Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis

Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.     

Modulation of cytokeratin expression during in vitro cultivation of human hepatic stellate cells: evidence of transdifferentiation from epithelial to mesenchymal phenotype

The embryonal origin of hepatic stellate cells (HSCs), the principal cells in hepatic fibrogenesis, is still intriguing. To explore the origin and the differentiation of HSCs, we studied the expression of cytokeratin 18 (CK18) and 19 (CK19), the standard markers of simple epithelial cells, in cultured human HSCs. Hepatic stellate cells were isolated from five normal human livers. In immunofluorescence staining, both clone C-51 anti-CK18 antibody and clone RCK108 anti-CK19 antibody labeled almost all stellate cells in the primary culture. Double immunofluorescence staining for cytokeratin/vimentin and cytokeratin/alpha-smooth muscle actin detected by confocal laser scanning microscopy clearly demonstrated the localization of cytokeratin immunoreactivity in human HSCs. During subsequent cultivation of human HSCs to the tenth passage, immunocytochemical staining and western blot analysis demonstrated gradually decreasing profiles of CK18 and CK19 expression. The progressive reduction of cytokeratin expression was further confirmed in a culture of clone cells originated from a single HSC. In conclusion, both CK18 and CK19 are expressed in cultured human HSCs, and the extent of their expression decreases gradually during prolonged cultivation. Our results suggest that HSCs may be of epithelial origin, and that they undergo the transdifferentiation from epithelial to mesenchymal phenotype during an activation process in vitro.     

Inside Back Cover: Development of a 3‐dimensional tissue lung phantom of a preterm infant for optical measurements of oxygen—Laser‐detector position considerations (J. Biophotonics 3/2018)

The picture depicts the different 3d‐printed organs, thorax, lungs, heart and bone. Assembled it is used as an optical phantom of a preterm infant for performing percutaneous optical measurements of the gas content in the lungs. In order to simulate the optical properties of the tissue, the heart and thorax can be filled with liquid phantoms, a mixture of Intralipid and Indian Ink. Further details can be found in the article by Jim Larsson et al. ( e201700097 ).

     

Stable nitrogen isotope analysis of dentine serial sections elucidate sex differences in weaning patterns of wild chimpanzees (Pan troglodytes)

Offspring provisioning is one of the most energetically demanding aspects of reproduction for female mammals. Variation in lactation length and weaning strategies between chimpanzees (Pan troglodytes), our closest living relative, and modern human societies have been reported. When and why these changes occurred is frequently debated. Our study used stable nitrogen isotope data of tooth root dentine from wild Western chimpanzees (Pan troglodytes verus) in Taï National Park, Côte d'Ivoire, to quantify weaning in these chimpanzees and explore if infant sex plays a role in maternal investment. We analyzed serial sections of deciduous lateral incisor root dentine from four Taï chimpanzees to establish the δ¹⁵N signal of nursing infants; we then analyzed serial sections of first permanent mandibular molar root dentine from 12 Taï chimpanzees to provide quantitative δ¹⁵N data on weaning in this population. Up to 2 years of age both sexes exhibited dentine δ¹⁵N values ≈2–3‰ higher than adult female Taï chimpanzees, consistent with a nursing signal. Thereafter a steady decrease in δ¹⁵N values consistent with the onset, and progression, of weaning, was visible. Sex differences were also evident, where male δ¹⁵N values decreased at a significantly slower rate compared to females. Confirmation of sex differences in maternal investment among Taï chimpanzees, demonstrates the viability of using isotope analysis to investigate weaning in non‐human primates. Additionally, assuming that behaviors observed in the Taï chimpanzees are illustrative of the ancestral pattern, our results provide a platform to enable the trajectory of weaning in human evolution to be further explored. Am J Phys Anthropol 153:635–642, 2014. © 2013 Wiley Periodicals, Inc.     

Overproduction of AgfA subunit of Salmonella enteritidis as a MBP-fusion protein in E. coli

To study the characteristics of recombinant thin aggregative fimbriae of salmonella and to develop a vaccine for salmonella infections, the AgfA subunit gene was amplified from Salmonella entiritidis using PCR. Maltose binding protein (MBP)-AgfA fusion protein was over-produced in E. coli and purified. Antibody against MBP-AgfA was prepared and its immunogenicity was studied.     

Complementary non-radioactive assays for investigation of human flap endonuclease 1 activity

FEN1, a key participant in DNA replication and repair, is the major human flap endonuclease that recognizes and cleaves flap DNA structures. Deficiencies in FEN1 function or deletion of the fen1 gene have profound biological effects, including the suppression of repair of DNA damage incurred from the action of various genotoxic agents. Given the importance of FEN1 in resolving abnormal DNA structures, inhibitors of the enzyme carry a potential as enhancers of DNA-interactive anticancer drugs. To facilitate the studies of FEN1 activity and the search for novel inhibitors, we developed a pair of complementary-readout homogeneous assays utilizing fluorogenic donor/quencher and AlphaScreen chemiluminescence strategies. A previously reported FEN1 inhibitor 3-hydroxy-5-methyl-1-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione displayed equal potency in the new assays, in agreement with its published IC₅₀. The assays were optimized to a low 4 µl volume and used to investigate a set of small molecules, leading to the identification of previously-unreported FEN1 inhibitors, among which aurintricarboxylic acid and NSC-13755 (an arylstibonic derivative) displayed submicromolar potency (average IC₅₀ of 0.59 and 0.93 µM, respectively). The availability of these simple complementary assays obviates the need for undesirable radiotracer-based assays and should facilitate efforts to develop novel inhibitors for this key biological target.     

Infection with the fungal endophyte Epichloë festucae may alter the allelopathic potential of red fescue

Red fescue (Festuca rubra) is a perennial grass used as both forage and turfgrass. Asymptomatic plants of this species are systemically infected by the fungal endophyte Epichloë festucae, which has a beneficial effect on the infected plants. The aim of this study was to determine the effect of the endophyte Epichloë festucae on the allelopathic potential of F. rubra against four associated pasture species that are also considered as weeds in lawns, Trifolium pratense, Trifolium repens, Lotus corniculatus and Plantago lanceolata. Two experiments were designed to evaluate the allelopathic effect of extracts from the roots and leaves of endophyte‐infected (E+) and non‐infected (E?) plants on the germination and seedling growth of the four target species. Regardless of the endophyte status of the host plant, leaf extracts elicited a stronger reduction in germination and seedling growth than root extracts. Extracts from E+ plants reduced the speed of germination index of Trifolium spp. to a greater extent than those from E? plants. Radicle length of the target species was the parameter most affected by the presence of the endophyte in F. rubra. Root extracts from E+ plants had a greater inhibitory effect on the radicle growth of the target species than did root extracts from E? plants. A greater concentration in total phenolic compounds was found in the roots of E+ plants than of E?; however, this difference was not observed in the leaves. Thus, the allelopathic potential of F. rubra is altered in infected plants.     

Further Daphniphyllum Alkaloids with Insecticidal Activity from the Bark of Daphniphyllum macropodum Miq.

Two new Daphniphyllum alkaloids, macropodumines J and K ( 1 and 2 , resp.), together with six known structurally related alkaloids, 3 – 8 , were isolated from the bark of Daphniphyllum macropodum Miq . The structures of the new compounds 1 and 2 were elucidated on the basis of a comprehensive analysis of their spectroscopic and chemical data. Macropodumine J ( 1 ) contains a CN group which is relatively rare in naturally occurring alkaloids. All isolated compounds were tested for their insecticidal activities against a number of insect species. Daphtenidine C ( 5 ) is the most active compound against Plutella xylostella. This is the first report of insecticidal properties of Daphniphyllum alkaloids.