Providencia alcalifaciens causes barrier dysfunction and apoptosis in tissue cell culture: potent role of lipopolysaccharides on diarrheagenicity.

Providencia alcalifaciens is a member of the Enterobacteriaceae family that occasionally causes diarrheagenic illness in humans via the intake of contaminated foods. Despite the epidemiological importance of P. alcalifaciens, little is known about its pathobiology. Here we report that P. alcalifaciens causes barrier dysfunction in Caco-2 cell monolayers and induces apoptosis in calf pulmonary artery endothelial cells. P. alcalifaciens infection caused a 30% reduction in transepithelial resistance in Caco-2 cell monolayers, which was greater than that for cells infected with Shigella flexneri or non-pathogenic Escherichia coli. As with viable bacteria, bacterial lysates treated with heat, benzonase or proteinase, but not with polymixin B, were also involved in the cellular response. TLR4 antibody neutralisation significantly restored the P. alcalifaciens-induced transepithelial resistance reduction in Caco-2 cells, suggesting that lipopolysaccharides (LPSs) might play a central role in this cellular response. Western blotting further indicated that P. alcalifaciens LPSs reduced occludin levels, whereas LPSs from Shigella or E. coli did not. Although the viability of Caco-2 cells was not altered significantly, the calf pulmonary artery endothelial cell line was highly sensitive to P. alcalifaciens infection. This sensitivity was indeed dependent on LPS, which induced rapid apoptosis. Together, these data show that P. alcalifaciens LPSs participate in epithelial barrier dysfunction and endothelial apoptosis. The findings give insight into the LPS-dependent cell signal events affecting diarrheagenicity during infection with P. alcalifaciens.

Risk factors for non-cancer health events in patients with head and neck squamous cell carcinoma.

BACKGROUND: Cancer progression and non-cancer-related morbidities can affect the quality of life and survival of patients with head and neck squamous cell carcinomas (HNSCC). The aim of this study was to investigate the risk factors for the development of non-cancer health events (NCHEs) in HNSCC. PATIENTS AND METHODS: The study involved 465 previously-untreated patients with HNSCC diagnosed between 2005 and 2009 at the Asan Medical Center. Non-cancer-associated morbidity was defined as readmission after treatment of HNSCC due to non-cancer-related causes. NCHEs were defined as the occurrence of non-cancer-associated morbidity or mortality. The incidence and risk factors for NCHEs were analyzed. RESULTS: During the median follow-up of 47.6 months, non-cancer morbidity and mortality occurred in 83 (17.8%) and 25 patients (5.4%), respectively. Thirteen patients (52%) died from non-cancer-related causes with no previous admission for non-cancer causes. Multivariate analysis showed that the incidence of NCHEs was significantly associated with a Charlson comorbidity index ≥1 and stage III/IV disease (P < 0.001). CONCLUSIONS: Patients with comorbidities and advanced diseases may be at higher risk of NCHEs. Because NCHEs are sometimes life-threatening, every effort should be made to avoid unexpected non-cancer-associated mortality in the HNSCC patients.

Allopurinol-induced aplastic anemia in a patient with chronic kidney disease.

Aplastic anemia is a rare complication of allopurinol use. We report an unusual case of aplastic anemia associated with allopurinol therapy for hyperuricemia in a patient with chronic kidney disease. A 37-year-old female patient diagnosed with Stage III chronic kidney disease was admitted with pancytopenia. She had a history of taking allopurinol for 5 months. Her bone marrow showed extremely decreased cellularity (< 20%) and there was no malignant cell infiltration. She was free of infections, including parvovirus B19, cytomegalovirus and Epstein-Barr virus. These results suggested a diagnosis of aplastic anemia. Allopurinol was discontinued immediately and treatment with blood transfusions and prednisolone was begun. After 6 months, the bone marrow cellularity improved to approximately 70%. Recently, it was suggested that decreased activity of multidrug resistance P-glycoprotein may play a role in acquired aplastic anemia. So we measured the inhibitory effect of allopurinol and oxypurinol on P-glycoprotein activity. But neither allopurinol nor oxypurinol inhibited P-glycoprotein activity.

Removal of a metallic foreign body embedded in the external nose via open rhinoplasty approach.

Most metallic foreign bodies are inert, but they can cause chronic inflammatory reactions and be a source of infection. Identification and removal of foreign bodies from wounds is often necessary. The present report describes two cases of a foreign body embedded in the external nose. Each case was successfully treated by an open rhinoplasty approach. This approach is an effective and safe method for removal of foreign bodies in the external nose. It provides a good surgical field and a better cosmetic outcome than a conventional incision.

Antibacterial compounds from Rose Bengal-sensitized photooxidation of beta-caryophyllene.

The bactericidal activity of beta-caryophyllene photooxidized in acetonitrile was examined for 5 Gram-positive and 4 Gram-negative foodborne bacteria. The beta-caryophyllene (5 x 10(-3) M) was photooxidized in acetonitrile containing Rose Bengal (6.25 x 10(-4) M) for 24 h under fluorescent light. The antimicrobial activities of samples were determined by the agar-disc diffusion method. Active compounds from the photooxidized beta-caryophyllene were isolated by silica gel open-column chromatography in conjunction with recyclic high-performance liquid chromatography (HPLC), and were identified by infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. The antimicrobial activity of the photooxidized beta-caryophyllene was strongly enhanced against Streptococcus aureus and Vibrio parahaemolyticus, relative to that of beta-caryophyllene, but was weakly enhanced against other tested bacteria. The photooxidized beta-caryophyllene contained 3 active compounds specific for these 2 bacteria, and the compounds were identified as 5-alpha-hydroxycaryophylla-4(12),8(13)-diene, 5-alpha-hydroxycaryophylla-3(4),8(13)-diene, and 5-beta-hydroxycaryophylla-3(4),8(13)-diene. The efficacies of these compounds were similar, but the efficacy of 5-beta-hydroxycaryophylla-3(4),8(13)-diene was slightly higher than that of the other 2 compounds. The results suggest that the antibacterial activities of beta-caryophyllene for S. aureus and V. parahaemolyticus could be enhanced by dye-sensitized photooxidation, and the photooxidized beta-caryophyllene and the isolated individual compounds could be useful antimicrobial agents to control the growth of S. aureus and V. parahaemolyticus in certain food systems.

Utility of FDG PET in patients with squamous cell carcinomas of the oral cavity.

BACKGROUND: The utility of (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET) in oral cavity cancer has received little attention in a clinician's perspective. We systematically evaluated the clinical roles of FDG PET in patients with oral cavity squamous cell carcinomas (SCCs). METHODS: Between August 2001 and February 2005, 82 new patients with resectable oral cavity SCCs underwent CT/MRI and FDG PET at initial staging and follow-up. The sensitivity and specificity of CT/MRI and FDG PET for neck metastases were compared with histopathologic reference of 67 patients who underwent neck dissection. The relationships between the maximal standardized uptake value (SUV) of primary tumors and clinicopathologic parameters, such as gender, age, tumor thickness, local invasiveness, T and N categories, tumor-node-metastasis stage, and histological grade, as well as with disease-free survival (DFS), were assessed. RESULTS: FDG PET was more sensitive than CT/MRI for detecting cervical metastases on a level-by-level basis (38/43 vs. 28/43; P=0.002). Age, T and N categories, tumor thickness (>8mm) and SUV (>5.0) were also significant variables of 3-year DFS in univariate analysis. T category was an independent determinant of DFS in multivariate analysis (P<0.05). During a mean follow-up of 36 months, FDG PET correctly diagnosed locoregional recurrences in 20 patients, distant metastases in six and second cancers in five. CONCLUSION: FDG PET may have potential roles in initial staging, survival prediction, and the detection of recurrences and second cancers.

Comparison of tear secretion and tear film instability after photorefractive keratectomy and laser in situ keratomileusis.

PURPOSE: To evaluate and compare tear secretion and tear film instability following photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, Yonsei University School of Medicine, Seoul, Korea. METHODS: In a prospective study, 36 eyes (21 patients) had PRK and 39 eyes (25 patients) had LASIK to correct myopia. Tear secretion and tear film instability were tested preoperatively and 3 and 6 months postoperatively using Schirmer test values, tear breakup time (BUT) scores, and tear osmolarity. RESULTS: Six months after surgery, the change in Schirmer test values from preoperative levels was -14.57% +/- 6.39% (SD) in the PRK eyes and -23.40% +/- 5.94% in the LASIK eyes and the change in BUT scores, -12.54% +/- 8.28% and -18.79% +/- 13.01%, respectively. The change in tear osmolarity was 14.95% +/- 6.46% and 35.63% +/- 8.51%, respectively. CONCLUSIONS: The decrease in tear secretion was greater after LASIK than after PRK at 6 months. Proper treatment of dry eye is required after LASIK and PRK, particularly in the LASIK postoperative period.

A novel strategy for designing irreversible inhibitors of metalloproteases: acetals as latent electrophiles that interact with catalytic nucleophile at the active site.

A new strategy for design of irreversible inactivators for carboxypeptidase A (CPA), a prototypic zinc protease, has been developed by exploiting the property of acetals to generate an oxacarbenium ion intermediate in the conversion into the corresponding carbonyl compounds. The design strategy is exemplified by 2-benzyl-5-alkyl-3,5-dioxapentanoic acids (1a-c). Interestingly, (R)-1b is slightly more potent than an (S)-1b as an inactivator of CPA.

Angiopoietin-1 inhibits irradiation- and mannitol-induced apoptosis in endothelial cells.

BACKGROUND AND PURPOSE: Angiopoietin-1 (Ang1) is a vasculogenic factor that signals through the endothelial cell-specific Tie2 receptor tyrosine kinase. We recently reported that Ang1 prevented apoptosis induced by serum deprivation in endothelial cells. In this study, we examined whether Ang1 prevents apoptosis in endothelial cells treated with irradiation or clinical concentrations of mannitol. METHODS AND RESULTS: ++Ang1 prevented irradiation- and mannitol-induced apoptosis in human umbilical vein endothelial cells in a dose-dependent manner. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the antiapoptotic effect of Ang1. Two phosphatidylinositol 3'-kinase (PI3-kinase)-specific inhibitors, wortmannin and LY294002, blocked the Ang1-induced antiapoptotic effect. The antiapoptotic potency of Ang1 was similar to or greater than that of vascular endothelial growth factor, basic fibroblast growth factor, and endothelin-1. Ang1 also prevented apoptosis in cultured endothelial cells from porcine pulmonary and coronary arteries and in endothelial cells of explanted rat aorta. CONCLUSIONS: Ang1 promotes the survival of endothelial cells in irradiation- and mannitol-induced apoptosis through Tie2 receptor binding and PI3-kinase activation. Pretreatment with Ang1 could be beneficial in maintaining normal endothelial cell integrity during intracoronary irradiation or systemic mannitol therapy.

Cell cycle regulators during human atrial development.

OBJECTIVES: The molecular mechanisms that regulate cardiomyocyte cell cycle and terminal differentiation in humans remain largely unknown. To determine which cyclins, cyclin dependent kinases (CDKs) and cyclin kinase inhibitors (CKIs) are important for cardiomyocyte proliferation, we have examined protein levels of cyclins, CDKs and CKIs during normal atrial development in humans. METHODS: Atrial tissues were obtained in the fetus from inevitable abortion and in the adult during surgery. Cyclin and CDK proteins were determined by Western blot analysis. CDK activities were determined by phosphorylation amount using specific substrate. RESULTS: Most cyclins and CDKs were high during the fetal period and their levels decreased at different rates during the adult period. While the protein levels of cyclin D1, cyclin D3, CDK4, CDK6 and CDK2 were still detectable in adult atria, the protein levels of cyclin E, cyclin A, cyclin B, cdc2 and PCNA were not detectable. Interestingly, p27KIP1 protein increased markedly in the adult period, while p21CIP1 protein in atria was detectable only in the fetal period. While the activities of CDK6, CDK2 and cdc2 decreased markedly, the activity of CDK4 did not change from the fetal period to the adult period. CONCLUSION: These findings indicate that marked reduction of protein levels and activities of cyclins and CDKs, and marked induction of p27KIP1 in atria, are associated with the withdrawal of cardiac cell cycle in adult humans.

Inhibition of papain with 2-benzyl-3,4-epoxybutanoic acid esters. Mechanistic and stereochemical probe for cysteine protease catalysis.

Papain, a prototypic cysteine protease was inactivated by methyl and benzyl esters of (2S,3S)-2-benzyl-3,4-epoxybutanoic acid. On the other hand, methyl ester of (2S,3R)-2-benzyl-3,4-epoxybutanoic acid was shown to be a competitive inhibitor for the enzyme. It was inferred from the inactivation stereochemistry that in the papain catalytic reaction the nucleophilic attack of the side chain thioalkoxide of Cys-25 on the scissile peptide bond of substrates occurs in the 're' fashion. The papain inactivating potency of (2S,3S)-2-benzyl-3,4-epoxybutanoic acid methyl ester was enhanced over three-fold in a pH 8.0 solution compared with in the neutral solution. This together with our previous observation with alpha-chymotrypsin and the recent theoretical treatment of the enzymic reaction of papain, suggest that in the inactivation of papain by oxirane containing inhibitors, the oxirane does not need to be activated by prior protonation as thought previously. The oxirane ring is sufficiently labile that the unprotonated oxirane moiety can undergo an electrophilic reaction with the Cys-25 thiolate.

Transfer of conjugative plasmid pAM beta 1 from Lactococcus lactis to mouse intestinal bacteria.

Conjugal transfer of plasmid pAM beta 1 from Lactococcus lactis to intestinal bacteria of BALB/c mice was studied. Plasmid transfer was observed to Enterococcus faecalis in vitro by a filter mating method with transfer frequencies of 2.3 x 10-3 and with lower frequencies to other species. In vivo, using gastric intubation with the pAM beta 1-bearing Lactococcus lactis as donor and Ent. faecalis as recipient, a few transconjugants were detected from faecal Ent. faecalis. However, when these mice were given erythromycin through drinking water, a large number of conjugated Ent. faecalis were detected in faeces. Plasmid transfer to Ent. faecalis occurred at high frequency, 1.2 x 10-3, in mice whose anus was artificially closed after gastric intubation with pAM beta 1-bearing Lactococcus lactis. These results demonstrate clearly that pAM beta 1 transfer occurs between Gram-positive bacteria in the gut of mice harbouring many species of bacteria.

The incidence of Listeria species in retail foods in Japan.

Meat, fish and vegetable products obtained at retail shops in or around Tokyo were examined for Listeria contamination. Listeria spp. were isolated from 43 (56.6%) out of 76 samples of meat products. L. monocytogenes occurred in 26 (34%) of the samples, L. monocytogenes was isolated from 7 (6.1%) out of 114 samples of fish and fish products including 'ready-to-eat' foods. Listeria was not isolated from any of 21 samples of vegetable and vegetable product including 'ready-to-eat' foods investigated.