An over expression APP model for anti-Alzheimer disease drug screening created by zinc finger nuclease technology.

Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid ß (Aß) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aß abundance. What is more, the activity of the three key secretases in Aß formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer's disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.

Pedobacter ginsengisoli sp. nov., a DNase-producing bacterium isolated from soil of a ginseng field in South Korea.

A Gram-negative, strictly aerobic, rod-shaped, non-motile, non-spore-forming bacterial strain, designated Gsoil 104T, was isolated from a soil sample from a ginseng field in Pocheon Province (South Korea) and was characterized taxonomically by using a polyphasic approach. On the basis of 16S rRNA gene sequence similarities, strain Gsoil 104T was shown to belong to the family Sphingobacteriaceae, being related to Pedobacter africanus DSM 12126T (97.0%), Pedobacter caeni LMG 22862T (96.9%), Pedobacter cryoconitis DSM 14825T (96.8%) and Pedobacter heparinus DSM 2366T (96.6%). The phylogenetic distance from any other Pedobacter species with a validly published name was greater than 3.4% (i.e.<96.6% 16S rRNA gene sequence similarity). DNA-DNA hybridization experiments showed that values for DNA-DNA relatedness between strain Gsoil 104T and its phylogenetically closest neighbours were below 37%. The G+C content of the genomic DNA was 43.6 mol%. The predominant respiratory quinone was MK-7. The major fatty acids were C16:1omega7c, iso-C15:0, C16:0, iso-C17:0 3-OH and iso-C15:0 2-OH. These chemotaxonomic data support the affiliation of strain Gsoil 104T to the genus Pedobacter. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 104T represents a novel species in the genus Pedobacter, for which the name Pedobacter ginsengisoli sp. nov. is proposed. The type strain is Gsoil 104T (=KCTC 12576T=LMG 23399T).

The effect of Nepeta cataria extract on adherence and enzyme production of Staphylococcus aureus.

Nepeta cataria L., commonly known as catnip, is a perennial herb with a considerable folkloric reputation. A diethyl ether extract of this plant has been shown to have antimicrobial activity against fungi and Gram-positive bacteria. The aim of this work was to study the activity of N. cataria extract on 44 Staphylococcus aureus strains, some resistant to methicillin, and S. aureus 6538P (American Type Culture Collection) by evaluating the effect of subminimum inhibitory concentrations on coagulase, DNAse, thermonuclease and lipase production, and on in-vitro adherence. DNAse, thermonuclease and lipase were inhibited by concentrations equal to 1/2 and 1/4 MIC. A reduction of adherence was also observed.

Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus.

The exact molecular mechanism of ischemic neuronal death still remains unclear from rodents to primates. A number of studies using lower species animals have suggested implication of apoptosis cascade, while using monkeys the authors recently claimed necrosis cascade by calpain-induced leakage of lysosomal cathepsins (calpain-cathepsin hypothesis). This paper is to study implications of apoptotic versus necrotic cascades for the development of hippocampal CA1 neuronal death in the primate brain undergoing complete global ischemia. Here, we focused on two terminal cell death effectors; caspase-activated DNase (CAD) and lysosomal enzyme DNase II, in the monkey CA1 sector undergoing 18 min ischemia. The expressions of their mRNA and proteins, and the subcellular localizations as well as ultrastructure and specific DNA gel electrophoresis were examined. Expression of CAD was much less in the normal brain, compared with the lymph node or heart tissues. On day 1 after ischemia, however, CAD mRNA and protein were significantly increased in the CA1 sector, and then CAD protein immunohistochemically showed a translocation from the perikarya into the nucleus. Activated DNase II protein was significantly increased on days 2 and 3 after ischemia, and also showed a similar translocation indicating lysosomal leakage. Although the post-ischemic CA1 neurons showed positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining on days 3-5, they showed eosinophilic coagulation necrosis on light microscopy, and frank membrane disruption and mild chromatin condensation on electron microscopy. Furthermore, DNA smear pattern typical for necrosis was observed instead of DNA laddering. These data altogether suggest that the post-ischemic CA1 neuronal death of the monkey occurs not by apoptosis but by necrosis with participations of lysosomal enzymes DNase II and cathepsins as well as CAD. The interactions between apoptotic (caspase-3 and CAD) and necrotic (calpain, cathepsin and DNase II) cascades should be studied further.

Biochemical properties and hormonal regulation of barley nuclease.

The amino acid composition and NH2-terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3'-nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2' and 5'-monophosphates, and deoxyribonucleoside 3' and 5'-monophosphates, and is also inactive towards the 3'-phosphoester linkage of nucleoside cyclic 2',3' and 3',5'-monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5' residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidylic acid greater than polyuridylic acid greater than polyadenylic acid much greater than polyguanylic acid. The enzyme also has preference for single-stranded nucleic acids. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5'-phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P.H. and Ho, T.-h. D. (1986) Plant Physiol. 82, 801-806]. Consistent with these results, gibberellic acid induces up to an eight-fold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine-linked, high-mannose oligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa.

Characterization of Campylobacter jejuni/coli-isolates from human faeces.

Campylobacter jejuni/coli strains were isolated from the faeces of 240 patients suffering from acute enteritis. The following characteristics were investigated: (i) growth at different temperatures, and on different substrates under either microaerophilic conditions or anaerobically, with fumarate or nitrate as terminal electron acceptors; (ii) production of H2S in cysteine-containing broth; (iii) hydrolysis of hippuric acid; (iv) DNase; (v) alkaline phosphatase; (vi) beta-lactamase; (vii) presence of menaquinone; and (viii) reduction of selenite. Based on characteristics (ii)-(v), the strains could be divided in 9 phenotypical groups. Most of the strains represented group 2 (DNase+, H2S+, hippurate hydrolysis+, alk. phosphatase-) (32%), and groups 8 (DNase-, H2S+, hippurate hydrolysis+, alk. phosphatase-) (32%). The other groups were of minor importance. On the other hand, most of the isolates from the United States (Weaver, 1981) fitted well into group 1 (DNase+, H2S+, hippurate hydrolysis+, alk. phosphatase+) which might demonstrate geographical variations among C. jejuni/coli.