Mating-Type Genes Control Sexual Reproduction, Conidial Germination, and Virulence in Cochliobolus lunatus.

Cochliobolus lunatus (anamorph: Curvularia lunata) is a major pathogenic fungus that causes the Curvularia leaf spot of maize. ClMAT1-1-1 and ClMAT1-2-1, the C. lunatus orthologs of C. heterostrophus ChMAT1-1-1 and ChMAT1-2-1, were investigated in the present study to uncover their functions in C. lunatus. Southern blot analysis showed that these mating-type MAT genes exist in the C. lunatus genome as a single copy. ClMAT1-1-1 and ClMAT1-2-1 were knocked out and complemented to generate ΔClmat1-1-1 and ΔClmat1-2-1 and ΔClmat1-1-1-C and ΔClmat1-2-1-C, respectively. The mutant strains had defective sexual development and failed to produce pseudothecia. There were no significant differences in growth rate or conidia production between the mutant and wild-type strains. However, the aerial mycelia and mycelial dry weight of ΔClmat1-1-1 and ΔClmat1-2-1 were lower than those of wild type, suggesting that MAT genes affect asexual development. ClMAT genes were involved in the responses to cell wall integrity and osmotic adaptation. ΔClmat1-2-1 had a lower conidial germination rate than the wild-type strain CX-3. The virulence of ΔClmat1-2-1 and ΔClmat1-1-1 was also reduced compared with the wild-type. Complementary strains could restore all the phenotypes.

First Report of Phomopsis longicolla Causing Leaf Spot on Soybean in China.

During July 2012, leaf spots affecting 60% of the leaves were observed on soybean cultivar He Feng 60 in fields near Shenyang City, Liaoning Province, leading to 5 to 10% yield loss. The leaf spots were associated with the leaf margins and were irregularly shaped, with brown to black margins and surrounded by a thin, yellow halo. Often, several spots merged to form large necrotic areas, which contained numerous pycnidia on the underside of the leaf. Small pieces (5 mm2) were excised from the margin of diseased and healthy tissue, surface-sterilized in 70% ethanol solution for 30 s and 0.1% mercuric chloride solution for 1 min, washed in three changes of sterile distilled water, and transferred to plates containing potato dextrose agar (PDA). Cultures were maintained in an incubator at 25°C with a 12 h dark/light photoperiod for 5 to 7 days. On PDA, colonies were white with yellow areas, floccose, dense, and moderately fast growing, attaining a diameter of 3.9 mm after 5 days and 9.0 mm after 14 days. Finally, large black stromata appeared after 28 days at 25°C. The conidiomata pycnidia were black, stomatic, globose, length 83.6 to 232 µm, width 37.9 to 146.3 µm and produced α-conidia that were unicellular, hyaline, sometimes two-guttulate, length 4.75 to 8.25 µm, width 1.50 to 3.00 µm. ß-Conidia were not observed. To confirm the morphological identification, the ribosomal internal transcribed spacers (ITS1-5.8S-ITS2) from isolates were sequenced (GenBank Accession No. KC460334). The PCR products were cloned into a pMD-19T Cloning Vector (Sangon Biotech, Shanghai, China). The clones were purified with TIANprep Mini Plasmid Kit (Tiangen Biotech, Beijing, China) to get the full-length ITS sequence. BLAST analysis of the isolates showed 100% nucleotide sequence identity with Phomopsis longicolla (AY745021). Four additional primer pairs-large subunit (NL1/NL4), beta-tublin gene (Bt2a/Bt2b), translation elongation factor 1α gene(EF1-728F/EF1-986R), and act gene(ACT-512F/ACT-783R) (1,2)-were amplified and sequenced as described above. The large subunit gene, ß-tubulin gene, and translation elongation factor 1α gene from isolates were sequenced (Sangon Biotech). BLAST analysis indicated that the isolates had 100% nucleotide sequence identity with P. longicolla (AB107259, HQ333514, and AF398896). Because the act gene sequence of P. longicolla was not in the NCBI database, this sequence had 94% nucleotide sequence identity with P. cuppatea (JN230389). To fulfill Koch's postulates, five leaves on five healthy soybean plants were inoculated with a conidial suspension (106/ml). Plants inoculated with sterile water served as the noninoculated controls. Plants were incubated in the greenhouse at 25°C. All the inoculated leaves developed pinhead spots on the leaves, gradually increasing to large brown spots. Spots were irregularly shaped, brown and necrotic in the center, and surrounded by a yellow halo. Black pycnidia appeared after 10 days, whereas the noninoculated control plants remained asymptomatic. P. longicolla was consistently recovered from all inoculated plants, except the control. Morphological description of isolates was similar to that of Hobbs (3). However, as described by Hobbs and others, P. longicolla conidiomata pycnidia have prominent necks more than 200 µm long, opening by apical ostioles; locules are uniostiolate or multiostiolate, globose, up to 500 µm wide. The pycnidia size of isolates by frozen section method was smaller than that of Hobbs. Based on morphological and sequence comparisons, the pathogen of leaf spot disease is caused by P. longicolla. This is the first reported leaf spot caused by P. longicolla on soybean in China. References: (1) T. Boekhou et al. Stud. Mycol. 38:75, 1995. (2) P. W. Crous et al. Stud. Mycol. 75:37, 2013. (3) T. W. Hobbs et al. Mycologia 77:535, 1985.

Effect of tetrandrine on neutrophilic recruitment response to brain ischemia/reperfusion.

AIM: To investigate the effect of tetrandrine (Tet) on neutrophilic recruitment response to bain ischemia/reperfusion (I/R). METHODS: Middle cerebral artery (MCA) ischemia (2 h)/reperfusion model was built on rats (male). Brain water content, neutrophilic recruitment, the expression of intercellular adhesion molecule-1 (ICAM-1) mRNA and activation of NF-kappaB in cellular nucleus after brain I/R were measured with dry-wet weight, 51Cr-labeled neutrophil, reverse transcriptase polymerase chain reaction, and electrophoretic mobility shift assay, respectively. RESULTS: Brain water and neutrophilic recruitment were parallelly increased from 3 h to 24 h after reperfusion (P < 0.01). The expression of ICAM-1 mRNA was detected at 1 h after reperfusion (P < 0.01), increased to the highest peak at 12 h (P < 0.01), and at 24 h decreased to level at 3 h. The activation of NF-kappaB was detected at 0.5 h after reperfusion (P < 0.01), increased to the highest peak at 6 h (P < 0.01), and at 24 h decreased to level at 1 h. Tet 10 and 20 mg/kg decreased brain water content, neutrophilic recruitment, the expression of ICAM-1 mRNA, and the activation of NF-kappaB at 6 h, 12 h, and 24 h after reperfusion. CONCLUSION: Tet inhibited neutrophilic recruitment, expression of ICAM-1 mRNA, and activation of NF-kappaB after brain I/R.

[Effect of the tetrandrine on morphine induced hyperactivity and reinforcement in mice].

OBJECTIVE: After single administration of morphine and the motion activity was measured by ambulometer, conditioned place-preference paradigm was used to study the reinforcing effect of morphine, climbing behavior was used to evaluate the relation with Dopaminergic system and immediate early expression of c-fos gene was in brain was showed by immunohistochemical method. RESULTS: Single administration of morphine could induce hyperactivity, repeated treatment would produce a conditioned place-preference response, tetrandrine 30 or 60 mg/kg hypodermic injection could inhibit the morphine induced hyperactivity, 60 mg/kg could inhibit the conditioned place-preference response but no influence on climbing behavior in mice was found. Tetrandrine could inhibit the c-fos gene expression in nucleus accumbens, ventral tegmental and prefrontal cortex in place-preference model formed by morphine. CONCLUSION: Tetrandrine could inhibit the hyperactivity and conditioned place preference response induced by morphine, it might relate to reducing the c-fos gene expression in special area of brain in mice.

[Expression of FAS and FASL genes in brain and effect of flunarizine on these expressions in rats after global ischemia-reperfusion].

AIM: To explore the expression of Fas and FasL genes after ischemia-reperfusion in rats and the effect of flunarizine. METHODS: Ischemia was induced by four-vessel occlusion for 30 min following reperfusion in rats. The biopsy tissues from brain were immunohistochemically assayed with Fas and FasL genes polyclonal antibody. RESULTS: The expression of Fas was increased as early as 6 h after the onset of reperfusion. The peak of the expression of Fas occurred 24-48 h after ischemia-reperfusion. The expression of FasL was observed 12 h after ischemia-reperfusion and peaked at 48-72 h. The expression of Fas and FasL gene was quite obvious in the cortex and hippocampus CA1, the more sensitive areas to ischemic injury. Flunarizine i.p. 10 mg.kg-1 and 20 mg.kg-1 obviously inhibited the expression of Fas and FasL in dose-dependent manner. CONCLUSION: Expression of Fas and FasL in cerebral cortex and hippocampus can be induced by global ischemia-reperfusion. Flunarizine significantly inhibited the expression of Fas and FasL genes following ischemia-reperfusion.

Effects of tetrandrine on changes of NMDA receptor channel in cortical neurons of rat induced by anoxia.

AIM: To study the effects of tetrandrine (Tet) on the changes of NMDA receptor channels in cortical neurons induced by anoxia. METHODS: Cell-attached configuration of patch-clamp techniques. Anoxia was produced by perfused cells with 95% N2 + 5% CO2 gassed bath solution. RESULTS: During anoxia, the open time constant (tau 2), open probability (Po) of 35-pS and 100-pS channels increased. Tet 7.5 mumol.L-1 reduced the Po of 35-pS and 100-pS channels, 15 and 30 mumol.L-1 inhibited open of 100-pS channel fully, and changed the open time constant of 35-pS from two to single exponential distribution. CONCLUSION: Tet inhibition of the open of NMDA receptor channels induced by anoxia was one of its protective mechanisms.

Regenerative changes in median nerve defects using various rabbit skeletal muscles.

We examined the morphological changes of median nerve regeneration which situated to pass through degenerative latissimus dorsi and brachial triceps muscles in rabbits. Morphological observation was performed at 7, 14, 28, 45, 60 and 180 days after the creation of defect of the bilateral median nerves. Regenerative nerve fibers were observed in the residual tubes of left degenerative muscle bridges. In this respect the regenerative effect of the latissimus dorsi was better than that of the brachial triceps. These results suggest that regular and longer muscle fibers as those of latissimus dorsi may contribute to the effective regeneration of nerve.

[Effect of verapamil on Ca2+ and Ca(2+) Mg(2+)-ATPase activity in rat brain synaptosomes].

To elicit the correlation between the adrenergic transmitter release and calmodulin (CaM), the effect of verapamil on the free Ca2+ concentration was measured with fluorescence analysis and Ca(2+) Mg(2+)-ATPase activity in rat synaptosomes were studied. When stimulated with high-K+ or norepinephrine, the concentration of free Ca2+ in rat synaptosome was increased by verapamil 10, 50, and 100 mumol.L-1. But the free Ca2+ concentration in the resting synaptosome was reduced by verapamil. The activity of Ca(2+) Mg(2+)-ATPase in synaptosome was remarkably inhibited by verapamil in a dose-dependent manner. These results support our hypothesis that CaM not only acts directly on the vesicles to enhance the transmitter release, but also acts on the activity of Ca(2+) Mg(2+)-ATPase to reduce the free Ca2+ in the cytosol, and indirectly inhibited the transmitter release.

[The abnormalities of the cellular immunity in rheumatoid synovium].

With the technique of immunohistochemical (ABC) method monoclonal antibodies were used to identify the lymphocyte subsets, macrophage and the expression of class II MHC (HLA-DR, HLA-DQ) antigens in the synovium cells from 18 RA patients and 8 patients with osteoarthritis as control. The results showed that the main cellular abnormality at the sublayer of the synovium was the appearance of lymphoid follicles which mainly consisted of the infiltration of T lymphocytes (77.8%).50.6% of them were CD4+, which mainly consisted by of CD45RO+ cells in the rheumatoid synovium on the consecutive sections. The increased ratio of CD4/CD8 in RA patients was significantly higher when compared with that in the controls (2.11 +/- 0.93 vs 0.63 +/- 0.13, P < 0.001). In advanced RA with fibrosis of joints, the ratio tended to decrease and was accompanied with reduction of infiltrated lymphocytes. Compared with T cells CD20+ B lymphocyte not only had a lower percentage (25.2%), but also showed a characteristic picture of locating in the centre of the follicles. The fact that most of the CD4+ T cells was helper memorized lymphocytes with CD4 phenotype of positive TAC(+) (interleukin-2 receptor) and that up to 54.2% was anti-HLA-DR and 54.1% anti-HLA-DQ monoclonal antibodies indicated that these T lymphocytes were activated in vivo. Cells with anti-CD68+ were seen all over the RA synovium. Class II HLA and CD68 molecule were also expressed on the endothelium cells of the small vessels. It is suggested that the activated lymphocytes, macrophages and endothelium cells and their abnormal distribution may indicate the abnormalities of the cellular immunity in rheumatoid synovium.

[Clinical analysis of 90 cases of eosinophilia and pulmonary infiltrates with peripheral eosinophilia].

90 patients diagnosed as eosinophilia and pulmonary infiltrates with peripheral eosinophilia in PUMC Hospital from 1957 to 1986 were reported. The cause of the eosinophilia was unknown in 42% of the patients. Among these patients, hypereosinophilic syndromes (HES) were suspected in 11 patients. The most common presenting symptoms of HES were fever, skin lesions gastrointestinal symptoms and hepatosplenomegaly, the mean peripheral blood eosinophil count was as high as 25.6 x 10(9) cells/L. Biopsies of bone marrow and involved organs revealed extensive eosinophilic infiltration and severe necrosis. In the past ten years, eosinophilia and pulmonary infiltrates caused by parasitic infections obviously decreased. The most common parasites were ascaris Lumbricoides, Ancylostoma duodenale and Clonorchis sinesis. The other main causes which induced pulmonary infiltrates were allergic bronchopulmonary mycosis (ABPM) and chronic eosinophilic pneumonia. The prevalence of ABPM increased recently; early diagnosis and effective therapy could prevent the irreversible damage resulting in airways obstruction and fibrosis.

Effects of ginsenosides on release of [3H]norepinephrine from rat vas deferens and portal vein.

The effects of ginsenosides (G) on the release of [3H]norepinephrine ([ 3H]NE) from the isolated rat vas deferens (RVD) and portal vein (RPV) preloaded with [3H]NE were studied, G (100 micrograms/ml) did not affect the spontaneous or high potassium (H-K+, 60 mmol/L)- and tyramine (Tyr 10 mumol/L)-evoked release of [3H]NE, but obviously blunted the phentolamine (Phe 10 mumol/L)-induced increase in [3H]NE release from RVD and enhanced the isoprenaline (Iso 0.1 mumol/L)-augmented [3H]NE release from RPV evoked by H-K+. It is still not known whether G can bind with adrenoceptors. We infer that G may act as a modulator in sensitizing both presynaptic alpha 2- and beta-receptors.

Effect of sodium depletion on the release of [3H]norepinephrine from central and peripheral tissue of Wistar-Kyoto and spontaneously hypertensive rats.

To study the relationship between sodium intake, the sympathetic nervous system, and hypertension, we studied the effects of a 7-9 day dietary restriction of sodium in three different ages of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Field-stimulated [3H]norepinephrine ( [3H]NE) release was measured in portal vein, anterior hypothalamus, and the A2 region of the nucleus tractus solitarius (NTS) of 5- to 6-, 10- to 11-, and 28- to 30- week-old SHR and age-matched WKY. A low-sodium diet (0.05% Na+, control 0.5% Na+) significantly lowered stimulated [3H]NE release from portal vein and anterior hypothalamus in SHR and WKY at all three ages. However, release from the A2 region was not altered by sodium restriction. The results of the present study suggest that lowered dietary sodium can selectively alter norepinephrine release in both the peripheral and central sympathetic nervous system of SHR and WKY. The results also suggest that the SHR at 5-6 weeks are more sensitive to altered dietary sodium than are age-matched WKY.

Effect of low sodium diet on the facilitatory effect of angiotensin on 3H-norepinephrine release in the rat portal vein.

The ability of angiotensin to enhance the field-stimulation induced release of 3H-norepinephrine from the superfused rat portal vein was examined in vessels obtained from animals fed a normal (0.5% Na+) or low sodium diet (0.05% Na+). Angiotensin was seen to enhance the field-stimulation (480 pulses, 2 Hz, 1 ms duration, supramaximal voltage) induced release of 3H-norepinephrine from vessels obtained from Sprague-Dawley, Wistar, Wistar-Kyoto (WKY) and the spontaneously hypertensive rats (SHR) maintained on a normal sodium diet. The effect of angiotensin was attenuated when examined in vessels obtained from animals maintained on the low sodium diet. The selectivity of the low sodium diet for angiotensin was demonstrated by a lack of effect of the low sodium diet in altering the facilitatory effect of isoproterenol on the release of 3H-norepinephrine and an enhanced response to the alpha 2-adrenoceptor-selective antagonist, yohimbine. The simultaneous treatment of rats with a low sodium diet plus captopril (estimated to be approximately 50 mg/kg/day for 7 days) prevented the attenuation of the angiotensin-induced enhancement of the release of 3H-norepinephrine seen by sodium alone. These results are consistent with the hypothesis that low sodium treatment increases circulating angiotensin levels which lead to a down-regulation of the angiotensin receptors located on adrenergic nerve varicosities.

Angiotensin facilitation of noradrenergic neurotransmission in central tissues of the rat: effects of sodium restriction.

This study investigated the ability of angiotensin II (Ang II) to facilitate the stimulation-induced release of [3H]norepinephrine [( 3H]NE) from two cardiovascular regulatory areas in normal and sodium-restricted rats. Ang II (10(-7) M) facilitated the field-stimulation-induced release of [3H]NE from the A2 area of the nucleus tractus solitarius but not from the anterior hypothalamus of Sprague-Dawley and Wistar rats. Placement of rats on a sodium-restricted diet abolished the facilitation of [3H]NE release due to Ang II. Captopril given during sodium restriction partially restored the facilitory effects of Ang II. In an effort to determine the interaction of Ang II and sodium reduction, the effects of chronic Ang II were studied. Seven-day intravenous Ang II infusions blocked the facilitory effect of Ang II on [3H]NE release in a manner similar to that seen with sodium restriction. These results suggest that low sodium diets may alter the facilitation of [3H]NE release by Ang II by interactions with the renin-angiotensin system.