Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene

The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His(6) secreted by S. cerevisiae migrated as a broad diffuse band on SDS-PAGE, with an apparent molecular weight higher than that in natural A. niger GOD. To investigate the effects of hyperglycosylation on the secretion efficiency and enzyme properties, GOD-His(6) was expressed and secreted in a S. cerevisiae mutant in which the PMR1 gene encoding Ca(++)-ATPase was disrupted. The pmr1 null mutant strain secreted an amount of GOD-His(6) per unit cell mass higher than that in the wild-type strain. In contrast to the hyperglycosylated GOD-His(6) secreted in the wild-type strain, the pmr1 mutant strain secreted GOD-His(6) in a homogeneous form with a protein band pattern similar to that in natural A. niger GOD, based on SDS-PAGE. The hyperglycosylated and pmr1Delta mutant-derived GOD-His(6) enzymes were purified to homogeneity by immobilized metal ion-affinity chromatography and their specific activities and stabilities were compared. The specific activity of the pmr1Delta mutant-derived GOD-His(6) on a protein basis was very similar to that of the hyperglycosylated GOD-His(6), although its pH and thermal stabilities were lower than those of the hyperglycosylated GOD-His(6).     

Rehmannia glutinosa ameliorates scopolamine-induced learning and memory impairment in rats

Many studies have shown that the steamed root of Rehmannia glutinosa (SRG), which is widely used in the treatment of various neurodegenerative diseases in the context of Korean traditional medicine, is effective for improving cognitive and memory impairments. The purpose of this study was to examine whether SRG extracts improved memory defects caused by administering scopolamine (SCO) into the brains of rats. The effects of SRG on the acetylcholinergic system and proinflammatory cytokines in the hippocampus were also investigated. Male rats were administered daily doses of SRG (50, 100, and 200 mg/kg, i.p.) for 14 days, 1 h before scopolamine injection (2 mg/kg, i.p.). After inducing cognitive impairment via scopolamine administration, we conducted a passive avoidance test (PAT) and the Morris water maze (MWM) test as behavioral assessments. Changes in cholinergic system reactivity were also examined by measuring the immunoreactive neurons of choline acetyltransferase (ChAT) and the reactivity of acetylcholinesterase (AchE) in the hippocampus. Daily administration of SRG improved memory impairment according to the PAT, and reduced the escape latency for finding the platform in the MWM. The administration of SRG consistently significantly alleviated memory-associated decreases in cholinergic immunoreactivity and decreased interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mRNA expression in the hippocampus. The results demonstrated that SRG had a significant neuroprotective effect against the neuronal impairment and memory dysfunction caused by scopolamine in rats. These results suggest that SRG may be useful for improving cognitive functioning by stimulating cholinergic enzyme activities and alleviating inflammatory responses.     

A hybrid peptide derived from cecropin-A and magainin-2 inhibits osteoclast differentiation

The adult skeleton is in a dynamic state, being continually broken down and reformed by the coordinated actions of osteoclasts and osteoblasts. Increased osteoclast activity may contribute to the development of osteoporosis. Therefore, the intervention of osteoclast-mediated bone resorption is considered as an effective therapeutic approach in the treatment of osteoporosis. In the course of searching for agents that inhibit osteoclast differentiation and activation, we found that a novel hybrid peptide P1 derived from cecropin-A and magainin-2 reduced osteoclast differentiation in various osteoclast culture systems. As this peptide had no cytotoxicity on various cultures of primary cells and established cell lines, its inhibitory effect on osteoclastogenesis was not due to general cytotoxicity. The effects of P1 on osteoclasts appear to be mediated through the inhibition of NF-kappaB and JNK activation induced by the osteoclastogenic cytokine, receptor activator of NF-kappaB ligand (RANKL). These results provide an evidence for the potential usefulness of P1 for the treatment of bone-resorbing diseases.     

C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria

PMAP-23 is a member of the cathelicidin family derived from pig myeloid cells and has potent antimicrobial activity. Amidation of the carboxyl terminus (C-terminus) of an antimicrobial peptide generally enhances its structural stability and antimicrobial activity or decreases its cytotoxicity. The aim of the present study was to investigate the effect of amidation on the mode of action in PMAP-23. Irrespective of amidation, PMAP-23 adopts a helix–hinge–helix structure in a membrane-mimetic environment. The antibacterial activities of PMAP-23C, which had a free C-terminus, and PMAP-23N, which had an amidated C-terminus, were similar against Gram-negative bacteria, reflecting a similar ability to neutralize lipopolysaccharide. However, PMAP-23N assumed a perpendicular orientation across the outer to the inner leaflet of the bacterial inner membrane, while PMAP-23C was orientated parallel to the lipid bilayer, as determined by following the blue shift in tryptophan fluorescence, as well as calcein release from liposomes and SYTOX Green uptake assays. These results suggest that N-terminal amidation of PMAP-23 provides structural stability and increases the peptide’s cationic charge, facilitating translocation into the bacterial inner membrane.     

Processing of Ada protein by two serine endoproteases Do and So from Escherichia coli

Two soluble serine proteases Do and So from Escherichia coli were found to distinctively cleave the purified, 39 kDa Ada protein into fragments with sizes of 12-31 kDa. Protease So appears to generate a C-terminal 19 kDa polypeptide, similarly to OmpT protease. In addition, the purified 19 kDa C-terminal half of Ada protein can be further processed mainly to an 18 kDa fragment by protease So and to a 12 kDa by protease Do. These results suggest that proteases Do and So are involved in endogenous cleavage of Ada protein, which may play a role in down-regulating the adaptive response to alkylating agents.     

Structural and functional characterization of osmotically inducible protein C (OsmC) from Thermococcus kodakaraensis KOD1

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T. kodakaraensis KOD1 was cloned and expressed in Escherichia coli. TkOsmC showed a homotetrameric structure based on gel filtration and electron microscopic analyses. TkOsmC has a significant peroxidase activity toward both organic and inorganic peroxides in high, but not in low temperature.     

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.