9-polylysine protein transduction domain: enhanced penetration efficiency of superoxide dismutase into mammalian cells and skin

Antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), have been considered to have a beneficial effect against various diseases that are mediated by the reactive oxygen species (ROS). Although a variety of modified recombinant antioxidant enzymes have been generated to protect against oxidative stresses, the lack of their transduction ability into cells resulted in a limited ability to detoxify intracellular ROS. To render the SOD enzyme capable of detoxifying intracellular ROS when added extracellularly, cell-permeable recombinant SOD proteins were generated. A human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) gene was fused with a gene fragment that encodes the 9 amino acids Tat protein transduction domain (RKKRRQRRR) of HIV-1 and lysine rich peptide (KKKKKKKKK) in a bacterial expression vector in order to produce a genetic in-frame Tat-SOD and 9Lys-SOD fusion protein, respectively. The expressed and purified Tat-SOD and 9Lys-SOD fusion proteins can transduce into human fibroblast cells, and they were enzymatically active and stable for 24 h. The cell viability of the fibroblast cells that were treated with paraquat, an intracellular superoxide anion generator, was increased by the transduced Tat-SOD or 9Lys-SOD. The transduction efficacy of 9Lys-SOD was more efficient than that of Tat-SOD. We evaluated the ability of the SOD fusion pmteins to transduce into animal skin. This analysis showed that Tat-SOD and 9Lys-SOD fusion proteins efficiently penetrated into the epidermis as well as the dermis of the subcutaneous layer, when sprayed on mice skin (judged by the immunohistochemistry and specific enzyme activities). The enzymatic activity of the transduced 9Lys-SOD was higher than that of Tat-SOD, indicating that the penetration of 9Lys-SOD was more efficient when put into the skin. These results suggest Tat-SOD and 9Lys-SOD fusion proteins can be used as anti-aging cosmetics, or in protein therapy, for various disorders that are related to this antioxidant enzyme and ROS.     

Postembryonic neurogenesis in the central nervous system of the tobacco hornworm,Manduca sexta. III. Spatial and temporal patterns of proliferation

Postembryonic neurogenesis leads to a dramatic increase in the number of functional neurons within the segmental ganglia of the moth, Manduca sexta. These adult-specific neurons are generated during larval life by segment-specific arrays of individually identifiable stem cells, or neuroblasts (Nbs). By the end of the feeding larval stage, each Nb has generated a discrete nest of progeny, which ranges in size from less than 10 to more than 70 progeny. The sizes of these identifiable nests of progeny vary in a segment-specific manner, with the thoracic nests containing a greater number of progeny compared with their homologues in the simpler abdominal ganglia. In order to describe those factors that influence the size of the postembryonic neuronal lineages, we examined the spatial and temporal pattern of postembryonic neurogenesis in the segmental ganglia of Manduca. The rates at which the identifiable nests accumulated progeny were estimated by counting the number of progeny within the nests, using sectioned material isolated from animals at stages ranging from embryonic hatching until the end of the feeding larval stage. All of the postembryonic Nbs began to generate progeny at around the time of the molt to the third larval instar. Each nest added progeny at a rate that was a characteristic of its identity and segment of origin. Although all of the nests within the thorax continued to accumulate progeny throughout the feeding larval stage, several of the abdominal nests showed little or no growth following the molt to the fifth larval instar. The thymidine analog 5-bromo 2-deoxyuridine (5-BrdU) was used to estimate the mitotic rates of the identifiable Nbs. The number of labeled progeny within a nest 24 h after application of 5-BrdU ranged from a low of 1 to 2 to a high of 11 to 13 labeled cells. In some instances there was a good correlation between the estimated mitotic rate of an identified Nb and the rate of growth of its associated nest of progeny. However, several of the identifiable nests accumulated progeny at a slower rate than predicted based on the estimated mitotic rate of the Nb. Cell death appears to be responsible for slowing the growth of the nests during the feeding larval stage. We estimate that 10% to 70% of the neurons generated during the feeding larval stage degenerate within 24 h of their birth. The level of cell death observed within a nest was dependent on both its identity and its segment of origin. © 1996 John Wiley & Sons, Inc.     

Dipeptidyl peptidase-4 inhibitor with β-amino amide scaffold: Synthesis, SAR and biological evaluation

Inhibitors of dipeptidyl peptidase-4 (DPP4) have been shown to be effective treatments for type 2 diabetes. Several series of β-amino amide containing piperazine derivatives have been prepared and evaluated as a inhibitor of DPP4. Finally compound 5m was selected for further evaluation.     

Molecular mechanism of macrophage activation by Exopolysaccharides from liquid culture of Lentinus edodes

Mushrooms are regarded as one of the well-known foods and biopharmaceutical materials with a great deal of interest. beta-Glucan is the major component of mushrooms that displays various biological activities such as antidiabetic, anticancer, and antihyperlipidemic effects. In this study, we explored the molecular mechanism of its immunostimulatory potency in immune responses of macrophages, using exopolysaccharides prepared from liquid culture of Lentinus edodes. We found that fraction II (F-II), with large molecular weight protein polysaccharides, is able to strongly upregulate the phenotypic functions of macrophages such as phagocytic uptake, ROS/NO production, cytokine expression, and morphological changes. F-II triggered the nuclear translocation of NF-kappaB and activated its upstream signaling cascades such as PI3K/ Akt and MAPK pathways, as assessed by their phosphorylation levels. The function-blocking antibodies to dectin-1 and TLR-2, but not CR3, markedly suppressed F-II-mediated NO production. Therefore, our data suggest that mushroom-derived beta-glucan may exert its immunostimulating potency via activation of multiple signaling pathways.     

Na/K-ATPase beta1-subunit associates with neuronal growth regulator 1 (NEGR1) to participate in intercellular interactions

Neuronal growth regulator 1 (NEGR1) is a GPI-anchored membrane protein that is involved in neural cell adhesion and communication. Multiple genome wide association studies have found that NEGR1 is a generic risk factor for multiple human diseases, including obesity, autism, and depression. Recently, we reported that Negr1^−/− mice showed a highly increased fat mass and affective behavior. In the present study, we identified Na/K-ATPase, beta1-subunit (ATP1B1) as an NEGR1 binding partner by yeast two-hybrid screening. NEGR1 and ATP1B1 were found to form a relatively stable complex in cells, at least partially co-localizing in membrane lipid rafts. We found that NEGR1 binds with ATP1B1 at its C-terminus, away from the binding site for the alpha subunit, and may contribute to intercellular interactions. Collectively, we report ATP1B1 as a novel NEGR1-interacting protein, which may help deciphering molecular networks underlying NEGR1-associated human diseases.     

Two species of goby, Boleophthalmus pectinirostris and Scartelaos sp., as the new second intermediate hosts of heterophyid fluke in Korea

A survey was performed in order to determine the infection status of the metacercariae of heterophyid fluke in two goby species, Boleophthalmus pectinirostris and Scartelaos sp., collected from Gangjin-gun, and Shinangun, Sooncheon-shi, Jeollanam-do, Republic of Korea. A total of three metacercariae of Heterophyopsis continua was found in only one B. pectinirostris (10.0%) from Gangjin-gun. Heterophyes nocens metacercariae were detected in 24 B. pectinirostris (96.0%) and 14 Scartelaos sp. (63.6%) from Shinan-gun. Heterophyopsis continua metacercariae were found in 11 B. pectinirostris (44.0%) and 21 Scartelaos sp. (95.5%) from Shinan-gun. Stictodora fuscata metacercariae were detected in 18 B. pectinirostris (72.0%) from Shinan-gun. No metacercariae were detected in 20 B. pectinirostris from Sooncheon-shi. From the above results, this study is the first to prove that B. pectinirostris and Scartelaos sp. serve as the second intermediate hosts of some heterophyid flukes in Korea.     

Rapid and simple purification of a novel extracellular β-amylase from Bacillus sp.

Bacillus sp. KYJ 963, a local isolate, produced an extracellular amylase with M _r=59 kDa. The amylase was easily purified by adsorption on soluble starch. The analyses of TLC and N-terminal amino acid sequence from the purified protein revealed that the enzyme was a novel -amylase which could not hydrolyze maltose or -cyclodextrin and its N-terminal amino acid sequence was A-V-N-G-Q-S-F-N-S-N-Y-K-T-Y-K-.     

Bioactive molecules from Nocardia: diversity,bioactivities and biosynthesis

Journal of Industrial Microbiology & Biotechnology - Nocardia spp. are catalase positive, aerobic, and non-motile Gram-positive filamentous bacteria. Many Nocarida spp. have been reported as...