[Inactivation of PMS2 gene by promoter methylation in nasopharyngeal carcinoma].

Objective: To investigate the inactivation of PMS2 gene mediated by promoter methylation and its regulatory mechanism in nasopharyngeal carcinoma (NPC). Methods: Fifty-four NPC tissues, 16 normal nasopharyngeal epithelia (NNE), 5 NPC cell lines (CNE1, CNE2, TWO3, HNE1 and HONE1) and 1 normal nasopharyngeal epithelial cell line (NP69) were collected.Methylation-specific PCR (MSP) was used to detect the PMS2 promoter methylation, semi-quantitative reverse transcription PCR (qRT-PCR) was applied to determine its mRNA expression, and immunohistochemistry (IHC) was used to detect the protein expression of PMS2. The expressions of PMS2 mRNA in CNE1 and CNE2 cells before and after treated with methyltransferase inhibitor 5-aza-2-deoxycytidine were analyzed by qRT-PCR. The impact of methylation and demethylation on the mRNA expression of PMS2, and the association of mRNA and protein expression of PMS2 with clinicopathological features of nasopharyngeal cancer were analyzed. Results: Methylation of PMS2 gene was detected in all of the five NPC cell lines, but not in normal nasopharyngeal epithelial NP69 cells. The methylation rate of PMS2 gene in NPC tissues was 63% (34/54), significantly higher than that of the normal nasopharyngeal epithelia (0/16, P<0.001). The expression levels of PMS2 mRNA and protein were significantly down-regulated in the 54 NPC tissues when compared with those in the 16 NNE tissues (P<0.001), and were also significantly lower in the 34 methylated NPC tissues than those in the 20 unmethylated NPC tissues (P<0.001). After treatment with 5-aza-2-deoxycytidine, the expression of PMS2 mRNA was restored in the CNE1 and CNE2 cells.However, the expressions of PMS2 mRNA and protein were not significantly correlated with patients' age, gender, TNM stage, histopathologic type or lymph node metastasis (P>0.05 for all). Conclusions: Promoter methylation-mediated inactivation of PMS2 gene participates in carcinogenesis and development of NPC. PMS2 may be a candidate tumor suppressor in the treatment for patients with inactivation of PMS2 promoter methylation.

First Report of Stem Canker Disease of Pitaya (Hylocereus undatus and H. polyrhizus) Caused by Neoscytalidium dimidiatum in Taiwan.

Pitaya (Hylocereus undatus and H. polyrhizus Britt. & Rose), a perennial succulent plant grown in the tropics, is becoming an emerging and important fruit plant in Taiwan. In September of 2009 and 2010, a number of pitaya plants were found to have a distinctive canker on stems. The disease expanded quickly to most commercial planting areas in Taiwan (e.g., Pintung, Chiayi, and Chunghua). Symptoms on the stem were small, circular, sunken, orange spots that developed into cankers. Pycnidia were erumpent from the surface of the cankers and the stems subsequently rotted. After surface disinfestation with 0.1% sodium hypochloride, tissues adjacent to cankers were placed on acidified potato dextrose agar (PDA) and incubated at room temperature for 1 week, after which colonies with dark gray-to-black aerial mycelium grew. Hyphae were branched, septate, and brown and disarticulated into 0- to 1-septate arthrospores. Sporulation was induced by culturing on sterile horsetail tree (Casuarina equisetifolia) leaves. Conidia (12.79 ± 0.72 × 5.14 ± 0.30 µm) from pycnidia were one-celled, hyaline, and ovate. The internal transcribed spacer (ITS) region of ribosomal DNA was PCR amplified with primers ITS1 and ITS4 (2) and sequenced. The sequence (GenBank Accession No. HQ439174) showed 99% identity to Neoscytalidium dimidiatum (Penz.) Crous & Slippers (GenBank Accession No. GQ330903). On the basis of morphology and nucleotide-sequence identity, the isolates were identified as N. dimidiatum (1). Pathogenicity tests were conducted in two replicates by inoculating six surface-sterilized detached stems of pitaya with either mycelium or conidia. Mycelial plugs from 2-day-old cultures (incubated at 25°C under near UV) were inoculated to the detached stems after wounding with a sterile needle. Conidial suspensions (103 conidia/ml in 200 µl) were inoculated to nonwounded stems. Noninoculated controls were treated with sterile medium or water. Stems were then incubated in a plastic box at 100% relative humidity and darkness at 30°C for 2 days. The symptoms described above were observed on inoculated stems at 6 to 14 days postinoculation, whereas control stems did not develop any symptoms. N. dimidiatum was reisolated from symptomatic tissues. To our knowledge, this is the first report of N. dimidiatum causing stem canker of pitaya. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.

First Report of Fruit Rot Disease of Mango Caused by Botryosphaeria dothidea and Neofusicoccum mangiferae in Taiwan.

Mango (Mangifera indica L.) is an economically important fruit crop in the tropical and subtropical areas of the world. In southern Taiwan, mango is grown on 18,000 ha of hilly land mainly located in Tainan, Kaohsiung, and Pingtung. Tons (180,000) of mango with a value of NT$6.6 billion (US$206 million) are produced annually. In 2008, mango fruit rot disease was observed 1 week after harvest on 30 to 72% of stored mangoes collected from seven orchards in southern Taiwan. The initial symptom was a small, brown lesion and rot symptoms advanced progressively. Two predominant fungi were isolated from the margin of lesions on acidified potato dextrose agar (PDA with lactic acid, pH 3.8). Isolates of each fungal type were transferred to 2% water agar containing sterilized pine needles and exposed to near UV light to induce sporulation. For the first fungus, conidia obtained from pycnidia were ovate, one-celled, and hyaline, with an average length and width of 12.93 ± 0.93 × 6.98 ± 0.40 µm and an average length/width ratio of 1.85. To confirm the identity of the fungus, PCR amplification by universal primers, ITS1/ITS4, and DNA sequencing of the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) were conducted. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (Accession No. GQ421486). It showed a sequence identity of 100% with Neofusicoccum mangiferae (Syd. & P. Syd.) Crous, Slippers & A. J. L. Phillips) (GenBank Accession No. AY615185). For the second fungus, conidia obtained from pycnidia were fusiform, one-celled, and hyaline, with an average length and width of 22.87 ± 1.32 × 6.42 ± 0.46 µm and a length/width ratio of 3.53. The ITS sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (Accession No. GQ421485). It showed a sequence identity of 100% with Botryosphaeria dothidea (Moug.: Fr.) Ces & De Not.) (GenBank Accession No. AY 786321). To test pathogenicity, four mango fruits were wounded with a sterile needle, inoculated with mycelium agar plugs (0.5 mm in diameter) excised from separate monoconidial cultures, and incubated in a plastic box with a 100% relative humidity for 2 days at room temperature. Brown lesions appeared on all wounded sites of each fungus 2 days postinoculation. In control experiments, sterile agar plugs were placed on the wounded mango fruits. These fruits remained completely free from symptoms throughout the experiment. The pathogen was reisolated from the lesions of inoculated fruits and identified as N. mangiferae and B. dothidea, thus fulfilling Koch's postulates. N. mangiferae and B. dothidea have been reported on mango trees in Australia and South Africa (1). To our knowledge, this is the first report of these fungi causing fruit rot of mango in Taiwan. References: (1) B. Slippers et al. Mycologia 97:99, 2005.

First Report of a Fruit Rot Disease of Avocado Caused by Neofusicoccum mangiferae.

Production of avocado (Persea americana) has increased significantly during the last 10 years in Taiwan and the area of cultivation is approximately 500 ha. The most important postharvest disease of avocado is anthracnose caused by Colletotrichum gloeosporioides (Penz.) in Taiwan (1). In 2008, a new disease was found to be infecting avocado fruit at some orchards in Tainan County of southern Taiwan. Infected avocados developed smooth, brown, circular spots first on the surface of harvested fruits. A fungus was always isolated from the margin of lesions and could also be found from symptomless fruit pedicles and stems. Fungal colonies cultured on acidified potato dextrose agar (PDA with lactic acid; pH 3.8) were initially colorless, turned dark gradually, and ultimately became gray to dark gray. After 4 days under fluorescent light at 25°C, pycnidia formed on PDA. Conidia obtained from fruiting bodies were ovate, one celled, and hyaline, with an average length and width of 12.9 (9.9 to 15.6) × 6.4 (5.2 to 7.2) µm. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (No. EU847427). It showed a sequence identity of 99% with Neofusicoccum mangiferae ((Syd. & P. Syd.) Crous, Slippers & A.J.L. Phillips) (GenBank No. AY615185). Thus, both morphological and molecular results confirmed the isolated fungus as N. mangiferae. Five avocado fruits were used to test the pathogenicity with three different treatment inoculation sites on each fruit. Wounded and unwounded sites on fruit were inoculated with mycelia agar plugs (0.5 mm in diameter) excised from a monoconidial culture and the fruit was kept in a plastic box with high humidity for 2 days at room temperature. Brown lesions appeared on all wounded sites 2 days postinoculation (dpi) and on unwounded sites at 4 dpi. The pathogen was reisolated from the lesions of inoculated fruits and found to be N. mangiferae, thus fulfilling Koch's postulates. In control experiments, sterile agar plugs were placed on the wounded avocado fruits. These fruits remained completely free from symptoms throughout the experiment. Several species of Botryosphaeria have been reported on avocado, including N. parvum (anamorph of B. parva), Fusicoccum aesculi (anamorph of B. dothidea), and Dothiorella aromatica (= F. luteum). To our knowledge, this is the first report of N. mangiferae causing fruit rot of avocado in Taiwan. Previously, N. mangiferae has been reported on mango trees worldwide, especially in Australia and Thailand (2). The presence of N. mangiferae in the subtropical area presents a serious disease problem not only to avocado but also to mango. References: (1) Y. P. Tsai, ed. List of Plant Diseases in Taiwan. 4th ed. Taiwan Phytopathological Society, 2002. (2) B. Slippers et al. Mycologia 97:99, 2005.

First Report of Lasiodiplodia Fruit Rot of Jackfruit in Taiwan.

Jackfruit (Artocarpus heterophyllus Lam.) is a tropical fruit that is native to India. Five diseases, including Rhizopus fruit rot and anthracnose fruit rot, have been recorded in Taiwan (2). In 2003, brown lesions were observed on mature or harvested fruits at the Chiayi Agricultural Experiment Branch. The disease caused fruits to collapse and was easily distinguished from anthracnose and Rhizopus fruit rot. In the field, Rhizopus fruit rot was characterized by black flocci sporangia and mycelia covering the flowers and young fruits. Lasiodiplodia fruit rot often occurred on mature or wounded fruit and diseased fruit were covered with gray or black flat mycelia under humid conditions. In the early stage of Lasiodiplodia fruit rot, tiny yellow-brown lesions appeared on the peel. The lesions could rapidly expand to 10 cm in diameter within 5 days and became dark brown with a light margin. The rot symptoms progressed quickly from the peel surface into the sarcocarps that eventually turned black and soft. A fungus was isolated from the margin of the lesions and cultured on acidified potato dextrose agar (PDA) (pH 3.8). The morphology of the fungus was similar to Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (synonym Botryodiplodia theobromae Pat.), which causes the stem-end rot of mango, papaya, and banana in Taiwan. The fungus grew well and produced pycnidia and conidia on PDA. Young conidia were ovate, hyaline, and thin walled without septa. Mature conidia (20 to 28 × 12 to 15 µm) were dark brown and thick walled with one median septum and longitudinal striations. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was submitted to GenBank (Accession No. EU 407235) and showed 100% sequence identity with that of Botryosphaeria rhodina (anamorph Lasiodiplodia theobromae; GenBank Accession No. DQ458890). On the basis of morphological and molecular criteria, the fungus was identified as L. theobromae (1). Three healthy jackfruit fruits were wounded and inoculated with 2 × 2 mm mycelial agar plugs of the fungus from a monoconidial culture. A sterile agar plug was placed on the wounded site as a control. The fruits were kept in a box to maintain high humidity for 2 days at room temperature. Brown lesions were observed on all inoculated sites 6 days post infection. The pathogen was reisolated from the lesions of inoculated fruits, fulfilling Koch's postulate. The experiment was repeated twice. To our knowledge, this is the first report of L. theobromae causing fruit rot of jackfruit in Taiwan. References: (1) B. C. Sutton. The Coelomycetes. Commonwealth Mycological Institute, Kew, UK, 1980. (2) Y. P. Tsai, ed. List of Plant Diseases in Taiwan. 4th ed. Taiwan Phytopathological Society, 2002.

Cryogenic blockade of the central nucleus of the amygdala attenuates aversively conditioned blood pressure and respiratory responses.

The central nucleus of the amygdala (ACE) was reversibly blocked during extinction of an aversively conditioned cardiorespiratory response in unanesthetized, freely moving cats. Cryoprobes were positioned bilaterally in the ACE of 4 cats and in the nucleus entopeduncularis of 1 cat. Blood pressure typically showed biphasic changes in response to the conditioned stimulus (CS) during non-cooling trials. Blood pressure initially dropped and then rose. Heart rate consistently dropped, and respiratory rate increased in response to the CS. ACE cooling did not alter the pre-CS baseline blood pressure, heart rate or respiratory timing, but changed the cardiorespiratory response to the CS. During ACE cooling, blood pressure and respiratory responses were greatly attenuated or abolished. No significant effect on the heart rate response was observed during ACE cooling. Cooling of a nearby structure, the nucleus entopeduncularis, did not affect blood pressure, heart rate or respiratory responses to the CS. These results support the hypothesis that the ACE plays a role in both cardiovascular and respiratory regulation during conditioned aversive responses. The study also suggests that, in cats, the predominant influence of the ACE on cardiovascular control is on blood pressure rather than on heart rate regulation.

Respiratory-related discharge of periaqueductal gray neurons during sleep-waking states.

Extracellular single-unit spontaneous activity was recorded from the periaqueductal gray region (PAG) in undrugged, freely moving cats. Two analyses, cross-correlation histograms and linear regression techniques, were used to examine state relationships of neuronal discharge with respiratory patterning. Of 68 cells recorded, 19 (28%) showed a timing relationship (breath-by-breath dependency), 14 of which were state-dependent. Twenty-three (34%) showed a tonic discharge correlation with the respiratory cycle, and activity of all these cells was state-dependent. These results suggest that a subset of PAG cells may play a role in state-related respiratory patterning.

Cardiovascular-related discharge of periaqueductal gray neurons during sleep-waking states.

The periaqueductal gray (PAG) contains topographically organized areas that, upon stimulation or lesion, lead to modifications in blood pressure and heart rate and redistribution of blood flow. We examined patterns of discharge of single neurons in the PAG in 5 undrugged, freely moving cats to determine if cardiovascular patterning changes observed during different sleep states were related to activity in this midbrain region. Cross-correlation histograms and linear regression techniques were used to calculate dependencies between neuronal discharge and cardiac activity. Fifty of 68 cells recorded (74%) showed a discharge timing relationship (cardiac cycle-by-cycle) and/or a tonic discharge correlation with the cardiac cycle. Nearly all (48 or 96%) of these dependencies were state related. The large proportion of neurons showing a state-related cardiac dependency suggests that the PAG may contribute to mediating different cardiac patterns observed in each state.

Dynamic properties of respiratory timing following cocaine administration.

We assessed the timing and amplitude characteristics of diaphragmatic muscle activity following administration of intravenous cocaine HCl (10 mg/kg) to awake, unrestrained cats. Cocaine produced a pronounced tachypnea which was interrupted by deep inspiratory efforts coincident with tonic-clonic movements over the first 10 min following cocaine administration. Following that period, diaphragmatic cycle rates slowly increased for up to 1 h and were interrupted occasionally by longer inspiratory efforts which were not associated with other overt motor activities. As respiratory rate increased, breath-to-breath variability decreased, and the incidence of deep inspiratory efforts decreased. As total cycle time decreased, the ratio of inspiratory time to expiratory time remained the same between precocaine and early, intermediate and late intoxication periods. The amplitude of diaphragmatic EMG activity increased with the extreme tachypnea. A number of neural mechanisms may mediate the changes in diaphragmatic muscle activity, including hyperthermia and alteration of rostral brain influences on brainstem timing mechanisms.