Intraductal Carcinoma of Salivary Gland Originating from an Intraparotid Lymph Node: A Case Report.

INTRODUCTION: Salivary gland intraductal carcinoma (IDC) is rare. We present the second case of IDC originating from an intraparotid lymph node (LN) with a more detailed description of the histogenesis, immunohistochemistry (IHC) and updated molecular information. CASE REPORT: An 87-year-old male had a tumour nodule over the left parotid tail for about 20 years. Physical examinations revealed a 4.5 cm soft, non-tender and fixed mass. After the left parotidectomy, pathology confirmed the diagnosis of IDC arising within an intraparotid lymph node. The cystic component of the tumour was lined by single to multilayered ductal cells with micropapillary growth pattern. The solid part showed intraductal proliferation of neoplastic cells in solid, cribriform, micropapillary and Roman bridge-like structure. By immunohistochemistry (IHC), the tumour cells were positive for S-100, CK (AE1/AE3), mammaglobin, SOX10, and estrogen receptor (ER), with myoepithelial cell rimming highlighted by positive p63 and calponin IHC stains. The prognosis of this patient is excellent after complete excision. DISCUSSION: The mechanism of salivary gland tumour arising in the intra-parotid gland LN was assumed to be related to salivary duct inclusion within the intraparotid gland LN which is a normal occurrence during embryology development. Although the terminology may raise some confusion about the relationship between IDC and conventional salivary duct carcinoma (SDA), they are different in immunophenotype and clinicopathologic features. IDC is characterised by S100 (+) ER (+) with predominant intraductal growth and excellent prognosis; while SDC features S100 (-) androgen receptor (+) with predominant invasive growth and aggressive behavior. Recent discovery of recurrent RET gene rearrangement in IDC but not SDC also supports that IDC is not precursor lesion of the SDC.

Incorporation of log odds of positive lymph nodes into the AJCC TNM classification improves prediction of survival in oral cancer.

OBJECTIVES: To assess the prognostic performance of a new N classification that incorporates the log odds of positive lymph nodes (LODDS) into the routinely used pathological N classification for oral squamous cell carcinoma (OSCC) patients. DESIGN: Retrospective cohort study utilising LODDS into pN category was performed, and the AJCC TNM stage and T-New N-M stage were compared with respect to 5-year disease-specific survival (DSS) rates. The discriminability was evaluated from the linear trend chi-square test, Akaike information criterion (AIC) and Harrell's c-statistic. SETTING: Medical centrer in Taiwan. PARTICIPANTS: A total of 463 patients received primary surgery and neck dissection between 2004 and 2013 for OSCC. MAIN OUTCOME MEASURES: The discriminability for 5-year DSS rates. RESULTS: The median follow-up period was 54 months, the mean patient age was 54 ± 11 years and 428 patients (92.4%) were male. The patients with higher LODDS had worse 5-year DSS rates. Incorporation of LODDS into the prognostic model based on the seventh edition of the TNM classification significantly improved discriminative performance for 5-year DSS with a lower AIC (1883 versus 1897), and higher prediction accuracy (Harrell's c-statistic: 0.768 versus 0.764). CONCLUSIONS: By utilising a merger of the LODDS and pN classifications to create a new N classification has better discriminatory and predictive ability than pathological TNM staging and could help identify high-risk patients for intense adjuvant therapy.

Evaluation of the in vitro anti-hyperglycemic effect of Cinnamomum cassia derived phenolic phytochemicals, via carbohydrate hydrolyzing enzyme inhibition.

Cinnamomum cassia (cinnamon) proanthocyanidins (PACs) are believed to have anti-hyperglycemic potential via stimulation of insulin sensitivity. The present study investigates the carbohydrate hydrolyzing enzyme inhibition of cinnamon PACs. Five grams of cinnamon bark powder were extracted in 100 mL acetone solution (CAE) (acetone: water: hydrochloric acid, 70:29.9:0.01) for 2 h at room temperature and in 100 mL deionized water for 30 min at 90 °C (CWE). PACs were purified from CAE using LH-20 (CAE-PAC) to be further evaluated. PAC contents were evaluated by 4-Dimethylaminocinnamaldehyde (DMAC) assay and yielded 795, 177 and 123 mg/g, for CAE-PAC, CAE and CWE respectively. The total phenolic contents of CAE and CWE were determined to be 152 and 134 mg/g respectively. All extracts were adjusted to the same PAC content (180, 90, 45 and 20 µg) and the inhibitory activity against rat α-glucosidase was determined. The CAE-PAC fraction had very low rat α-glucosidase inhibitory activity, CAE had the highest (IC50 0.474 mg/mL total phenolic (TP) basis) followed by CWE (IC50 0.697 mg/mL TP basis). The specific maltase and sucrase inhibitory activities were determined and CAE (IC50 0.38 and 0.10 mg/mL TP basis) had higher inhibition than CWE (IC50 0.74 and 0.37 mg/mL TP basis). Results suggest that the observed bioactivity is not PAC dependent and that CAE has a higher anti-hyperglycemic potential than CWE via inhibition of carbohydrate hydrolyzing enzymes.

The potential for intelligent decision support systems to improve the quality and consistency of medication reviews.

WHAT IS KNOWN AND OBJECTIVE: Drug-related problems (DRPs) are of serious concern worldwide, particularly for the elderly who often take many medications simultaneously. Medication reviews have been demonstrated to improve medication usage, leading to reductions in DRPs and potential savings in healthcare costs. However, medication reviews are not always of a consistently high standard, and there is often room for improvement in the quality of their findings. Our aim was to produce computerized intelligent decision support software that can improve the consistency and quality of medication review reports, by helping to ensure that DRPs relevant to a patient are overlooked less frequently. A system that largely achieved this goal was previously published, but refinements have been made. This paper examines the results of both the earlier and newer systems. METHODS: Two prototype multiple-classification ripple-down rules medication review systems were built, the second being a refinement of the first. Each of the systems was trained incrementally using a human medication review expert. The resultant knowledge bases were analysed and compared, showing factors such as accuracy, time taken to train, and potential errors avoided. RESULTS AND DISCUSSION: The two systems performed well, achieving accuracies of approximately 80% and 90%, after being trained on only a small number of cases (126 and 244 cases, respectively). Through analysis of the available data, it was estimated that without the system intervening, the expert training the first prototype would have missed approximately 36% of potentially relevant DRPs, and the second 43%. However, the system appeared to prevent the majority of these potential expert errors by correctly identifying the DRPs for them, leaving only an estimated 8% error rate for the first expert and 4% for the second. WHAT IS NEW AND CONCLUSION: These intelligent decision support systems have shown a clear potential to substantially improve the quality and consistency of medication reviews, which should in turn translate into improved medication usage if they were implemented into routine use.

Targeted inhibition of mitochondrial Hsp90 suppresses localised and metastatic prostate cancer growth in a genetic mouse model of disease.

BACKGROUND: The molecular chaperone heat shock protein-90 (Hsp90) is a promising cancer drug target, but current Hsp90-based therapy has so far shown limited activity in the clinic. METHODS: We tested the efficacy of a novel mitochondrial-targeted, small-molecule Hsp90 inhibitor, Gamitrinib (GA mitochondrial matrix inhibitor), in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. The TRAMP mice receiving 3-week or 5-week systemic treatment with Gamitrinib were evaluated for localised or metastatic prostate cancer, prostatic intraepithelial neoplasia (PIN) or localised inflammation using magnetic resonance imaging, histology and immunohistochemistry. Treatment safety was assessed histologically in organs collected at the end of treatment. The effect of Gamitrinib on mitochondrial dysfunction was studied in RM1 cells isolated from TRAMP tumours. RESULTS: Systemic administration of Gamitrinib to TRAMP mice inhibited the formation of localised prostate tumours of neuroendocrine or adenocarcinoma origin, as well as metastatic prostate cancer to abdominal lymph nodes and liver. The Gamitrinib treatment had no effect on PIN or prostatic inflammation, and caused no significant animal weight loss or organ toxicity. Mechanistically, Gamitrinib triggered acute mitochondrial dysfunction in RM1 cells, with loss of organelle inner membrane potential and release of cytochrome-c in the cytosol. CONCLUSIONS: The Gamitrinib has pre-clinical activity and favourable tolerability in a genetic model of localised and metastatic prostate cancer in immunocompetent mice. Selective targeting of mitochondrial Hsp90 could provide novel molecular therapy for patients with advanced prostate cancer.

Detection and characterization of enterovirus associated with herpangina and hand, foot, and mouth disease in Seoul, Korea.

BACKGROUND: Human enteroviruses (HEVs) are a major cause of herpangina, HFMD (hand, foot, and mouth disease), and other neurological diseases in Seoul, Korea. METHODS: A total of 56 specimens from hospitalized patients collected from February to December 2009 (37 females and 19 males) in Seoul were tested for HEV from stool, throat swab, and vesicle swab samples taken from patients with herpangina or HFMD using cell culture and RT-PCR in 2009. By the 1D gene, encoding the VP1 capsid protein, seven different HEV genotypes were detected with Coxsackievirus A2, A4, A5, A9, A16 (CA), Coxsackievirus B1 (CB), and Enterovirus 71 (EV71). The most prevalent genotype was CA16 (6, 10.7%), followed by CA2 (4, 7.1%), CA5 (4, 7.1%), EV71 (2, 3.6%), CA4 (1, 1.8%), CA9 (1, 1.8%), and CB1 (1, 1.8%). The 1D gene sequences of two EV71 strains were closely related with one another (98.5% nucleotide similarity) and belonged to the C4 genotype. CONCLUSIONS: It is important to continuously survey the genetic characteristics of EV71 and CA16 from patients, which will provide useful data that aids in our understanding of HFMD infections in Seoul, Korea and may contribute to future control.

Comparison of therapeutic results in sudden sensorineural hearing loss with/without additional hyperbaric oxygen therapy: a retrospective review of 465 audiologically controlled cases.

OBJECTIVE: To investigate the necessity of routine application of hyperbaric oxygen therapy for sudden sensorineural hearing loss. DESIGN/SETTING AND PARTICIPANTS: A retrospective chart review looked at 465 patients, with 353 of them receiving pharmacologic treatments alone. Among these patients, 76 underwent systemic steroid treatment only (steroid group) and 277 received systemic steroids and dextran (steroid-dextran group). The remaining 112 patients were treated with hyperbaric oxygen in addition to pharmacologic agents (steroid-dextran-hyperbaric oxygen group). MAIN OUTCOME MEASURES: The outcome was determined by comparing the difference of pure-tone thresholds and absolute hearing gains after treatment calculated at each audiometric octave frequency or grouped frequencies of audiograms. On the basis of the severity of initial hearing loss, patients were classified at three scales of hearing impairments measured in decibels hearing level (dBHL): ≦ 70 dBHL, less severe; 71-90 dBHL, severe; and ≧ 91 dBHL, profound. The outcomes of their hearing recovery were classified into three recovery grades: good, fair and poor. RESULTS: In those patients with initial hearing loss >90 dBHL, the addition of hyperbaric oxygen to steroid-dextran gave a significant hearing gain difference (P = 0.030) by showing a greater hearing gain of 24.5 ± 2.7 dB compared with steroid only (12.9 ± 3.7 dB) or steroid-dextran (15.6 ± 2.7 dB). This outcome was confirmed when we compared the outcome using the recovery grading; steroid-dextran-hyperbaric oxygen group showed that more patients with initial profound (≧ 91 dBHL) hearing loss responded to hyperbaric oxygen treatment by exhibiting good and fair recoveries (2% and 70%) as compared with steroid only (0% and 42%) or steroid-dextran (8% and 46%) groups (P = 0.043), while the patients with initial severe (71-90 dBHL) and less severe (≦ 70 dBHL) hearing loss responded to the addition of hyperbaric oxygen treatment with less favourable recoveries. Furthermore, the addition of dextran in steroid-dextran group showed no significant benefit compared with the steroid group (P = 0.435). CONCLUSIONS: When applied as an adjuvant to pharmacologic agents, hyperbaric oxygen benefits patients with initial profound sudden sensorineural hearing loss. Therefore, we recommend the routine application of hyperbaric oxygen in conjunction with pharmacologic agents for those patients. The addition of dextran to steroid has no benefit and cannot be recommended.

Hyperbaric oxygen ameliorates delayed neuropsychiatric syndrome of carbon monoxide poisoning.

OBJECTIVES: Delayed neuropsychiatric syndrome (DNS) is characterized by mental impairment, motor dysfunction, dementia, or psychosis that develops between a few days and weeks after acute carbon monoxide (CO) poisoning. One possible mechanism responsible for CO-mediated encephalopathy involves oxidative stress, such as lipid peroxidation, caused by the cellular uptake of CO and which leads to an inflammatory cascade. There is no current effective treatment for DNS. We applied 8-40 sessions of hyperbaric oxygen therapy (HBO2) to patients with DNS and evaluated its effectiveness. METHODS: After admission, all patients were administered piracetam or bromocriptine, or both, and received HBO2. Neuropsychiatric tests included EEG, mini-mental status examination (MMSE), brain MRI, event-related potential (ERP), and brain perfusion scan (brain SPECT). Results of these tests were compared before and after HBO2, and the clinical features were monitored during this period. RESULTS: The symptoms of DNS for all patients improved significantly after HBOT. Although white matter changes remained evident in the brain MRI scans, other examinations such as EEG, MMSE, ERP, and 99mTc-ECD brain SPECT were nearly normal after HBOT. CONCLUSION: Our results suggest that HBO2 decreases the severity of impairment in patients with DNS. Although a large randomized trial is required to address the efficacy of this therapy, therapeutic application of HBO2 may be recommended in patients with DNS after CO poisoning.

Three-body nonmesonic weak decay of the (Lambda)12C hypernucleus.

We have measured the branching ratio of the three-body process in the nonmesonic weak decay of Lambda12C to be 0.29+/-0.13. This result was obtained by reproducing the nucleon and the nucleon pair yields introducing a measured final state interaction. At the same time, we have determined the absolute decay widths, Gamma(n) and Gamma(p), along with Gamma2N, whose relative ratio has been a long-standing puzzle. Including the three-body process, we have successfully reproduced the nucleon energy distribution, the coincidence two-nucleon angular correlation, and the momentum sum distribution simultaneously.

Responses of wild plant species to polycyclic aromatic hydrocarbons in soil.

Responses of plants to polycyclic aromatic hydrocarbons (PAHs) contamination were determined with fifty-five Korean wild plants. Responsiveness of species was evaluated based on germination and shoot weight and shoot length of plants grown in soil spiked with four PAHs (pyrene, fluorene, phenanthrene and fluoranthene). Seeds of test plants were germinated with mixtures of PAHs of 0, 10, 30, 100, 300 mg kg(-1) spiked in soil. Seed germination of test plants changed when subjected to PAHs. As compared to control germination percentages ranged from 0 (completely inhibited) to 242.9% (highly promoted) of control at 300 mg kg(-1) of PAHs. In germination responses, Fabaceae plants were much less affected (105% of control) compared to species belonging to Caryophyllaceae (18.7% of control), which showed highly susceptible responses. Results demonstrated that seed germination was affected by species-specific responses to PAHs. In seedling growth experiments on Bromus tectorum and Veronica persica, species classified as highly susceptible in germination experiments, a low No Observed Effect Concentration (NOEC) of 10 mg kg(-1) was observed. On the other hand, NOEC was 100 mg kg(-1) in Bromus japonicus and Cerastium holosteoides var. hallaisanense, which were also classified as highly susceptible by the germination experiment. However, most species classified as susceptible showed high NOEC of greater than 10 mg kg(-1). EC(50) values of test species ranged from 2.87 x 10(2) (Humulus japonicus) to 8.05 x 10(81) mg kg(-1) (Bidens bipinnata) based on shoot length. The wide range of EC(50) for shoot weight suggests that shoot weight is more appropriate as an endpoint for PAHs toxicity than shoot length for determining the susceptibility of plant species to PAHs. It was confirmed that dose-response of plants to PAHs spiked soil can be used to estimate critical concentration of PAHs inhibiting early establishment of plants in contaminated fields.

Drug resistance in extra-pulmonary tuberculosis in South Korea: comparison with pulmonary tuberculosis.

OBJECTIVE: To compare the prevalence of and trends in drug resistance in extra-pulmonary tuberculosis (EPTB) and pulmonary tuberculosis (PTB). METHODS: We retrospectively analysed the results of phenotypic drug susceptibility testing (DST) in culture-confirmed TB patients from January 2010 to December 2014 at seven university hospitals in South Korea. RESULTS: Of 5599 patients included, 320 (5.7%) were classified in the EPTB group and 5279 (94.3%) in the PTB group. The proportion of EPTB among all TB cases had gradually increased from 2010 to 2014 (P = 0.004). Among both new and previously treated patients, there were no significant differences in rates of resistance to any kind of anti-tuberculosis drug between the EPTB and PTB groups. The trends in drug resistance rates among new patients were similar in both the EPTB and PTB groups. The rates of multidrug-resistant TB among new patients gradually decreased in both groups (P = 0.031 and P = 0.001, respectively). CONCLUSION: The prevalence of and trends in drug resistance among new patients are not significantly different between patients with EPTB and those with PTB.

Lack of carcinogenicity of lyophilized Agaricus blazei Murill in a F344 rat two year bioassay.

The Brazilian mushroom Agaricus blazei Murill has antimutagenic, antioxidant, immunostimulatory and antitumorigenic activities, and is increasingly consumed as a health food worldwide. We undertook the present study to evaluate the chronic toxicity and oncogenicity of A. blazei Murill in F344 rats. To establish a no-observed-adverse-effect level (NOAEL), four treatment groups of 100 rats each (50 males and 50 females) were fed a powder diet containing lyophilized A. blazei aqueous extract at 0, 6250, 12,500, and 25,000 ppm for up to 2 years. During this period, there was no remarkable change in mean body weight, body weight gain, hematologic or serum chemistry parameters, or absolute or relative organ weights in control or treatment groups. Mortality in male treatment groups (26%, 16%, and 30%), however, was significantly lower than in controls (48%). Histopathological studies showed no increased incidence of tumors in any treatment group, and total tumor incidence across all groups was comparable to historical data. In conclusion, an A. blazei Murill lyophilized powder diet even at 25,000 ppm (1176 mg/kgb x w x /day for male rats and 1518 mg/kgb.w./day for female rats) resulted in no remarkable carcinogenic effects in F344 rats over a 2-year period. Therefore, the dietary NOAEL is 25,000 ppm.

Temperature and salt effects on proteolytic function of turnip mosaic potyvirus nuclear inclusion protein a exhibiting a low-temperature optimum activity.

The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a protease responsible for processing the viral polyprotein into functional proteins. The NIa protease exhibits an unusual optimum proteolytic activity at about 16 degrees C. In order to understand the origin of the low-temperature optimum activity, the effects of temperature and salt ions on the catalytic activity and the structure of the NIa protease have been investigated. The analysis of the temperature dependence of k(cat) and K(m) revealed that K(m) decreases more drastically than k(cat) as temperature decreases. The thermodynamic analysis showed that the decrease of K(m) is driven entropically, suggesting a possibility that the substrate binding might need a large entropy cost. The secondary structure of the NIa protease was significantly perturbed at temperatures between 20 and 40 degrees C and the protease was unfolded at very low concentrations of guanidine hydrochloride with a transition midpoint of 0.8 M. These results suggest that the NIa protease is highly flexible in structure. Interestingly, salt ions including NaCl, KCl, CaCl(2) and MgCl(2) stimulated the proteolytic activity by 2-6-fold and increased the optimum temperature to 20-25 degrees C. This stimulatory effect of the salt ions was due to the lowering of K(m). The salt ions promoted the structural rigidity as evidenced in the higher resistance to the heat-induced unfolding in the presence of the salt ions. The increase in rigidity may lead to the lowering of K(m) possibly by reducing the entropic cost for substrate binding. Taken together, these results suggest that the NIa protease is highly flexible in structure and the low-temperature optimum activity might possibly be attributed to lowered entropy cost for substrate binding at lower temperatures.

4-Week repeated intravenous dose toxicity study of a new camptothecin anticancer agent CKD-602 in dogs.

CKD-602 is a new camptothecin derivative antitumor agent with a formula (7-[2-(N-isopropylamino)ethyl]-(20S)-camptothecin) developed by Chong Kun Dang Pharmaceutical Company in Korea. In the present study, the subacute toxicity of CKD-602 was investigated after 4-week repeated intravenous administration of the test chemical in beagle dogs. The test chemical was administered intravenously at dose levels of 0, 0.001, 0.005, or 0.01 mg/kg/day for 4 weeks to male and female dogs (n = 3 for male and female dogs for each dose). During the test period, clinical signs, mortality, body weights, food and water consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights and histopathology were examined. In the high dose group, an increase in the incidence of abnormal clinical signs and a decrease in food and water intake and body weight gain were observed in both sexes. Hematological investigations revealed decreased white blood cells (WBC) in both sexes and reduced red blood cells (RBC), hemoglobin and hematocrit in females. Histopathological examinations revealed an increase in the incidence of atrophy of the sternal and femoral marrow and spleen in both sexes and atrophy of the thymus and mesenteric lymph node in males. No treatment-related adverse effects were observed in both sexes of the low and middle dose groups. In conclusion, the 4-week repeated intravenous dose of CKD-602 to beagle dogs caused increases in the clinical signs and histopathological changes, and decreases in the body weight gain, food and water intake, RBC, hemoglobin, hematocrit and WBC at the dose level of 0.01 mg/kg/day. In the present experimental conditions, the target organs were determined to be bone marrow, blood cells, spleen, thymus, and mesenteric lymph node. The no-observed-adverse-effect levels (NOAEL) for males and females were considered to be 0.005 mg/kg/day, respectively.

Systemic administration of lipopolysaccharide induces release of nitric oxide and glutamate and c-fos expression in the nucleus tractus solitarii of rats.

There is increasing recognition that communication pathways exist between the immune system and brain, which allows bidirectional regulation of immune and brain responses to infection. The endotoxin lipopolysaccharide (LPS) has been reported to elicit release of cytokines and expression of inducible nitric oxide synthase (iNOS) in peripheral organs. Whereas LPS given systemically causes endotoxic shock, little is known about its central nervous system action, particularly the induction of iNOS. Nitric oxide (NO) and glutamate in the nucleus tractus solitarii (NTS) are important mediators of central cardiovascular regulation. We have previously demonstrated that intravenous injections of LPS increased the NO precursor L-arginine-induced depressor effect in the NTS. The present study investigated further the effects of LPS on the release of NO and glutamate in the NTS and the expression of c-fos, an immediate early response gene product, in neural substrates for central cardiovascular control. In vivo microdialysis coupled with chemiluminescence and electrochemical detection techniques were used to measure extracellular levels of NO and glutamate in the rat NTS. Immunohistochemistry was used for the examination of c-fos protein expression. We found that intravenous infusion of LPS (10 mg/kg) produced a biphasic depressor effect, with an early, sharp hypotension that partially recovered in 15 minutes and a secondary, more prolonged hypotension. In the NTS, a progressive increase of extracellular glutamate and NO levels occurred 3 and 4 hours after LPS was given, respectively. The effects of LPS on the induction of delayed hypotension and NO formation in the NTS were abolished by pretreatment with the iNOS inhibitor aminoguanidine. Finally, c-fos protein expression in the NTS and related structures for cardiovascular regulation was observed after LPS challenge. Taken together, these data suggest that an endotoxin given systemically can elicit delayed increases of glutamate release and iNOS-dependent NO production in the NTS and activate the central neural pathway for modulating cardiovascular function.

Anti-intercellular adhesion molecule-1 antibody and intercellular adhesion molecule-1 gene deficiency do not prevent pulmonary neutrophil recruitment in polymicrobial sepsis.

The intercellular adhesion molecule (ICAM)-1 is expressed constitutively in normal lungs and increased in pulmonary inflammation. Whether increased ICAM-1 expression in the lung contributes to neutrophil sequestration during lung inflammation in sepsis is unclear. We tested this hypothesis in mice after systemic sepsis from cecal ligation and puncture (CLP). ICAM-1 expression in mouse CLP lung tissue was found to increase with time. The time course of lung ICAM-1 up-regulation correlated with increases in lung myeloperoxidase (MPO) activity and neutrophil sequestration by light microscopy. The monoclonal IgG2b rat anti-mouse antibody, an anti-ICAM-1 antibody (YN1/1.7), administered intravenously at doses of 3, 10, or 30 mg/kg, however, did not decrease the lung MPO levels compared with nonimmune rat IgG. In support of these findings, lung MPO content in ICAM-1-deficient mice that underwent CLP was significantly higher than similarly treated ICAM-1-sufficient mice. Our results suggest that neutrophil sequestration in the mouse lung after CLP is not dependent on ICAM-1.

Hyperbaric oxygen attenuates lipopolysaccharide-induced acute lung injury.

OBJECTIVES: To study the effect of hyperbaric oxygen therapy in alleviating acute lung injury induced by lipopolysaccharide (LPS) in rats. DESIGN AND INTERVENTIONS: The rats received an intraperitoneal injection of LPS (15 mg/kg). Animals were either breathing air at 1 ATA or subjected to hyperbaric oxygen (HBO(2)) therapy. The HBO(2) therapy was carried out in a hyperbaric chamber at a pressure of 3 ATA for 90 min. In another two groups, LPS-treated rats also received intraperitoneal injection of N(omega)-nitro-L-arginine (LNAME, 25 mg/kg) or L-N(6)-(iminoethyl)lysine (LNIL, 10 ml/kg). Another two groups of LPS-treated rats were subjected to HBO(2) exposure after the injection of L-NAME or L-NIL. MEASUREMENTS AND MAIN RESULTS: The bronchoalveolar lavage (BAL) was done into the left lung at 7.5 h after intraperitoneal injection of LPS. Parts of the right lung were excised for myeloperoxidase measurement, whereas the rest was collected for wet/dry ratio determination. LPS significantly increased the nitrite/nitrate (NO(x)(-)) concentration (34.4+/-15.7 vs 4.5+/-3.1 microM), LDH activity (66+/-17 vs 46+/-15 mAbs/min), and protein concentration (373+/-119 vs 180+/-90 mg/l) in the BAL fluid. Treatment with HBO(2) immediately after the injection of LPS enhanced the increase of NO(x)(-) production, but reduced the LDH and protein in BAL fluid to the control levels. Pretreatment with either L-NAME or L-NIL abolished the increase of NO(x)(-) in the BAL fluid and further elevated the LDH level and protein concentration. CONCLUSION: Our results suggested that HBO(2) alleviates the LPS-induced acute lung injury, which may be related to the enhancement of nitric oxide production.

Molecular cloning, expression, and purification of nuclear inclusion A protease from tobacco vein mottling virus.

The gene encoding the C-terminal protease domain of the nuclear inclusion protein a (NIa) of tobacco vein mottling virus (TVMV) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue. The 27-kDa protease was purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography. The purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, Ac-Glu-Asn-Asn-Val-Arg-Phe-Gln-Ser-Leu-amide and Ac-Arg-Glu-Thr-Val-Arg-Phe-Gln-Ser-Asp-amide, containing the junction sequences between P3 protein and cylindrical inclusion protein and between nuclear inclusion protein b and capsid protein, respectively. The Km and k(cat) values were about 0.2 mM and 0.071 s(-1), respectively, which were approximately five-fold lower than those obtained for the NIa protease of turnip mosaic potyvirus (TuMV), suggesting that the TVMV NIa protease is different in the binding affinity as well as in the catalytic power from the TuMV NIa protease. In contrast to the NIa proteases from TuMV and tobacco etch virus, the TVMV NIa protease was not autocatalytically cleaved into smaller proteins, indicating that the C-terminal truncation is not a common phenomenon occurring in all potyviral NIa proteases. These results suggest that the TVMV NIa protease has a unique biochemical property distinct from those of other potyviral proteases.

Characterization of active-site residues of the NIa protease from tobacco vein mottling virus.

Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased kcat marginally by about 4.7-fold and increased Km by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in kcat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pKa values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.