Bacterial Symbionts of a Devastating Coffee Plant Pest,the Stinkbug Antestiopsis thunbergii (Hemiptera: Pentatomidae)

Stinkbugs of the genus Antestiopsis, so-called antestia bugs or variegated coffee bugs, are notorious pests of coffee plants in Africa. We investigated the symbiotic bacteria associated with Antestiopsis thunbergii, a major coffee plant pest in Rwanda. PCR, cloning, sequencing, and phylogenetic analysis of bacterial genes identified four distinct bacterial lineages associated with A. thunbergii: a gammaproteobacterial gut symbiont and symbionts representing the genera Sodalis, Spiroplasma, and Rickettsia. In situ hybridization showed that the gut symbiont densely occupied the lumen of midgut crypts, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont sparsely and sporadically infected various cells and tissues. Diagnostic PCR survey of 154 A. thunbergii individuals collected at 8 localities in Rwanda revealed high infection frequencies (100% for the gut symbiont, 51.3% for the Sodalis symbiont, 52.6% for the Spiroplasma symbiont, and 24.0% for the Rickettsia symbiont). These results suggest that the gut symbiont is the primary symbiotic associate of obligate nature for A. thunbergii, whereas the Sodalis symbiont, the Spiroplasma symbiont, and the Rickettsia symbiont are the secondary symbiotic associates of facultative nature. We observed high coinfection frequencies, i.e., 7.8% of individuals with quadruple infection with all the symbionts, 32.5% with triple infections with the gut symbiont and two of the secondary symbionts, and 39.6% with double infections with the gut symbiont and any of the three secondary symbionts, which were statistically not different from the expected coinfection frequencies and probably reflected random associations. The knowledge of symbiotic microbiota in A. thunbergii will provide useful background information for controlling this devastating coffee plant pest.     

Ifit1 Inhibits Japanese Encephalitis Virus Replication through Binding to 5′ Capped 2′-O Unmethylated RNA

The interferon-inducible protein with tetratricopeptide (IFIT) family proteins inhibit replication of some viruses by recognizing several types of RNAs, including 5′-triphosphate RNA and 5′ capped 2′-O unmethylated mRNA. However, it remains unclear how IFITs inhibit replication of some viruses through recognition of RNA. Here, we analyzed the mechanisms by which Ifit1 exerts antiviral responses. Replication of a Japanese encephalitis virus (JEV) 2′-O methyltransferase (MTase) mutant was markedly enhanced in mouse embryonic fibroblasts and macrophages lacking Ifit1. Ifit1 bound 5′-triphosphate RNA but more preferentially associated with 5′ capped 2′-O unmethylated mRNA. Ifit1 inhibited the translation of mRNA and thereby restricted the replication of JEV mutated in 2′-O MTase. Thus, Ifit1 inhibits replication of MTase-defective JEV by inhibiting mRNA translation through direct binding to mRNA 5′ structures.     

Heterologous production,purification, and immunodetection of Magnaporthe oryzae avirulence protein AVR-Pia

The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H₂O₂ and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4?kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7?ng/g rice sheath.     

Purification and Some Properties of “Methemoglobin Reductase”

A procedure for obtaining the electrophoretically and ultracentrifugally homogenous preparation of “methemoglobin reductase” from erythrocytes of blue-white dolphin was developed. Method consists of DEAE-cellulose adsorption, fractionation with ammonium sulfate, Sephadex G-75 gel filtration and DEAE-Sephadex A-50 column chromatography. There were obtained three preparations of enzyme. All these preparations strongly reduced methemoglobin, metmyoglobin and cytochrome c in the presence of methyleneblue when NADPH or NADH was used as the cofactor. The activity of NADPH as the cofactor was higher than that of NADH. The enzyme contained neither flavin nor heme, and molecular weight was 23,000 ~ 28,000.     

Lutein Dispersed in Acetone Aqueous Solution; Spectroscopic Analysis and Electron Microscope Observation

The sizes and shapes of lutein aggregates in acetone aqueous solution were examined by spectroscopic analyses and electron microscopic observation, since lutein dispersed into acetone aqueous solution and provided a much simpler system than others. This system is suitable for basic research on lutein aggregates. The lutein aggregate gave maximum molar ellipticity in acetone concentrations from 35 to 45% aqueous solution. When the acetone concentration increased, CD spectra were reversed in 45% concentration from a negative exciton chirality to the positive. Since the aggregates were most slender in these acetone concentrations, it was found that the length of the lutein aggregate was closely related to the molar ellipticity. The apparent widths of the aggregates became large with increases of the acetone concentration after the reversion of CD spectra, where the molar ellipticities were diminished. These results are similar to those for the lutein aggregates in dilute surfactant solution, and this system is clearer and simpler for electron microscopic observation than those in surfactant solutions.     

Involvement of ERK Phosphorylation of Trigeminal Spinal Subnucleus Caudalis Neurons in Thermal Hypersensitivity in Rats with Infraorbital Nerve Injury

To evaluate the involvement of the mitogen-activated protein kinase (MAPK) cascade in orofacial neuropathic pain mechanisms, this study assessed nocifensive behavior evoked by mechanical or thermal stimulation of the whisker pad skin, phosphorylation of extracellular signal-regulated kinase (ERK) in trigeminal spinal subnucleus caudalis (Vc) neurons, and Vc neuronal responses to mechanical or thermal stimulation of the whisker pad skin in rats with the chronic constriction nerve injury of the infraorbital nerve (ION-CCI). The mechanical and thermal nocifensive behavior was significantly enhanced on the side ipsilateral to the ION-CCI compared to the contralateral whisker pad or sham rats. ION-CCI rats had an increased number of phosphorylated ERK immunoreactive (pERK-IR) cells which also manifested NeuN-IR but not GFAP-IR and Iba1-IR, and were significantly more in ION-CCI rats compared with sham rats following noxious but not non-noxious mechanical stimulation. After intrathecal administration of the MEK1 inhibitor PD98059 in ION-CCI rats, the number of pERK-IR cells after noxious stimulation and the enhanced thermal nocifensive behavior but not the mechanical nocifensive behavior were significantly reduced in ION-CCI rats. The enhanced background activities, afterdischarges and responses of wide dynamic range neurons to noxious mechanical and thermal stimulation in ION-CCI rats were significantly depressed following i.t. administration of PD98059, whereas responses to non-noxious mechanical and thermal stimulation were not altered. The present findings suggest that pERK-IR neurons in the Vc play a pivotal role in the development of thermal hypersensitivity in the face following trigeminal nerve injury.     

Discriminant function analysis for evaluating the status of coronary heart disease risk

We investigated to discriminate those individuals categorized by 1. obesity, 2. hypercholesterolemia, 3. hypertension, 4. low maximal oxygen uptake, 5. an abnormal electrocardiogram reflecting ischemic patterns, and/or 6. real sedentary life, from relatively healthier individuals without coronary heart disease (CHD) risk factors. One hundred and six Japanese women, aged 30 to 72 years, all of whom were in the postabsorptive state, were recruited in a series of tests for anthropometric and physiologic profiles both during the resting state and during the submaximal-maximal cycling exercise. Subjects were categorized into two groups--those who possessed four or more of the above 1, 2, 3, 4, 5, and 6 (high-CHD-risk group, n = 15) and apparently healthy individuals with a minimum number of risk factors (low-CHD-risk group, n = 83). Analyses of the data revealed that a combination of 8 variables extracted from among original 25 variables accurately classified 13/15 (87%) of high-CHD-risk group and 77/83 (93%) of low-CHD-risk group (mean = 90/98 or 92%) into their respective groups. The 8 variables were double product, Katsura index, waist girth, chest girth, TG, TC, and skinfold thicknesses at the subscapular and abdominal sites. Subsequent t-test identified significant differences between groups not only for VO2max, SBP and TC but also for DBP, LDLC, TG, Hb, HR, and HRmax. Most of these differences were of a much greater magnitude compared to the existing difference in chronological age. These findings suggest the usefulness and importance of anthropometric and blood lipid variables in the explanation of differences in the health status between high-CHD-risk women and their counterparts.     

Establishment of strongly neutralizing monoclonal antibody to human interleukin-6 and its epitope analysis

Three monoclonal antibodies against human interleukin-6 were established and characterized. One antibody was shown to strongly neutralize both the Ig-inducing and hybridoma/plasmacytoma growth activity of interleukin-6. The results of its epitope analysis using protease treated interleukin-6 and immobilized antibody indicated that this neutralizing antibody binds to a peptide corresponding to Leu151-Lys171 of interleukin-6 molecule. Further analysis using synthetic peptides showed that a shorter peptide corresponding to Ala153-Thr162 can also inhibit the binding of the antibody to interleukin-6. These results suggest that this carboxyl-terminal region plays a crucial role in interleukin-6 functions.