Activation of defense responses in Chinese cabbage by a nonhost pathogen, Pseudomonas syringae pv. tomato

Pseudomonas syringae pv. tomato (Pst) causes a bacterial speck disease in tomato and Arabidopsis. In Chinese cabbage, in which host-pathogen interactions are not well understood, Pst does not cause disease but rather elicits a hypersensitive response. Pst induces localized cell death and H2O2 accumulation, a typical hypersensitive response, in infiltrated cabbage leaves. Pre-inoculation with Pst was found to induce resistance to Erwinia carotovora subsp. carotovora, a pathogen that causes soft rot disease in Chinese cabbage. An examination of the expression profiles of 12 previously identified Pst-inducible genes revealed that the majority of these genes were activated by salicylic acid or BTH; however, expressions of the genes encoding PR4 and a class IV chitinase were induced by ethephon, an ethylene-releasing compound, but not by salicylic acid, BTH, or methyl jasmonate. This implies that Pst activates both salicylate-dependent and salicylate-independent defense responses in Chinese cabbage.     

Astrocytic Expressions of Phosphorylated Akt,GSK3β and CREB Following an Excitotoxic Lesion in the Mouse Hippocampus

Glycogen synthase kinase 3β (GSK3β) is believed to play important roles in the regulation of synaptic plasticity, cell survival and circadian rhythms in the mature CNS. However, although several studies have been focused on the GSK3β, little is known about GSK3β changes in glial cells under neuropathological conditions. In this study, we evaluated the expressions of molecules associated with the GSK3β signaling pathway, following the induction of an excitotoxic lesion in mouse brain by kainic acid (KA) injection, which caused pyramidal cell degeneration in the hippocampal CA3 region. In injured hippocampi, Ser47-Akt (protein kinase B, PKB) phosphorylation increased from 4 h until 1 day post-injection (PI). Ser9-GSK3β and Ser133-cAMP responsive element-binding protein (CREB) phosphorylations showed similar spatiotemporal patterns in hippocampi at 1 day until 3 days PI. Double immunohistochemistry also showed that these phosphorylated forms of Akt, GSK3β and CREB were expressed in astrocytes. For the first time, our data demonstrate the injury-induced astrocytic changes in the levels of phosphorylation of Akt, -GSK3β and -CREB in vivo, which may reflect mechanisms of glial cells protection or adaptive response to damage. DW Kim and JH Lee contributed equally to this work.     

Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library

A metagenomic library was constructed using total genomic DNA extracted from the mud in the west coast of Korea and was used together with a fosmid vector, pCC1FOS in order to uncover novel gene sources. One clone from approximately 30,000 recombinant Escherichia coli clones was identified that showed proteolytic activity. The gene for the proteolytic enzyme was subcloned into pUC19 and sequenced, and a database search for homologies revealed it to be a zinc-dependent metalloprotease. The cloned gene included the intact coding gene for a novel metalloproteinase and its own promoter. It comprised an open reading frame of 1,080 base pairs, which encodes a protein of 39,490 Da consisting of 359 amino acid residues. A His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent metalloproteases, was found in the deduced amino acid sequence of the gene, suggesting that the enzyme is a zinc-dependent metalloprotease. The purified enzyme showed optimal activity at 50°C for 1 h and pH 7.0. The enzyme activity was inhibited by metal-chelating reagents, such as EDTA, EGTA and 1,10-phenanthroline. The enzyme hydrolyzed azocasein as well as fibrin. Thus, the enzyme could be useful as a therapeutic agent to treat thrombosis. The sequence reported in this paper has been deposited in the GenBank database (Accession number: EF100137).     

Impaired long-term memory and long-term potentiation in N-type Ca2+ channel-deficient mice

Voltage-dependent N-type Ca(2+) channels, along with the P/Q-type, have a crucial role in controlling the release of neurotransmitters or neuromodulators at presynaptic terminals. However, their role in hippocampus-dependent learning and memory has never been examined. Here, we investigated hippocampus-dependent learning and memory and synaptic plasticity at hippocampal CA3-CA1 synapses in mice deficient for the alpha(1B) subunit of N-type Ca(2+) channels. The mutant mice exhibited impaired learning and memory in the Morris water maze and the social transmission of food preference tasks. In particular, long-term memory was impaired in the mutant mice. Interestingly, among activity-dependent long-lasting synaptic changes, theta burst- or 200-Hz-stimulation-induced long-term potentiation (LTP) was decreased in the mutant, compared with the wild-type mice. This type of LTP is known to require brain-derived neurotrophic factor (BDNF). It was found that both BDNF-induced potentiation of field excitatory postsynaptic potentials and facilitation of the frequency of miniature excitatory postsynaptic currents (mEPSCs) were reduced in the mutant. Taken together, these results demonstrate that N-type Ca(2+) channels are required for hippocampus-dependent learning and memory, and certain forms of LTP.     

Airway exposure levels of lipopolysaccharide determine type 1 versus type 2 experimental asthma

Allergic asthma is characterized by airway inflammation initiated by adaptive immune responses to aeroallergens. Recent data suggest that severe asthma may be a different form of asthma rather than an increase in asthma symptoms and that innate immune responses to LPS can modulate adaptive immune responses to allergens. In this study, we evaluated the hypothesis that airway exposure to different doses of LPS induces different form of asthma. Our study showed that neutrophilic inflammation and IFN-gamma expression were higher in induced sputum from severe asthma patients than from mild to moderate asthmatics. Animal experiments indicated that allergen sensitization with low-dose LPS (0.1 microg) induced type 2 asthma phenotypes, i.e., airway hyperresponsiveness, eosinophilic inflammation, and allergen-specific IgE up-regulation. In contrast, allergen sensitization with high-dose LPS (10 microg) induced asthma phenotypes, i.e., airway hyperresponsiveness and noneosinophilic inflammation that were not developed in IFN-gamma-deficient mice, but unaffected in the absence of IL-4. During the allergen sensitization period, TNF-alpha expression was found to be enhanced by both low- and high-dose LPS, whereas IL-12 expression was only enhanced by high-dose LPS. Interestingly, the asthma phenotypes induced by low-dose LPS, but not by high-dose LPS, were completely inhibited in TNF-alpha receptor-deficient mice, whereas the asthma phenotypes induced by high-dose LPS were abolished in the homozygous null mutation of the STAT4 gene. These findings suggest that airway exposure levels of LPS induces different forms of asthma that are type 1 and type 2 asthma phenotypes by high and low LPS levels, respectively.     

Complete sequence of the mitochondrial genome of Taenia saginata: comparison with T. solium and T. asiatica

The complete sequence of the Taenia saginata mitochondrial genome was determined, and its organization and structure were compared to other human-tropic Taenia tapeworms for which complete mitochondrial sequence data were available. The mitochondrial genome was 13,670 bp long, contained 12 protein-coding genes, two ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs (tRNAs). It did not encode the atp8 gene. Overlapping regions were found between nad4L and nad4, nad1 and trnN, and cox1 and trnT. The ATG initiation codon was used for 10 protein-coding genes, and the GTG initiation codon was used for the remaining 2 genes (nad4 and atp6). The size of the protein-coding genes of the three human Taenia tapeworms did not vary, except for Taenia solium nad1 (891 aa) and nad4 (1212 aa) and Taenia asiatica cox2 (576 aa). The tRNA genes were 57-75 bp long, and the predicted secondary structures of 18 of these genes had typical clover-leaf shapes with paired dihydrouridine (DHU) arms. The genes in all human Taenia tapeworms for the two mitochondrial rRNA subunits rrnL and rrnS are separated by trnC. The putative T. saginata rrnL and rrnS are 972 and 732 bp long, respectively. The non-coding regions of the mt genome of T. saginata consisted of 2 regions: a short non-coding region (SNR, 66 nucleotides) and a long non-coding region (LNR, 159 nucleotides). The overall sequence difference in the full mitochondrial genome between T. saginata and T. asiatica was 4.6%, while T. solium differed by 11%. In conclusion, the complete sequence of the T. saginata mitochondrial genome will serve as a resource for comparative mitochondrial genomics and systematic studies of the parasitic cestodes.     

Production and characterization of monoclonal antibodies to porcine brain pyridoxal kinase.

Six monoclonal antibodies that recognize porcine brain pyridoxal kinase have been selected and designated as PK67, PK86, PK91, PK144, PK252 and PK275. A total of six monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which four inhibited the enzyme activity. When total proteins of porcine brain homogenate separated by SDS-PAGE were subjected to monoclonal antibodies, a single reactive protein band of molecular weight 39 kDa which comigrated with purified porcine pyridoxal kinase was detected. Using the anti-pyridoxal kinase antibodies as probes, the cross reactivities of brain pyridoxal kinase from human and other mammalian tissues and from avian sources were also investigated. Among human and all animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 39 kDa. These results indicate that mammalian brains contain only one major type of immunologically similar pyridoxal kinase, although some properties of the enzymes reported previously differed from one another.