, and 6 weeks postpartum using bioelectrical impedance analysis. Weight decreased during this postpartum period (P < 0.001); the postpartum weight retention from prepregnancy to 6 weeks postpartum was 4.43 ± 4.0 kg. Among various body composition components, ECW, ICW, total body water, and fat-free mass (FFM) decreased postpartum. However, fat mass (FM) and visceral fat area, the components that experienced the greatest changes, increased postpartum. Our results demonstrate that the postpartum period is associated with a preferential accumulation of adipose tissue in the visceral compartment, even though overall body weight is decreased. Further studies are needed to evaluate the changes in body composition over longer time periods and their long-term effects on health.     

Random BAC FISH of monocot plants reveals differential distribution of repetitive DNA elements in small and large chromosome species

BAC FISH (fluorescence in situ hybridization using bacterial artificial chromosome probes) is a useful cytogenetic technique for physical mapping, chromosome marker screening, and comparative genomics. As a large genomic fragment with repetitive sequences is inserted in each BAC clone, random BAC FISH without adding competitive DNA can unveil complex chromosome organization of the repetitive elements in plants. Here we performed the comparative analysis of the random BAC FISH in monocot plants including species having small chromosomes (rice and asparagus) and those having large chromosomes (hexaploid wheat, onion, and spider lily) in order to understand a whole view of the repetitive element organization in Poales and Asparagales monocots. More unique and less dense dispersed signals of BAC FISH were observed in species with smaller chromosomes in both the Poales and Asparagales species. In the case of large-chromosome species, 75-85% of the BAC clones were detected as dispersed repetitive FISH signals along entire chromosomes. The BAC FISH of Lycoris did not even show localized repetitive patterns (e.g., centromeric localization) of signals.