HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci,induces a hypersensitive response in tobacco wildfire-resistant Nicotiana tabacum ‘N509’

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram-negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease-resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E-deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum ‘N509’. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C-terminal truncated HopAZ1 abolished HopAZ1-dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.     

Autophagy-Related Atg8 Localizes to the Apicoplast of the Human Malaria Parasite Plasmodium falciparum

Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite Plasmodium falciparum (PfAtg8). PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although Plasmodium parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.     

The periodic loss of metabolically unstable DNA during replication of chromosomal DNA of CHO cells

The fate of ³H-thymidine incorporated into newly synthesized DNA of CHO cells was analyzed by either the estimation of the incorporated radioactivity per cell or sedimentation in alkaline sucrose gradient. Under conditions in which DNA synthesis proceeded continuously, of incorporated radioactivity was periodically lost and regained during a 90 min chase, corresponding to a cyclic change in the sedimentation profiles. When DNA synthesis was inhibited by hydroxyurea no cyclic change of the incorporated radioactivity was observed. The cyclic changes were regarded as the result of an actual metabolic change in³H-labelled DNA probaly joining to one of the newly formed sister strands of DNA and the loss of radioactivity seems to require active continued DNA synthesis.     

Recognition of heteropolysaccharide alginate by periplasmic solute-binding proteins of a bacterial ABC transporter

Alginate is a heteropolysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G). The Gram-negative bacterium Sphingomonas sp. A1 directly incorporates alginate into the cytoplasm through the periplasmic solute-binding protein (AlgQ1 and AlgQ2)-dependent ABC transporter (AlgM1-AlgM2/AlgS-AlgS). Two binding proteins with at least four subsites strongly recognize the nonreducing terminal residue of alginate at subsite 1. Here, we show the broad substrate preference of strain A1 solute-binding proteins for M and G present in alginate and demonstrate the structural determinants in binding proteins for heteropolysaccharide recognition through X-ray crystallography of four AlgQ1 structures in complex with saturated and unsaturated alginate oligosaccharides. Alginates with different M/G ratios were assimilated by strain A1 cells and bound to AlgQ1 and AlgQ2. Crystal structures of oligosaccharide-bound forms revealed that in addition to interaction between AlgQ1 and unsaturated oligosaccharides, the binding protein binds through hydrogen bonds to the C4 hydroxyl group of the saturated nonreducing terminal residue at subsite 1. The M residue of saturated oligosaccharides is predominantly accommodated at subsite 1 because of the strict binding of Ser-273 to the carboxyl group of the residue. In unsaturated trisaccharide (ΔGGG or ΔMMM)-bound AlgQ1, the protein interacts appropriately with substrate hydroxyl groups at subsites 2 and 3 to accommodate M or G, while substrate carboxyl groups are strictly recognized by the specific residues Tyr-129 at subsite 2 and Lys-22 at subsite 3. Because of this substrate recognition mechanism, strain A1 solute-binding proteins can bind heteropolysaccharide alginate with different M/G ratios.     

Type 2 diabetes mellitus in obese mouse model induced by monosodium glutamate.

The number of diabetic patients is increasing every year, and new model animals are required to study the diverse aspects of this disease. An experimental obese animal model has reportedly been obtained by injecting monosodium glutamate (MSG) to a mouse. We found that ICR-MSG mice on which the same method was used developed glycosuria. Both female and male mice were observed to be obese but had no polyphagia, and were glycosuric by 29 weeks of age, with males having an especially high rate of incidence (70.0%). Their blood concentrations of glucose, insulin, total cholesterol, and triglycerides were higher than in the control mice at 29 weeks. These high concentrations appeared in younger males more often than in females, and were severe in adult males. Also, the mice at 54 weeks of age showed obvious obesity and increased concentrations of glucose, insulin, and total cholesterol in the blood. The pathological study of ICR-MSG female and male mice at 29 weeks of age showed hypertrophy of the pancreatic islet. This was also observed in most of these mice at 54 weeks. It was recognized as a continuation of the condition of diabetes mellitus. From the above results, these mice are considered to be useful as new experimental model animals developing a high rate of obese type 2 (non-insulin dependent) diabetes mellitus without polyphagia.     

Involvement of basal metabolic rate in determination of type of cold tolerance

This study aimed to assess the relationship between basal metabolic rate (BMR) and metabolic heat production, and to clarify the involvement of BMR in determining the phenotype of cold tolerance. Measurements of BMR, maximum oxygen uptake, and cold exposure test were conducted on ten males. In the cold exposure test, rectal (T(rec)) and mean skin temperatures (T(ms)), oxygen uptake, and blood flow at forearm (BF(arm)) were measured during exposure to cold (10 degrees C) for 90 min. Significant correlations were observed between BMR and increasing rate of oxygen uptake, as well as between decreasing rate of BF(arm) and increasing rate of oxygen uptake at the end of cold exposure. These findings suggested that individuals with a lower BMR were required to increase their metabolic heat production during cold exposure, and that those with a higher BMR were able to moderate increased metabolic heat production during cold exposure because they were able to reduce heat loss. This study showed that BMR is an important factor in determining the phenotype of cold tolerance, and that individuals with a low BMR showed calorigenic-type cold adaptation, whereas subjects with a high BMR exhibited adiabatic-type cold adaptation by peripheral vasoconstriction.     

Nuclear DNA fragmentation during cell death of short-lived ray tracheids in the conifer <Emphasis Type="Italic">Pinus densiflora</Emphasis>

One key event in the programmed cell death is nuclear DNA fragmentation. We investigated the timing of nuclear DNA fragmentation during the cell death of short-lived ray tracheids in Pinus densiflora using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Fluorescence due to TUNEL was detected only in deformed nuclei that lacked obvious chromatin in ray tracheids that were adjacent to ray tracheids that no longer contained nuclei. Our observations revealed that nuclear DNA fragmentation occurred only at the final stage of cell death in ray tracheids in situ.     

Effects of Chronic Administration of Interferon α A/D on Serotonergic Receptors in Rat Brain

The effects of chronic administration of interferon (IFN; recombinant human IFN -A/D) on serotonergic binding sites in rat brain were investigated. IFN was injected daily for 2 weeks at a dose of 100000 I.U./kg, (i.p.) in male Wistar rats. IFN did not alter either [³H]ketanserin binding to 5-HT_2A receptors or [³H]paroxetine binding to 5-HT transporters. Scatchard analysis of [³H]8-hydroxy-dipropylaminotetraline (8-OH-DPAT) binding to 5-HT_1A receptors demonstrated the presence of high- and low-affinity binding sites in both treatment and control groups. IFN significantly increased both Kd and Bmax measures of [³H]8-OH-DPAT binding at low-affinity binding sites, but not at the high-affinity sites. These results suggest that IFN affects the low-affinity 5-HT_1A receptors sites and may be involved in the development of IFN-induced psychiatric disturbances.     

Characterization of the NikA histidine kinase implicated in the phosphorelay signal transduction of Aspergillus nidulans, with special reference to fungicide responses

We recently compiled a complete list of phosphorelay signal transduction components in the model filamentous fungus Aspergillus nidulans. In this study, we characterized a histidine protein kinase (designated NikA) that is found in many fungi, with special reference to responses to potent fungicides (iprodione and fludioxonil). We provided evidence that not only NikA, but also two downstream response regulators (SskA and SrrA) are crucially implicated in the mode of action of these fungicides, and also that the further downstream HogA-MAPK cascade is exaggerated abnormally (or ectopically) in hyphae by the fungicides in a manner dependent on the NikA-SskA phosphorelay.