Effective Management of Citrus Melanose Based on Combination of Ecofriendly Chemicals.

Citrus melanose, caused by the ascomycete fungus Diaporthe citri, is one of the most important diseases in China that affects not only the production but also the quality of citrus. In China, mancozeb is recommended to control melanose disease at the dose of 1.34 g/liter. However, it is widely applied in practice at the dose of 2.66 g/liter or even 4 g/liter, because reduced efficacy of the recommended dose was observed in regions severely damaged by melanose. In this study, some ecofriendly chemicals for melanose management were evaluated. First, the sensitivity to fungicides was screened in the laboratory based on the inhibition of mycelial growth and conidial germination of D. citri. Results showed that both quinone outside inhibitor (QoI) fungicides kresoxim-methyl and trifloxystrobin inhibited conidial germination of D. citri up to 100% at 0.1 µg/ml. The in vivo control efficacy on detached fruit indicated that treatments with elastic nanocopolymer film at 2 g/liter, mancozeb at 1 g/liter, and kresoxim-methyl at 0.1 g/liter significantly inhibited the infection process compared with the control treatment of mineral oil alone. In field trials, the efficacy of kresoxim-methyl at 0.1 g/liter and elastic nanocopolymer film at 2 g/liter mixed with mancozeb at 1 g/liter was equal to that of mancozeb at 2.66 g/liter. The use of mancozeb could be reduced greatly, and the newly developed fungicide combinations are more environmentally friendly due to the low toxicity of both QoI fungicides and elastic nanocopolymer film. The newly developed method with ecofriendly chemicals should play an important role in the management of citrus melanose in the future.

[Early changes within the lymphocyte population are associated with the long term prognosis in severely injured patients].

OBJECTIVE: To investigate the relationship between early lymphocyte responses and the prognosis in severely injured patients. METHODS: Consecutive patients with severe trauma who were treated in Peking University People's Hospital Trauma Medical Center between June 2017 and June 2020 were enrolled in this restropective chart-review study. According to the responses of lymphocyte after severe injury, the patients were divided into three groups, group 1: lymphopenia-returned to normal; group 2: persistent lymphopenia; group 3: never lymphopenic, and the outcome of 28 d were recorded. Clinical data such as gender, age, base excess, mechanism of injury, Glasgow coma scale (GCS), injury severity score (ISS) and massive blood transfusion were collected. Perform statistical analysis on the collected clinical data to understand the trend of lymphocyte changes in early trauma and the relationship with prognosis. In order to eliminate the interference of age, stratification was carried out according to whether the age was ≥ 65 years old, in different age groups, they were grouped according to whether the length of stay was ≥ 28 d, and the relationship between lymphocyte trend and length of stay was discussed. RESULTS: A total of 83 patients were included, 66 males and 17 females. The main injury mechanisms were traffic accident injuries and high-altitude fall injuries. The average ISS was (30±11) points. 65 patients had lymphopenia on the day of injury, 32 of them returned to normal on the 5th day, and the rest did not recover; the other 18 patients had normal lymphocyte levels after injury. Patients which are failure to normalize lymphopenia within the first 5 days following admission was related with the long hospitalization time and higher 28 d mortality rate. After further stratification by age, failure to normalize lymphopenia within the first 5 days following admission in the elderly group (age ≥65 years) was a risk factor for prolonged hospital stay (≥28 d), P=0.04. While in younger group, a high level of neutrophils within the first 5 d following admission was a risk factor for bad outcome. CONCLUSION: A failure to normalize lymphopenia in severely injured patients is associated with significantly higher mortality and longer hospital stay. This study reveals lymphocytes can be used as a reliable indicator for the prognostic evaluation.

[Risk factors for mechanical ventilation in patients with severe multiple trauma].

OBJECTIVE: To eludicate the risk factors of mechanical ventilation and prolonged mechanical ventilation in patients with severe multiple injuries. METHODS: Consecutive patients with severe multiple injures who were treated in Peking University People's Hospital Trauma Medical Center between December 2016 and December 2019 were enrolled in this restropective chart-review study. According to mechanical ventilation and ventilatory time, the patients were divided into mechanical ventilation (MV) group and non-mechanical ventilation (NMV) groups, prolonged mechanical ventilation (PMV) group and shortened mechanical ventilation (SMV) groups. Clinical data such as gender, age, base excess, mechanism of injury, Glasgow Coma Scale (GCS), abbreviated injury scale (AIS) and injury severity score (ISS) were collected. To indentify the risk factors of mechanical ventilation and prolonged mecha-nical ventilation, univariate and multivariate Logistic analyses were carried out. RESULTS: In the present study, 112 patients (82 male, 30 female) with severe multiple injuries having a median age of 52 (range: 16-89 years) and a median ISS of 34 (range: 16-66) were enrolled. The primary mechanism of injury was traffic accident injury and falling injury. In the study, 62 and 50 patients were assigned to MV and NMV groups, respectively. Logistic analysis showed that GCS (OR=0.72, 95%CI: 0.53-0.92, P=0.03), base excess (OR=0.56, 95%CI: 0.37-0.88, P=0.002) and multiple rib fracture (OR=1.72, 95%CI: 1.60-2.80, P=0.012) were independent significant risk factors for mechanical ventilation after severe multiple injuries. Within the mechanical ventilation group, 38 and 24 patients were assigned to PMV and SMVgroups, respectively. Compared with the SMV group, the PMV group had a higher ISS and higher rate of severe head trauma. The length of hospital stay of PMV group was longer than that of SMV groups. Meanwhile, the incidence of tracheotomy in PMV group was high. CONCLUSIONS: GCS, base excess and rib fracture might be independent risk factors for mechanical ventilation. Higher ISS and lower GCS might prolong the ventilatory time and the length of hospital stay. Meanwhile, the incidence of tracheotomy was high in PMV group because of the longer ventilatory time and poor consciousness.

[Application of multidisciplinary team (MDT) in the treatment of severe trauma].

OBJECTIVE: To explore the effect of multi-disciplinary team (MDT) in general hospitals on severe trauma patients. METHODS: This study reviewed the treatment of patients with severe trauma in trauma center of Peking University People's Hospital from March 2017 to April 2019. The baseline information: the patients' gender, age, injury mechanism, etc.; the start indicators: the Glasgow coma scale (GCS), trauma index (TI), injury severity score (ISS); the start related indicators: time for activation, time for MDT to arrive, time for CT scan, time for damage control surgery; patient treatment and prognosis: ICU (intensive care unit) length of stay, number of cured and discharged patients, number of dead cases, number of patients transferred to rehabilitation hospital, were all analyzed. It discussed the composition of MDT, the initiation scheme, the indicators of initiation of MDT for severe trauma, and analyzed the correlation between the application of MDT and the prognosis of patients. RESULTS: From March 2017 to April 2019, 112 trauma patients were treated by MDT in Peking University People's Hospital. There were 69 males and 43 females. The minimum age was 15 years, the maximum age was 89 years, most of them were 36-55 years old. The main injury mechanism was traffic accident injury. The GCS, TI, ISS were 13.0±2.9, 13.0±2.8, and 21.5±11.9, respectively. It took 3.7±0.8 minutes to start the call, 6.1±0.9 minutes for MDT personnel to arrive at the emergency rescue area, 23.8±3.0 minutes for fast CT and 92.6±15.4 minutes for injury control operation. All the hospitalized patients were treated effectively. ICU (Intensive care unit) hospitalization time was 12.6±6.7 days. 55 discharged patients were cured, 5 died (1 died of hemorrhagic shock, 4 died of severe brain injury) and 52 transferred to rehabilitation hospital. CONCLUSION: The treatment of severe trauma patients by MDT in trauma center of general hospitals can greatly improve the ability and level of treatment of severe trauma patients, make up for the lack of treatment of severe trauma especially multiple trauma patients in large general hospitals, and improve the treatment effect of severe trauma patients. It provides a reference model for large general hospitals to treat patients with severe trauma and multiple trauma and for the construction of trauma centers.

First Report of Dimethachlon Resistance in Field Isolates of Sclerotinia sclerotiorum on Oilseed Rape in Shaanxi Province of Northwestern China.

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen causing diseases in a wide range of plants, including oilseed rape (3). Substantial economic losses caused by S. sclerotiorum have been reported in the United States, Canada, Brazil, South Africa, Hungary, India, Nepal, and Japan (1). Application of fungicides is the principal tool for controlling S. sclerotiorum because of lack of high level of host resistance. Dicarboximide fungicides such as dimethachlon have been widely used to control S. sclerotiorum in recent years in China and field isolates with reduced sensitivity to dimethachlon have been reported in Jiangsu Province of eastern China (2). In order to understand the current status of dimethachlon resistance in S. sclerotiorum isolates of northwestern China, 196 and 344 isolates of S. sclerotiorum collected from oilseed rape fields in 10 counties throughout Shaanxi Province in 2011 and 2012, respectively, were assayed for sensitivity to dimethachlon using 5 µg ml-1 dimethachlon as a discriminatory dose. Mycelial plugs (6 mm in diameter) cut from the margin of a 48-h-old colony were placed in the center of petri dishes containing potato dextrose agar (PDA) amended with 5 µg ml-1 dimethachlon; PDA without fungicide served as the control. Cultures were incubated at 26°C and colony growth was measured after 72 h of incubation. Isolates that showed growth on PDA amended with fungicide were tentatively considered resistant to dimethachlon, whereas the completely inhibited isolates were considered sensitive. Results showed that 1.02% or 2 isolates of the 196 isolates collected in 2011 and 3.78% or 13 isolates of the 344 isolates collected in 2012 were resistant to dimethachlon. For all the isolates considered resistant and 42 randomly selected sensitive isolates, 50% effective concentrations (EC50) were determined on PDA amended with a series of dimethachlon concentrations. The average EC50 value of dimethachlon for sensitive isolates was 0.29 ± 0.02 µg ml-1 Resistance ratios (EC50 of resistant isolate / average EC50 of sensitive isolates) for the two resistant isolates detected in 2011 were 10.28 and 23.83, respectively, whereas resistance ratios for the 13 resistant isolates detected in 2012 ranged from 24.90 to 101.97. The average EC50 value of dimethachlon for the 13 resistant isolates detected in 2012 was 19.05 µg ml-1, and EC50 values for the two resistant isolates detected in 2011 were 2.98 and 6.91 µg ml-1, respectively. These results indicated that both resistance frequency and resistance level increased from 2011 to 2012. Bioassay results of three resistant isolates indicated that there was positive cross-resistance between dimethachlon and other dicarboximide fungicides such as iprodione and procymidone. To our knowledge, this is the first report of dimethachlon resistance in S. sclerotiorum in Shaanxi Province of northwestern China. The molecular mechanism of dimethachlon resistance in field isolates of S. sclerotiorum remains to be studied. Although resistance frequency is low at present, dimethachlon resistance should be kept in mind and fungicide resistance management tactics such as use of biological control agents, fungicide tank-mixing, or alternating dimethachlon with other fungicides having different modes of action is recommended in controlling S. sclerotiorum. References: (1) M. D. Bolton et al. Mol Plant Pathol. 7:1, 2006. (2) H. X. Ma et al. Plant Dis. 93:36, 2009. (3) L. H. Prudy. Phytopathology 69:875, 1979.

[Analysis of risk factors associated with weaning from mechanical ventilation in critical patients with major abdominal surgery].

OBJECTIVE: To analyze the risk factors involved in the weaning from mechanical ventilation in critical patients who underwent major abdominal surgery. METHODS: This retrospective study was conducted at Department of Critical Care Medicine in Peking University People's Hospital. The subjects included all critical ill patients who underwent major abdominal surgery from January 2011 to December 2013. Clinical and laboratory parameters in perioperative period were investigated for the risk factors involved in the weaning from mechanical ventilation. RESULTS: In this study, 381 patients were included, of whom, 274 were successfully weaned. We found old age, lower left ventricular ejection fraction (LVEF) before surgery, with the complication of myocardial injury after noncardiac surgery (MINS) and lower serum albumin level after surgery were the independent risk factors of weaning from mechanical ventilation. And the days of intensive care unit and 28-day mortality of patients who successfully weaned were better than patients who failed to wean. CONCLUSION: Old age, lower LVEF before surgery, with the complication of MINS and lower serum albumin level after surgery were independent risk factors of weaning in critical patients who underwent major abdominal surgery.

First Report of the ß-tubulin E198A Allele for Fungicide Resistance in Monilinia fructicola from South Carolina.

Resistance to methyl benzimidazole carbamates (MBCs) in Monilinia fructicola, the causal agent of brown rot of stone fruits, is known to be present in South Carolina peach orchards, but the molecular mechanism of resistance has not been investigated. Nine isolates were collected from peach in five counties in South Carolina and examined in petri dish assays on potato dextrose agar (PDA) for resistance to the MBC fungicide thiophanate-methyl (Topsin-M 70WP; Ceraxagri, King of Prussia, PA) at the discriminatory dose of 50 µg/ml. Isolates that grew on the fungicide-amended medium were considered highly resistant (HR). The ß-tubulin gene from four sensitive (S) and five HR M. fructicola isolates was PCR-amplified with primer pair TubA and TubR1 as described previously (1). Sequence analysis revealed several silent mutations in introns and exons in S and HR isolates and the presence of the previously described E198A allele in HR but not S isolates (1). Nucleotide sequences of the ß-tubulin gene from three S (BS, S2, MfEgpc1) and two HR isolates (MfPdt6 and BR2) were submitted to GenBank under accession numbers HM051379, HM051380, HM051381, HM051382, and HM051383, respectively. To our knowledge, this is the first report of the E198A in M. fructicola isolates from South Carolina and the East Coast. This allele is responsible for high levels of MBC resistance in M. fructicola (1). A previously reported PCR-based method using primers HRF+HRR designed to detect the E198A mutation in M. fructicola HR isolates (1) was improved by adding primer TR739 (5'-TCA CGA CGA ACA ACA TCA AGA-3') to the PCR cocktail. This additional internal primer amplified a 222-bp fragment from all S and HR isolates and therefore provided a useful, additional control. The confirmation of the E198A allele in M. fructicola isolates provides another useful tool to detect MBC resistance in commercial peach orchards in South Carolina. Reference: (1) Z. H. Ma et al. Appl. Environ. Microbiol. 69:7145, 2003.

Correlations of CYP11B2 gene polymorphisms with eclampsia.

OBJECTIVE: The aim of this study was to explore the relationship between CYP11B2 gene polymorphisms and eclampsia. PATIENTS AND METHODS: A total of 400 pregnant women treated in our hospital were enrolled in this study, including 200 normal pregnant women (pregnancy group) and 200 pregnant women with eclampsia (eclampsia group). Peripheral blood was collected from subjects of the two groups. Subsequently, genomic deoxyribonucleic acids (DNAs) were extracted and amplified via polymerase chain reaction (PCR) for detection of CYP11B2 rs4543, rs3802228 and rs104894072 polymorphisms. The expression level of CYP11B2 gene was measured as well. Additionally, the correlations of CYP11B2 gene polymorphisms with blood pressure and coagulation and renal function indexes were analyzed. RESULTS: The distribution of alleles of rs4543 locus in CYP11B2 gene was significantly different between eclampsia group and pregnancy group (p=0.027). The frequency of the allele C was significantly lower in eclampsia group than that of pregnancy group (p<0.05). There was a statistically significant difference in the genotype distribution of CYP11B2 rs3802228 (p=0.000) and rs104894072 (p=0.000) between eclampsia group and pregnancy group (p<0.05). Meanwhile, the frequency of AA genotype of rs3802228 and TG genotype of rs104894072 was remarkably higher in eclampsia group than that in pregnancy group (p<0.05). The distribution of the locus rs104894072 (p=0.044) in dominant model and rs3802228 (p=0.002) in recessive model in eclampsia group was different from that in pregnancy group (p<0.05). Eclampsia group showed remarkably elevated frequency of TT + TG of the locus rs104894072 in dominant model and lowered frequency of AG + GG of the locus rs3802228 in recessive model (p<0.05). Similarly, a significant difference was observed in the distribution of the haplotypes CGG (p=0.001) and TGT (p=0.048) in CYP11B2 gene between eclampsia group and pregnancy group (p<0.05). The linkage disequilibrium of the loci rs3802228 and rs104894072 was relatively high (D'=0.382). The polymorphism of the locus rs104894072 in CYP11B2 gene had an evident relation to CYP11B2 gene expression (p<0.05). Meanwhile, the expression of CYP11B2 gene was markedly higher in patients with GG genotype in eclampsia group (p<0.05). The polymorphism of CYP11B2 rs4543 was notably associated with PT level of patients in eclampsia group (p=0.000). Conversely, rs3802228 polymorphism was correlated with 24 h urine protein level (p=0.000). Besides, the proportion of patients with CGG haplotype was significantly larger among patients with systolic blood pressure of 140-160 mmHg (p<0.05). In addition, the proportion of patients with TGT haplotype was evidently greater among patients with systolic blood pressure >180 mmHg in eclampsia group (p<0.05). CONCLUSIONS: CYP11B2 gene polymorphisms are significantly correlated with the development and progression of eclampsia.

Cadherin 6 is activated by Epstein-Barr virus LMP1 to mediate EMT and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy, which is notorious among head-and-neck cancers with its metastatic feature. Epstein-Barr virus (EBV) infection plays a fundamental role in NPC development with the mechanism is not well understood. Here we demonstrate that EBV oncoprotein LMP1 drives EMT and metastasis of NPC by reactivating the adhesion molecule, cadherin 6 (CDH6), which normally occurs in embryogenesis with unknown role in NPC. CDH6 was found to be upregulated in LMP1-positive NPC tissues, and was identified as a target of the epithelium-specific miR-203. LMP1-activated NF-κB transcriptionally repressed the miR-203 expression by binding to the promoter region of miR-203 gene. CDH6 activation in turn induced EMT and promoted metastasis in NPC. CDH6 depletion, NF-κB inhibitor and miR-203 overexpression were able to impair the EMT effects. The miR-203 downregulation in NPC tissues was strongly associated with metastasis clinically. The CDH6 activator, Runt-related transcription factor 2 (RUNX2), was also activated by EBV in the event. For both CDH6 and RUNX2 are components at TGF-ß downstream, CDH6 became a node protein for the interplay of multiple signalings including NF-κB and TGF-ß. Therefore, the switch-on of miR-203 was important for nasopharyngeal epithelial cells to maintain normal phenotype. This study demonstrates that EBV has evolved sophisticated strategies by driving epithelial cells to obtain malignant features, particularly in NPC metastasis, providing novel biomarkers for the therapy and prognosis of EBV-associated NPC.

A baculovirus vector derived from immediately early gene promoter of Autographa californica nuclear polyhedrosis virus.

A transfer vector was constructed in which the neomycin resistance (neo) gene was under the control of a copy of Autographa californica nuclear polyhedrosis virus (AcMNPV) IE1 gene promoter at the p10 locus. After cotransfection of Spodoptera frugiperda (Sf9) cells with the transfer vector and infectious AcMNPV DNA, the recombinant virus-containing neo gene was selected by serial passage of the mixed progenies from cotransfection. This was done at low MOI in the presence of G418 in growth medium and was followed by limited dilution. RNA dot hybridization showed that the neo gene was transcribed from immediately early phase to very late phase, in infected Sf9 cells. These results demonstrate that a new baculovirus vector system had been constructed in infected cells. Furthermore, a new method for selection of positive recombinant baculovirus had been developed.

Development and use of a polymerase chain reaction-based diagnostic test for the causal mutation of progressive retinal atrophy in Cardigan Welsh Corgis.

OBJECTIVE: To develop an allele-specific polymerase chain reaction (ASPCR)-based diagnostic test for the mutation in the cyclic guanosine monophosphate phosphodiesterase alpha subunit gene (PDE6A) that causes the rcd3 form of progressive retinal atrophy (PRA) in Cardigan Welsh Corgis. ANIMALS: 1 affected homozygote, 1 unaffected carrier, 1 genotypically normal dog, and 500 unknown-PRA status Cardigan Welsh Corgis. PROCEDURE: Control blood samples were collected from Cardigan Welsh Corgis of known PRA status (ie, affected homozygote, unaffected carrier, and a genotypically normal dog) for test development. Test blood samples were collected from 500 Cardigan Welsh Corgis of unknown PRA status. Genomic DNA was used as a template in ASPCR. One pair of primers was designed to specifically amplify only the mutant allele, and another set to amplify only the wildtype allele. The PCR conditions were adjusted to ensure each reaction was 100% specific. RESULTS: The PCR conditions were identified so that each ASPCR only amplified the allele it was designed to amplify. Of the 500 Cardigan Welsh Corgis tested using the newly developed ASPCR, 457 were homozygous for the normal allele (genotypically normal), 43 were heterozygous (phenotypically normal carriers), and none were homozygous for the mutant allele. CONCLUSION AND CLINICAL RELEVANCE: A rapid, ASPCR diagnostic test able to detect the PDE6A gene mutation responsible for the rcd3 form of PRA in Cardigan Welsh Corgis was developed. The test provides a useful service for Cardigan Welsh Corgi breeders and will enable them to prevent the birth of homozygote mutant dogs.

[Effect of diazepam on delayed nausea and vomiting caused by anticancer agents].

We conducted an evaluation of the usefulness of antiemetics (5-Hydroxy-tryptamine 3 receptor antagonism, 5HT3RA) combined with diazepam for delayed nausea and vomiting due to anticancer agents in 17 patients with various malignancies (such as lung Ca, breast Ca, esophagus Ca, gastric Ca, colon Ca, and non Hodgkin's disease) for whom chemotherapy was performed with different regimens in the Dept. of Oncologic Chemotherapy, People's Hospital, Beijing Medical University. Antiemetics (5HT3RA) combined with diazepam were given only to cases that had symptoms of nausea and vomiting induced by anticancer agents in the 1st course and invalidity with antiemetics (5HT3RA) alone in this study. Antiemetic (5HT3RA) agents + Dexamethasone were dosed before chemotherapy and also diazepam 5 mg orally after 24 hours (namely, when nausea was observed). Nausea was reduced and vomiting decreased after the antiemetic treatment with 5HT3RA + Dexamethasone and diazepam. These results indicated that 5HT3RA and diazepam combination therapies were more effective than 5HT3 RA + Dexamethasone alone for delayed nausea and vomiting. Further, the antiemetics had characters that a short adminiter time, few times and a take not over dose. The only side effect related to this antiemetic therapy was light somnolence. Antiemetics combined with diazepam might be a useful therapy against delayed nausea and vomiting induced by anticancer agents.

[Construction of fusion recombinant plasmid coding for RGD peptide and urokinase B chain, its expression in Escherichia coli and preliminary characterization of its biological activity].

A synthetic RGD (Arg-Gly-Asp) peptide-coding sequence was fused with urokinase B-chain cDNA and then cloned into the prokaryotic expression vector pBV220. The fused gene was expressed in E. coli DH5 alpha under the control of PRPL promoter by 42 degrees C induction. The expression level of the fusion protein was over 9.2% of the total bacterial proteins as a or of inactive inclusion body. The purified fusion protein was obtained with similar antigenicity as urokinase shown by Western blotting. Its in vitro fibrinolysis and anti-platelet aggregation activity was also evaluated by bioassay.

Stimulation of RTH1 nuclease of the yeast Saccharomyces cerevisiae by replication protein A.

The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage. Replication protein A (RPA) is also an integral part of the DNA replication and repair processes. We have investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity. Initial in vitro studies, which employed a "flap" or a "pseudo Y" substrate containing a short 14 bp duplex region, showed the effect of RPA to be minimal or inhibitory. As RPA inhibition is unwarranted for a protein participating in the DNA replication process, we have further investigated the mechanism of such inhibition. Alternate flap and pseudo Y substrates with a long duplex region (50 bp) were prepared using M13mp19 ssDNA and synthetic oligonucleotides. Yeast RPA stimulated the endonuclease activity of RTH1 endonuclease with these substrates in a dose-dependent manner. Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the "load time" of RTH1 nuclease onto the DNA substrate decreased from approximately 5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reaction, the initial rate of the reaction increased 10-fold in the presence of yRPA. Further analysis of the interaction of RPA with various endonuclease substrates indicated that RPA has a weak helix destabilizing effect and could melt small, 14 bp, regions of duplex DNA. RTH1 endonuclease cleaves the DNA strand at the junction of single- and double-stranded DNA; consequently, the observed inhibition with small duplex substrates was likely due to duplex melting. Our studies also demonstrated that RPA stimulated the RNase H activity of RTH1 nuclease significantly. In both instances (RTH1 endonuclease and RNase H), the stimulation may involve a specific interaction of RPA with the RTH1 nuclease rather than a structural positioning of the DNA substrate by RPA.

Purification and characterization of the DNA polymerase alpha associated exonuclease: the RTH1 gene product.

We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae. Earlier, we identified a 5' --> 3' exonuclease activity that copurified with yeast DNA polymerase alpha-primase in a multiprotein complex [Biswas, E. E., et al. (1993) Biochemistry, 32, 3020-3027]. Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease. The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes. During our studies, we have discovered certain unique features of the mechanism of action of the native RTH1 nuclease. Studies presented here indicated that the exonuclease had specific pause sites during its 5'-3' exonuclease nucleotide excision. These pause sites were easily detected with long (approximately 50 bp) oligonucleotide substrates during exonucleolytic excision by the formation of a discontinuous ladder of excision product. We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pathways. Alignment of the pause sites with the nucleotide sequence of the oligonucleotide substrate indicated that the pause sites were dependent on the nucleotide sequence. Our analysis revealed that RTH1 nuclease pauses predominantly at G:C rich sequences. With poly(dA):oligo(dT)50 as substrate, the exonucleolytic products formed a continuous ladder with no evidence of pausing. The G:C rich DNA sequences are thermodynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at these sites.

Origin and modulation of ACh release from rat airway cholinergic nerves.

The release of acetylcholine (ACh) from airway parasympathetic nerves was studied in rat trachea. We established stimulus parameters, examined the role of extracellular Ca2+, and investigated the origin of the released ACh by use of vesamicol, an inhibitor of ACh uptake in synaptic vesicles. The role of muscarinic autoreceptors and prostanoids on ACh release was also studied. Tracheal rings were incubated in Krebs-Henseleit solution containing neostigmine and guanethidine with or without atropine. ACh release was measured by high-performance liquid chromatography with electrochemical detection. ACh release was dependent on frequency (0.5-16 Hz), voltage (10-25 V), and pulse duration (0.5-4 ms). At 4 Hz, one-fifth of electrical field stimulation-induced ACh release was extracellular Ca2+ independent and vesamicol resistant, indicating its nonvesicular origin. Three-fifths were Ca2+ dependent and vesamicol sensitive, indicating that it was newly synthesized, and one-fifth was Ca2+ dependent but vesamicol resistant, indicating its origin from prestored vesicles. At 16 Hz, two-fifths were nonvesicular and three-fifths were newly synthesized. Blockade of the muscarinic autoreceptor by atropine potentiated the release of ACh four- to fivefold. Neither of the cyclooxygenase inhibitors indomethacin or meclofenamate nor exogenous prostaglandin E2 affected ACh release, indicating that inhibitory prostanoids do not modulate ACh release.

Potentiation of acetylcholine release from tracheal parasympathetic nerves by cAMP.

We tested the hypothesis that increasing intracellular levels of adenosine 3', 5'-cyclic monophosphate (cAMP) increases acetylcholine (ACh) release from airway parasympathetic nerves. Muscle strips from equine trachea were preincubated for 60 min with 10(-7)M atropine, 10(-6)M neostigmine, and 10(-5) M guanethidine. The ACh release was evoked by electrical field stimulation (EFS, 20 V, 0.5 ms, 0.5 Hz) and measured by high-performance liquid chromatography with electrochemical detection. Agents known to increase cAMP, i.e., forskolin (10(-6) - 10(-4) M), 8-bromoadenosine 3', 5'-cyclic monophosphate (8-BrcAMP; 10(-5)-10(-3) M), and 3-isobutyl-1-methylxanthine (IBMX ; 10(-5)-10(-3)M) was potentiated by IBMX but not mimicked by 1,9 dideoxyforskolin. To determine if the augmentation of Ach release facilitated EFS-induced ACh release in a concentration-dependent manner. Forskolin-induced augmentation of ACh release induced by activation of beta 2-adrenoceptors is mediated via cAMP-dependent pathways, we also examined the additive effects of 8-BrcAMP, forskolin, and IBMX with 10(-6)M isoproterenol (ISO), the concentration that maximally augments ACh release. Neither forskolin nor 8-BrcAMP potentiated the maximal augmentation produced by ISO, but inhibition of phosphodiesterase with IBMX (10(-4) and 10(-3)M) augmented the maximal effect of ISO. These observations indicate that neuronal cAMP is a physiological modulator of ACh release from airway parasympathetic nerves and mediates ISO-induced augmentation of ACh release. Bronchodilators that increase cAMP may therefore paradoxically augment ACh release while relaxing smooth muscle.

Excitatory prejunctional beta 2-adrenoceptor distribution within equine airway cholinergic nerves.

We examined the effect of activation of beta 2-adrenoceptor (AR) by isoproterenol (ISO) on acetylcholine (ACh) release evoked by electrical field stimulation (EFS: 20 V, 0.5 Hz, 0.5 msec) from cholinergic nerves in five regions of equine airways. We also tested if the effect of ISO was dependent on epithelium or prostanoids by examining the effect of ISO on ACh release in the presence and absence of epithelium or cyclooxygenase inhibition. Trachealis strips or bronchial rings were preincubated for 60 min with 10(-7) M atropine, 10(-6) M neostigmine, and 10(-5) M guanethidine. The ACh amount was measured by high-performance liquid chromatography with electrochemical detection. Isoproterenol (10(-8)-10(-6) M) augmented ACh release throughout the whole airway in a concentration-dependent manner. Rubbing off the epithelium potentiated EFS-induced ACh release but neither epithelium removal nor cyclooxygenase inhibition affected the magnitude of ISO-induced augmentation of ACh release. These results indicate that in equine airway parasympathetic nerves: (1) excitatory prejunctional beta 2-adrenoceptors are distributed throughout the tracheobronchial tree; (2) the function of this excitatory beta 2-adrenoceptor is independent of endogenous prostanoids and epithelium; and (3) there is an epithelium-derived factor that inhibits ACh release.

Modulation of ACh release from airway cholinergic nerves in horses with recurrent airway obstruction.

To evaluate the functional status of neuronal alpha2-adrenoceptors (ARs) and beta2-ARs on ACh release in horses with recurrent airway obstruction (RAO), we examined the effects of the physiological agonists epinephrine (Epi) and norepinephrine (NE) and the beta2-agonists RR- and RR/SS-formoterol on ACh release from airway cholinergic nerves of horses with RAO. Because SS-formoterol, a distomer of the beta2-agonist, increases ACh release from airways of control horses only after the autoinhibitory muscarinic receptors are blocked by atropine, we also tested the hypothesis that if there is an M2-receptor dysfunction in equine RAO, SS-formoterol should increase ACh release even in the absence of atropine. ACh release was evoked by electrical field stimulation and measured by HPLC. Epi and NE caused less inhibition of ACh release in horses with RAO than in control horses. At the catecholamine concentration achieved during exercise (10(-7) M), the inhibition induced by Epi and NE was 10.8 +/- 13.2 and 3.4 +/- 6.8%, respectively, in equine RAO versus 41.0 +/- 6.4 and 27.1 +/- 5.6%, respectively, in control horses. RR- and RR/SS-formoterol (10(-8) to 10(-5) M) increased ACh release to a similar magnitude as that in control horses. These results indicate that neuronal beta2-ARs are functioning; however, the alpha2-ARs are dysfunctional in the airways of horses with RAO in response to circulating catecholamines. SS-formoterol (10(-8) to 10(-5) M) facilitated ACh release in horses with RAO even in the absence of atropine. Addition of atropine did not cause significantly more augmentation of ACh release over the effect of SS-formoterol alone. The magnitude of augmentation in horses with RAO in the absence of atropine was similar to that in control horses in the presence of atropine. The latter observations could be explained by neuronal muscarinic-autoreceptor dysfunction in equine RAO.