Bis(triethoxysilyl)ethane (BTESE)-Organosilica Membranes for H2O/DMF Separation in Reverse Osmosis (RO): Evaluation and Correlation of Subnanopores via Nanopermporometry (NPP), Modified Gas Translation (mGT) and RO Performance.

A 40 cm length Bis(triethoxysilyl)ethane (BTESE) membrane having different pore sizes was successfully prepared by changing the number of coating times for gas permeation (GP) and organic solvent reverse osmosis (OSRO) separation study. It was found that BTESE-6 membranes prepared through six-time coating consisted of small-sized pores in the range 0.56 to 0.64 nm estimated using modified Gas Translation (mGT) method and 0.59 to 0.67 nm estimated by nanopermporometry (NPP) method, respectively. These membranes demonstrated a high DMF rejection, RDMF > 95% with total flux, Jv total > 5 kg m-2 h-1 at operating condition feed pressure, Pf: 8 MPa; feed temperature, Tf : 50 °C; and feed flowrate, Qf : 30 mL/min; and they exhibited a high degree selectivity of He/SF6 in the range of ~ 260-3400 at a permeation temperature 200 °C. On the other hand, the larger pore sizes of the BTESE-4 membranes (pore size estimates > 0.76 nm to 1.02 nm) exhibited low DMF rejection and a low degree selectivity of He/SF6 around ~30% and 25, respectively, at the same operating condition as BTESE-6. Both GT and NPP methods can be considered as an indicator of the measurement membrane pore size. From this study, it was found that He and SF6 gases can be some of the potential predictors for water and DMF permeance. Furthermore, by comparing our OSRO membrane with other PV membranes for DMF/H2O separation, our BTESE-6 membranes still exhibited high flux in the range of 3-6 kg m-2 h-1 with a separation factor H2O/DMF in the range of 80-120.

X-ray structure of a novel endolysin encoded by episomal phage phiSM101 of Clostridium perfringens.

Gram-positive bacteria possess a thick cell wall composed of a mesh polymer of peptidoglycans, which provides physical protection. Endolysins encoded by phages infecting bacteria can hydrolyse peptidoglycans in the bacterial cell wall, killing the host bacteria immediately. The endolysin (Psm) encoded by episomal phage phiSM101 of enterotoxigenic Clostridium perfringens type A strain SM101 exhibits potent lytic activity towards most strains of Clostridium perfringens. Psm has an N-terminal catalytic domain highly homologous to N-acetylmuramidases belonging to the glycoside hydrolase 25 family, and C-terminal tandem repeated bacterial Src homology 3 (SH3_3) domains as the cell wall-binding domain. The X-ray structure of full-length Psm and a catalytic domain of Psm in complex with N-acetylglucosamine were determined to elucidate the catalytic reaction and cell wall recognition mechanisms of Psm. The results showed that Psm may have adopted a neighbouring-group mechanism for the catalytic hydrolysing reaction in which the N-acetyl carbonyl group of the substrate was involved in the formation of an oxazolinium ion intermediate. Based on structural comparisons with other endolysins and a modelling study, we proposed that tandem repeated SH3_3 domains of Psm recognized the peptide side-chains of peptidoglycans to assist the catalytic domain hydrolysing the glycan backbone.

Full mini nutritional assessment and prognosis in elderly patients with pulmonary tuberculosis.

OBJECTIVE: Malnutrition is observed frequently in elderly patients with pulmonary tuberculosis (TB). Full Mini Nutritional Assessment (full MNA) is a useful method of measuring nutrition status for elderly person. The objective of this study is to examine the relationship between full MNA and the mortality of elderly patients with pulmonary TB. METHODS: We evaluated 53 elderly patients with pulmonary TB. The nutrition risk assessment was carried out using full MNA. RESULTS: A receiver operating characteristic (ROC) curve was generated for further analysis of the prognostic value of full MNA score. The area under the curve was 0.856 (95% confidence interval [CI], 0.751-0.961). We used the maximum Youden index to obtain optimal cutoff values for full MNA score for prognostic assessment in elderly patients with pulmonary TB. For predicting the risk of mortality, the optimal cutoff value for full MNA score was 13.75. Based on this cutoff value, the Cox proportional hazard model was applied to assess the ability of full MNA score < 14 to predict the prognosis of elderly patients with pulmonary TB. Multivariate analysis identified age (hazard ratio [HR] = 1.114, 95% CI, 1.018-1.219, p = 0.019) and full MNA score < 14 (HR = 9.038, 95% CI, 1.064-76.768, p = 0.044) to be significant independent prognostic factors for survival. CONCLUSION: Severe malnutrition, as defined by full MNA score < 14, was a predictor of high mortality.

The prognostic significance of nutritional status using malnutrition universal screening tool in patients with pulmonary tuberculosis.

BACKGROUND: Malnutrition is frequently observed in patients with pulmonary tuberculosis (TB). The present study aimed to examine the relationship between nutritional status using Malnutrition Universal Screening Tool (MUST) and the mortality of patients with pulmonary TB. METHODS: Fifty-seven patients with pulmonary TB were analyzed. Nutrition assessment was carried out using MUST. The Cox proportional hazard model was applied to assess the ability of MUST to predict prognosis. Receiver operating characteristic curve analysis was used to assess MUST score as a prognostic indicator in pulmonary TB patients. To obtain optimal cut-off values for MUST score for prognostic assessment in TB patients, we used the maximum Youden Index. RESULTS: For predicting the risk of mortality, the optimal cut-off value for MUST score was 3.5. Univariate and multivariate analyses identified age and MUST score ≥ 4 as significant independent prognostic factors for survival. The patients with MUST score ≤ 3 had a median survival of 481 days (95% CI: 453 to 510) and that for the patients with MUST score ≥ 4 was 304 days (95% CI: 214 to 394); the difference was statistically significant (P = 0.001). CONCLUSION: MUST appears to be a reliable tool for nutritional risk assessment of patients with pulmonary TB. In addition, MUST may be a useful prognostic indicator of survival in patients with pulmonary TB.

Methotrexate and gemcitabine combination chemotherapy for the treatment of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumor of serosal surfaces with a poor prognosis. Methotrexate and gemcitabine have exhibited single-agent activity in MPM. We evaluated the feasibility of sequential administration of these agents in the treatment of MPM. A total of 21 patients with MPM received a 30-min infusion of 100 mg/m2 methotrexate and, 30 min later, a 30-min infusion of 800 mg/m2 gemcitabine. Twenty-four hours following the administration of methotrexate, leucovorin rescue therapy was initiated (10 mg/m2 leucovorin administered 4 times at 6-h intervals). These treatments were administered weekly, with 4 weekly administrations constituting a cycle of therapy. A total of 88 cycles were administered to the 21 patients, with each patient receiving 1-10 cycles (median, 4.2 cycles). Eight patients (38.1%) exhibited a partial response, 10 patients (47.6%) had stable disease and 3 patients (14.3%) had progressive disease. The median overall survival was 19.4 months (range, 02-41 months). One-year and 2-year survival rates were 61.9 and 38.1%, respectively. Hematological toxicity was considered acceptable, with grade 3-4 toxicities occurring in 3 (14.3%) patients. Non-hematologic toxicity was generally mild. There was no treatment-related mortality. Our results suggest that methotrexate and gemcitabine combination therapy is feasible and effective in the treatment of MPM. This regimen may offer an alternative to platinum-based chemotherapy and a prospective trial including a larger cohort of patients is recommended to confirm these results.

A single injection of the anabolic bone agent, parathyroid hormone-collagen binding domain (PTH-CBD), results in sustained increases in bone mineral density for up to 12 months in normal female mice.

Parathyroid hormone (PTH) is the most effective osteoporosis treatment, but it is only effective if administered by daily injections. We fused PTH(1-33) to a collagen binding domain (PTH-CBD) to extend its activity, and have shown an anabolic bone effect with monthly dosing. We tested the duration of action of this compound with different routes of administration. Normal young C57BL/6J mice received a single intraperitoneal injection of PTH-CBD (320 µg/kg). PTH-CBD treated mice showed a 22.2 % increase in bone mineral density (BMD) at 6 months and 12.8 % increase at 12 months. When administered by subcutaneous injection, PTH-CBD again caused increases in BMD, 15.2 % at 6 months and 14.3 % at 12 months. Radiolabeled PTH-CBD was concentrated in bone and skin after either route of administration. We further investigated skin effects of PTH-CBD, and histological analysis revealed an apparent increase in anagen VI hair follicles. A single dose of PTH-CBD caused sustained increases in BMD by >10 % for 1 year in normal mice, regardless of the route of administration, thus showing promise as a potential osteoporosis therapy.

Usefulness of the Malnutrition Screening Tool in patients with pulmonary tuberculosis.

OBJECTIVE: To examine the relation between the Malnutrition Screening Tool (MST) and the mortality of patients with pulmonary tuberculosis (TB). METHODS: Fifty-two patients with pulmonary TB were analyzed. Nutritional assessment was carried out using the MST. The MST incorporates three components: presence of weight loss (score 0 or 2), amount of weight lost (score 1-4), and poor food intake or poor appetite (score 0 or 1). A score ≥2 means that the patient is at risk for malnutrition. The Cox proportional hazard model was applied to assess the ability of the MST to predict prognosis. Receiver operating characteristic curve analysis was used to assess the MST score as a prognostic indicator in patients with pulmonary TB. To obtain optimal cutoff values for the MST score for the prognostic assessment in patients with TB, the maximum Youden index was used. RESULTS: For predicting the risk of mortality, the optimal cutoff value for the MST score was 2.5. Univariate and multivariate analyses identified age and a MST score ≥3 as significant independent prognostic factors for survival. The patients with a MST score <3 had a median survival of 453 d and those with a MST score ≥3 had a median survival of 242 d; the difference was statistically significant (P = 0.001). CONCLUSION: The MST appears to be a reliable tool for the nutritional risk assessment of patients with pulmonary TB. This risk assessment tool can play a valuable role in quickly identifying patients at an increased risk of death and providing adequate nutritional support.

Treatment for chemotherapy-induced alopecia in mice using parathyroid hormone agonists and antagonists linked to a collagen binding domain.

Parathyroid hormone (PTH) agonists and antagonists have been shown to improve hair growth after chemotherapy; however, rapid clearance and systemic side-effects complicate their usage. To facilitate delivery and retention to skin, we fused PTH agonists and antagonists to the collagen binding domain (CBD) of Clostridium histolyticum collagenase. in-vitro studies showed that the agonist fusion protein, PTH-CBD, bound collagen and activated the PTH/parathyroid hormone-related peptide receptor in SaOS-2 cells. The antagonist fusion proteins, PTH(7-33)-CBD and PTH([-1]-33)-CBD, also bound collagen and antagonized PTH(1-34) effect in SaOS-2 cells; however, PTH(7-33)-CBD had lower intrinsic activity. Distribution studies confirmed uptake of PTH-CBD to the skin at 1 and 12 hr after subcutaneous injection. We assessed in vivo efficacy of PTH-CBD and PTH(7-33)-CBD in C57BL/6J mice. Animals were depilated to synchronize the hair follicles; treated on Day 7 with agonist, antagonist, or vehicle; treated on Day 9 with cyclophosphamide (150 mg/kg i.p.) or vehicle; and sacrificed on Day 39. Normal mice (no chemo and no treatment) showed rapid regrowth of hair and normal histology. Chemo+Vehicle mice showed reduced hair regrowth and decreased pigmentation; histology revealed reduced number and dystrophic anagen/catagen follicles. Chemo+Antagonist mice were grossly and histologically indistinguishable from Chemo+Vehicle mice. Chemo+Agonist mice showed more rapid regrowth and repigmentation of hair; histologically, there was a normal number of hair follicles, most of which were in the anagen phase. Overall, the agonist PTH-CBD had prominent effects in reducing chemotherapy-induced damage of hair follicles and may show promise as a therapy for chemotherapy-induced alopecia.

Development and characterization of a xylose-inducible gene expression system for Clostridium perfringens.

A xylose-inducible gene expression vector for Clostridium perfringens was developed. Plasmid pXCH contains a chromosomal region from Clostridium difficile (xylR-P(xy)(lB)): xylR, encoding the xylose repressor, xylO, the xyl operator sequence, and P(xylB), the divergent promoter upstream of xylBA encoding xylulo kinase and xylose isomerase. pXCH allows tightly regulated expression of the chloramphenicol acetyltransferase reporter and the α-toxin genes in response to the inducer concentration. Thus, pXCH could constitute a new valuable genetic tool for study of C. perfringens.

Identification and characterization of a putative endolysin encoded by episomal phage phiSM101 of Clostridium perfringens.

Clostridium perfringens produces potent toxins and histolytic enzymes, causing various diseases including life-threatening fulminant diseases in humans and other animals. Aiming at utilizing a phage endolysin as a therapeutic alternative to antibiotics, we surveyed the genome and bacteriophage sequences of C. perfringens. A phiSM101 muramidase gene (psm) revealed by this study can be assumed to encode an N-acetylmuramidase, since the N-terminal catalytic domain deduced from the gene shows high homology of those of N-acetylmuramidases. The psm gene is characteristic in that it is present in phiSM101, an episomal phage of enterotoxigenic C. perfringens type A strain, SM101, and also in that homologous genes are present in the genomes of all five C. perfringens toxin types. The psm gene was cloned and expressed in Escherichia coli as a protein histidine-tagged at the N-terminus (Psm-his). Psm-his was purified to homogeneity by nickel-charged immobilized metal affinity chromatography and anion-exchange chromatography. The purified enzyme lysed cells of all C. perfringens toxin types but not other clostridial species tested, as was shown by a turbidity reduction assay. These results indicate the Psm-his is useful as a cell-wall lytic enzyme and also suggest that it is potentially useful for biocontrol of this organism.

Subjective global assessment in patients with pulmonary tuberculosis.

BACKGROUND: Malnutrition is observed frequently in patients with pulmonary tuberculosis (TB). Subjective global assessment (SGA) is a subjective method of measuring nutrition status. Few studies have investigated the prognostic role of SGA in patients with pulmonary TB. METHODS: The authors evaluated 39 patients with pulmonary TB. The SGA classification technique was performed; patients were classified as well nourished (A), moderately malnourished (B), or severely malnourished (C). RESULTS: The mean patient age was 67.7 ± 19.0 years, and the majority of patients were male (64.1% ). Twelve patients (30.1% ) were categorized as SGA class A, 14 patients (35.9% ) as class B, and 13 patients (33.3% ) as class C. The SGA-A group had a median survival of 438 days (95% confidence interval, 366-509), the median survival of the SGA-B group was 344 days (251-436), and the median survival of the SGA-C group was 118 days (37-198); these survival rates were significantly different (P < .001). CONCLUSION: SGA appears to be a useful tool for nutrition assessment of patients with pulmonary TB. In addition, SGA may be a prognostic indicator of survival in patients with pulmonary TB.

High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens.

Clostripain (CLO) produced by Clostridium histolyticum is an arginine-specific endopeptidase with the potential for applicability to diverse medical and industrial uses. In this study, we developed an expression system allowing high-level production and efficient purification of recombinant CLO (rCLO). Our expression system comprises pCLO, an rCLO expressing vector, and Clostridium perfringens 13Δ6, an in-frame deletion strain as to six genes encoding major virulence factors and secretory proteins. rCLO was purified from the culture supernatant of C. perfringens 13Δ6/pCLO by ammonium sulfate precipitation, hydroxyapatite chromatography, and affinity chromatography on benzamidine-Sepharose. From 200 ml of culture supernatant 4.5 mg of purified rCLO was obtained. N-Terminal amino acid sequencing and molecular mass determination of the purified rCLO and commercially available CLO revealed that the two enzymes have identical subunits, a 38.1-kDa heavy chain and a 15.0-kDa light chain, indicating that rCLO is processed in the same manner as CLO. Analysis of the enzymatic activities toward N-benzoyl-L-arginine p-nitroanilide and acyl-L-lysine p-nitroanilide showed that rCLO and CLO exhibit strict specificity for arginine at the P1 position, and that the specific activity of the former is approximately 2-fold higher than that of the latter. These results indicate that the new method involving a virulence-attenuated C. perfringens strain is useful for preparing large amounts of high-grade rCLO.

Development and application of a method for counterselectable in-frame deletion in Clostridium perfringens.

Many pathogenic clostridial species produce toxins and enzymes. To facilitate genome-wide identification of virulence factors and biotechnological application of their useful products, we have developed a markerless in-frame deletion method for Clostridium perfringens which allows efficient counterselection and multiple-gene disruption. The system comprises a galKT gene disruptant and a suicide galK plasmid into which two fragments of a target gene for in-frame deletion are cloned. The system was shown to be accurate and simple by using it to disrupt the alpha-toxin gene of the organism. It was also used to construct of two different virulence-attenuated strains, ΗΝ1303 and HN1314: the former is a disruptant of the virRS operon, which regulates the expression of virulence factors, and the latter is a disruptant of the six genes encoding the α, θ, and κ toxins; a clostripain-like protease; a 190-kDa secretory protein; and a putative cell wall lytic endopeptidase. Comparison of the two disruptants in terms of growth ability and the background levels of secreted proteins showed that HN1314 is more useful than ΗΝ1303 as a host for the large-scale production of recombinant proteins.

Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture.

Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (Clp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38 kDa) and light (16 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity toward N-benzoyl-l-arginine p-nitroanilide (Bz-l-arginine pNA), as demonstrated by assaying culture supernatants differing in the relative ratio of the two light chains. Although the purified Clp preferentially degraded Bz-dl-arginine pNA rather than Bz-dl-lysine pNA, it degraded the latter more efficiently than did Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. Clp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis.

High-level expression of his-tagged clostridial collagenase in Clostridium perfringens.

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.

[Expression of mesothelin as a potential diagnostic marker in mesothelioma].

Histological discrimination of mesothelioma from adenocarcinoma is often difficult, therefore, many investigators have tried immunohistochemical, ultrastructual, and molecular methods. Economically, immunohistological studies are more excellent, compared with ultrastractual and molecular biological methods. Immunohistologically, many well known markers are divided into two category; adenocarcinoma-related markers, which expressed by adenocarcinoma, and mesothelioma-related makers, which are positive for mesothelioma. CEA, TTF-1, and Ber-Ep4 are well known adenocarcinoma-related markers, mesothelin, TM, HBME-1 and calretinin, have been used as mesothelioma-related markers. Most previous reports associated with discrimination of adenocarcinoma and mesothelioma mentioned that a diagnosis of epithelioid mesothelioma would be excluded by the presence of adenocarcinoma-related antibody. The positive ratio of mesothelioma-related antibodies is lower than that of adenocarcinoma-related antibodies. Only a single mesothelioma-related marker cannot lead to a diagnosis of epithelioid mesothelioma and a correct diagnosis can be made by combination of several makers, which contain both mesothelioma-related markers and adenocarcinoma-related markers. We immunohistologically examined 41 cases of mesothelioma and 16 cases of adenocarcinoma of the lung, and re-evaluated the use of immunohistochemical markers, compared with previous reports. Reactivity for mesothelin was obtained in 19 (73%) of the epithelioid mesotheliomas, but none (0%) of the lung adenocarcinomas. None of the sarcomatoid mesotheliomas exhibited positivity for this marker, nor was any reactivity seen in the spindle cell component of the biphasic mesotheliomas. These findings indicate that, in some instances, mesothelin immunostaining can assist in the diagnosis of mesothelioma.

Construction and characterization of a clostripain-like protease-deficient mutant of Clostridium perfringens as a strain for clostridial gene expression.

The inherent difficulty of expressing clostridial AT-rich genes in a heterologous host has limited their biotechnological application. We previously reported a plasmid for high-level expression of clostridial genes in Clostridium perfringens (Takamizawa et al., Protein Expr Purif 36:70-75, 2004). In this study, we examined the extracellular proteases of C. perfringens strain 13. Zymographic analysis and caseinase assaying of a culture supernatant showed that it contained a protease activated by dithiothreitol and Ca(2+), suggesting that clostripain-like protease (Clp) is the most likely candidate for the major extracellular protease. Disruption of the clp gene by homologous recombination markedly decreased the level of caseinase activity in the culture supernatant. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Clp(-) mutant but not the wild type strain increased the levels of many polypeptides in the culture supernatant after the late exponential growth phase. Such polypeptides included both cytoplasmic and secretory proteins, suggesting proteins secreted or released into the medium were degraded by Clp. To assess the effects of Clp on the productivity and stability of recombinant proteins, 74-kDa NanI sialidase was expressed in the two strains. The mutant strain produced a higher level of NanI activity than the wild type strain. Furthermore, under the conditions where Clp was activated, NanI was degraded easily in the latter culture but not in the former one. These results indicate that the Clp(-) mutant could serve as a useful strain for efficiently expressing and preparing protease-free clostridial proteins.

[A case of primary lung cancer with cervical tuberculous lymphadenitis].

A 66-year-old woman was admitted due to right cervical lymphadenopathy and an abnormal chest radiograph. Acid-fast bacilli smear of fine needle aspiration from a right cervical lymph node was positive. Histopathological examination of the specimen obtained by percutaneous right cervical lymph node biopsy showed necrotizing epithelioid granulomas and no malignant cells. Therefore, right cervical tuberculous lymphadenitis was diagnosed. Partial lung resection of the right S4 was carried out by video-assisted thoracoscopic surgery and primary lung cancer was diagnosed. To our knowledge, there has been no previous report of both primary lung cancer and cervical tuberculous lymphadenitis being present at the time of the first examination. We report this very rare case.

[Optimum preparation of levocarnitine chloride solution in the hospital pharmacy].

Levocarnitine chloride is used for the therapeutic purpose of levocarnitine deficiency. For infants, however, levocarnitine chloride tablets must be crushed to avoid difficulties associated with swallowing, and also to administer an appropriately low dosage. Since the tablet is extremely hygroscopic and sour, it is dissolved in water containing simple syrup after crushing. In this study we investigated the stability of the drug after dissolution to optimize its preparation for clinical use. It was shown to be stable for at least 90 days after preparation, and microbes did not grow in 1-10% (w/v) solutions (pH 2.0-2.5) regardless of the presence or absence of simple syrup. Furthermore, the autoclaved levocarnitine chloride solution was as stable as the non-autoclaved one. In conclusion, the method employed in our hospital for the preparation of levocarnitine chloride for infants is appropriate and is recommended as a standard medicine supply method among different facilities.

Changes in ganglioside content affect the binding of Clostridium perfringens epsilon-toxin to detergent-resistant membranes of Madin-Darby canine kidney cells.

Epsilon-toxin (ET) of Clostridium perfringens, which causes fatal enterotoxemia in ungulates, was previously shown to bind to and form a heptameric pore within the detergent-resistant membranes (DRMs) of MDCK cells. Depletion of cholesterol has also been shown to decrease the cytotoxicity of ET and its heptamerization. In this study, we investigated the effects of changes in sphingolipids, other DRM components of MDCK cells, on the cells' susceptibility to ET. Treatment with fumonisin B1 and PDMP, inhibitors of sphingolipid and glycosphingolipid syntheses, respectively, increased the susceptibility, while D609, a sphingomyelin synthesis inhibitor, had the opposite effect. The exogenous addition of ganglioside G(M1) dramatically decreased the ET binding, heptamerization and cytotoxicity. These effects were shown not to be due to ET binding to G(M1) or to denaturation of ET. We also found that the ET cytotoxicity towards MDCK cells decreased with an increase in culture time. In accordance with the resistance observed for prolonged cultured cells, G(M3), a major ganglioside component, increased and sialidase treatment increased their susceptibility. These results suggest that membrane-anchored sialic acid of G(M3) within DRMs inhibits ET binding, leading to prevention of the heptamerization of ET and cell death. It is also suggested that sialidase produced by this organism aids the targeting of ET to MDCK cells.