Dodecylmaltoside Modulates Bicellular Tight Junction Contacts To Promote Enhanced Permeability.

Intestinal permeation enhancers are a crucial component of many oral formulations, without which many drugs would show an insufficient absorption in the gut. The present study sought to provide a better understanding of the molecular interaction of such absorption enhancers with the intestine, by investigating the effect of the surfactant-like permeation enhancer dodecylmaltoside (DDM) on Caco-2 cells. The extent to which the action of DDM is apportioned between the para- and transcellular routes was addressed by examining the transport of relevant marker compounds ([3H]-mannitol and [3H]-propranolol, respectively). In the case of [3H]-mannitol, a robust permeation enhancement was achieved with 0.5 mM DDM (∼6-fold), whereas little effect was seen on the permeation of [3H]-propranolol. Concomitantly measured TEER values revealed a rapid onset of action of DDM with a swift recovery and complete restitution (>90%) within 4 h after washout. To localize the site(s) of action of DDM at the absorptive surface of Caco-2 cells, sulfo-NHS-SS-biotin, a membrane-impermeable compound, was applied apically. In the presence of 0.5 mM DDM, translocated biotin was found to be accumulated toward bicellular contacts, whereas no biotin permeation was observed in untreated control cells. Western blot analysis of DDM-treated and untreated Caco-2 cells revealed an interaction of DDM with specific tight junction associated proteins, resulting in a reduction of claudin-3 and -4 and also occludin, as well as a depletion of claudin-2 from lipid rafts. Collectively, the results presented provide a more in depth understanding of the molecular mechanism(s) underlying the permeation-enhancing actions of DDM.

Hepatotoxicity of sub-nanosized platinum particles in mice.

Nano-sized materials are widely used in consumer products, medical devices and engineered pharmaceuticals. Advances in nanotechnology have resulted in materials smaller than the nanoscale, but the biologic safety of the sub-nanosized materials has not been fully assessed. In this study, we evaluated the toxic effects of sub-nanosized platinum particles (snPt) in the mouse liver. After intravenous administration of snPt (15 mg/kg body weight) into mice, histological analysis revealed acute hepatic injury, and biochemical analysis showed increased levels of serum markers of liver injury and inflammatory cytokines. In contrast, administration of nano-sized platinum particles did not produce these abnormalities. Furthermore, snPt induced cytotoxicity when directly applied to primary hepatocytes. These data suggest that snPt have the potential to induce hepatotoxicity. These findings provide useful information on the further development of sub-nanosized materials.

Effect of 70-nm silica particles on the toxicity of acetaminophen, tetracycline, trazodone, and 5-aminosalicylic acid in mice.

Exposure to nano-sized particles is increasing because they are used in a wide variety of industrial products, cosmetics, and pharmaceuticals. Some animal studies indicate that such nanomaterials may have some toxicity, but their synergistic actions on the adverse effects of drugs are not well understood. In this study, we investigated whether 70-nm silica particles (nSP70), which are widely used in cosmetics and drug delivery, affect the toxicity of a drug for inflammatory bowel disease (5-aminosalicylic acid), an antibiotic drug (tetracycline), an antidepressant drug (trazodone), and an antipyretic drug (acetaminophen) in mice. Co-administration of nSP70 with trazodone did not increase a biochemical marker of liver injury. In contrast, co-administration increased the hepatotoxicity of the other drugs. Co-administration of nSP70 and tetracycline was lethal. These findings indicate that evaluation of synergistic adverse effects is important for the application of nano-sized materials.

The safety of a mucosal vaccine using the C-terminal fragment of Clostridium perfringens enterotoxin.

The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a claudin-4 binder. Very recently, we found that nasal immunization of mice with C-CPE-fused antigen activated antigen-specific humoral and mucosal immune responses and that the deletion of the claudin-4-binding domain attenuated the immune responses. C-CPE-fusion strategy may be useful for mucosal vaccination. C-CPE is a fragment of enterotoxin, and the safety of C-CPE-fused protein is very important for its future application. In the present study, we investigated whether C-CPE-fused antigen induces immune responses without mucosal injury by using ovalbumin (OVA) as a model antigen. Immunohistochemical analysis showed that claudin-4 was expressed in epithelial cell sheets bordering the nasal cavity. Nasal immunization with C-CPE-fused OVA dose-dependently elevated the OVA-specific serum IgG titer, which was 1000-fold greater than the titer achieved by immunization with OVA or a mixture of OVA and C-CPE at 5 microg of OVA. Nasal immunization with C-CPE-fused OVA (5 microg of OVA) activated Th1 and Th2 responses. Histological analysis showed no mucosal injury in the nasal cavity or nasal passage. C-CPE-fused OVA exhibited mucosal vaccination without mucosal injury. These findings indicate thatclaudin-4-targeting using C-CPE can be a potent strategy for mucosal vaccination.

Functional transition of Pak proto-oncogene during early evolution of metazoans.

Proto-oncogenes encode signaling molecular switches regulating cellular homeostasis in metazoans, and can be converted to oncogenes by gain-of-function mutations. To address the molecular basis for development of the regulatory system of proto-oncogenes during evolution, we screened for ancestral proto-oncogenes from the unicellular choanoflagellate Monosiga ovata by monitoring their transforming activities, and isolated a Pak gene ortholog encoding a serine/threonine kinase as a 'primitive oncogene'. We also cloned Pak orthologs from fungi and the multicellular sponge Ephydatia fluviatilis, and compared their regulatory features with that of M. ovata Pak (MoPak). MoPak is constitutively active and induces cell transformation in mammalian fibroblasts, although the Pak orthologs from multicellular animals are strictly regulated. Analyses of Pak mutants revealed that structural alteration of the auto-inhibitory domain (AID) of MoPak confers higher constitutive kinase activity, as well as greater binding ability to Rho family GTPases than the multicellular Paks, and this structural alteration is responsible for cell transformation and disruption of multicellular tissue organization. These results show that maturation of AID function was required for the development of the strict regulatory system of the Pak proto-oncogene, and suggest a potential link between the establishment of the regulatory system of proto-oncogenes and metazoan evolution.

Suppression of tumorigenicity, but not anchorage independence, of human cancer cells by new candidate tumor suppressor gene CapG.

Previously, we isolated a series of cell lines from a human diploid fibroblast lineage as a model for multistep tumorigenesis in humans. After passaging a single LT-transfected fibroblast clone, differently progressed cell lines were obtained, including immortalized, anchorage-independent and tumorigenic cell lines. In the present paper, we analysed the gene expression profiles of these model cell lines, and observed that expression of the CapG protein was lost in the tumorigenic cell line. To examine the possibility that loss of CapG protein expression was required for tumorigenic progression, we transfected CapG cDNA into the tumorigenic cell line and tested for tumor-forming ability in nude mice. Results showed that ectopic expression of CapG suppressed tumorigenicity, but not growth in soft agar or liquid medium. We also found that certain cancer cell lines including stomach cancer, lung cancer and melanoma had also lost CapG expression. One such cancer cell line AZ521 also became non-tumorigenic after the introduction of CapG cDNA. Moreover, we showed that CapG expression was repressed in small-cell lung cancer tissues. Together, our findings indicated that CapG is a new tumor suppressor gene involved in the tumorigenic progression of certain cancers.

Multi-functional gene ASY/Nogo/RTN-X/RTN4: apoptosis, tumor suppression, and inhibition of neuronal regeneration.

ASY, also designated Nogo, RTN-X, or RTN4, is a reticulon family protein containing two transmembrane domains and a C-terminal double lysine endoplasmic reticulum (ER) retrieval motif. Three protein variants are synthesized from the cognate mRNAs produced by alternative splicing, and are expressed in almost all tissues, localizing predominantly in the ER. The ASY protein induces apoptosis in various cancer cells when overexpressed, whereas normal cells are relatively resistant to ASY-dependent apoptosis. Furthermore, transcription of this gene is suppressed in certain types of cancers, suggesting that ASY may act to suppress tumor development. Although the physiological function of this protein has not been well defined, Nogo-A protein, the large variant of ASY, has been shown to inhibit neuronal regeneration in the central nervous system. Therefore, the products of this gene may be multi-functional, regulating apoptosis, tumor development, and neuronal regeneration.

[The effects of sleep position change in sleep apnea syndrome].

Changing the sleep position from supine to non-supine is a simple but effective treatment for some patients with sleep apnea syndrome. In the present study, we compared the clinical data for good responders (GRs), those who responded well to the sleep position change, with those for poor responders (PRs), and also evaluated the effect of surgical treatment on the positional improvement of ventilation in the PR group. Forty-one adult patients with sleep apnea syndrome (mean age: 47.1 years, mean obesity index: 125.8%) were divided into two groups based on their polysomnographic responses to sleep position change. Thirty-two patients were classified as the GR group, whose apnea-hypopnea index (AHI) in the non-supine position declined to less than half of their AHI in the supine position, and nine whose non-supine AHI remained greater than half of their supine AHI were classified as the PR group. The AHI and the desaturation index (DI) for the PR group were significantly higher than those for the GR group, and the obesity index was also higher in the PR group. For the eight PRs who had surgical therapy, their polysomnographic data improved much more in the non-supine position than in the supine position. Of six patients whose total AHI was still 10 or more after surgery, four changed into GRs. The combination of surgery and sleep position change seemed to be an effective treatment even for patients with a little improvement before surgery.

Comparison of portal pressure with intravascular esophageal variceal pressure (IEVP) directly measured with a flexible indwelling needle.

Intravascular esophageal variceal pressure (IEVP) was measured using a flexible indwelling needle and compared with wedged hepatic venous pressure (WHVP) in 38 patients with liver cirrhosis. There was a high correlation between IEVP and WHVP; the former was lower than the latter, indicating the presence of a pressure gradient. Bleeders with a history of esophageal variceal rupture showed higher IEVP value than non-bleeders without a history of rupture, even though no difference in WHVP was observed. IEVP in the post-endoscopic injection sclerotherapy (EIS) group, even in those with endoscopic findings of large varices, was lower than that in the untreated groups. These results suggest that IEVP is a factor in the hemodynamics of patients with portal hypertension and that its measurement may be helpful in elucidating the pathophysiology of esophageal varices.

[Propranolol for prophylaxis of first hemorrhage in cirrhotic patients with esophageal varices--a controlled study comparing with sclerotherapy].

To compare the efficacy of oral propranolol and sclerotherapy in the prevention of first hemorrhage from esophageal varices, 65 cirrhotic patients with moderate to large esophageal varices and no history of bleeding were included in the prospective controlled trial. After randomization, 33 patients received propranolol at a does reducing the heart rate by 25%; 32 patients were treated with intra-variceal and extra-variceal injection of ethanolamine oleate. On entry to the trial, the two groups were comparable in terms of clinical and biological parameters. The patients were observed for up to 60 months, with an average of 31 months. Nine patients bled (5 in propranolol and 4 in sclerotherapy) during follow-up. No significant difference were observed between propranolol and sclerotherapy in the cumulative bleeding rate and survival. The multivariate Cox model indicated that drug compliance in the propranolol group and high portal pressure in the sclerotherapy group were factors predictive of the first hemorrhage. These data support that propranolol and sclerotherapy are of comparable value in preventing the first hemorrhage in cirrhotic patients with esophageal varices.

[Prospective controlled study of elective sclerotherapy plus oral propranolol for prevention of recurrent bleeding in cirrhotics with recent variceal hemorrhage].

In a prospective, randomized controlled trial, 43 patients with cirrhosis and variceal hemorrhage were allocated after control of the bleeding to treat by elective sclerotherapy alone (n = 23) or by oral propranolol after elective sclerotherapy (n = 20). The dose of oral propranolol was based on a reduction of the resting pulse rate by 25%. The end points of the study were rebleeding or death. Both treatment groups were comparable with respect to origin and severity of liver disease, size of esophageal varices and portal pressure at entry. The mean follow up was 27 +/- 19 months for all patients. Patients treated sclerotherapy alone had more rebleeding (n = 11) did than those in the sclerotherapy plus propranolol (n = 3). The cumulative percentages of patients free of rebleeding 1 and 2 years after inclusion were 77 and 66% in sclerotherapy alone, and 100 and 85% in sclerotherapy plus propranolol; the difference between the two groups was significant. No statistically significant effect on mortality was seen. These data support that oral propranolol after sclerotherapy reduces the risk of recurrent bleeding in cirrhotics treated elective sclerotherapy.