MMP8 increases tongue carcinoma cell-cell adhesion and diminishes migration via cleavage of anti-adhesive FXYD5.

Matrix metalloproteinases (MMPs) modify bioactive factors via selective processing or degradation resulting in tumour-promoting or tumour-suppressive effects, such as those by MMP8 in various cancers. We mapped the substrates of MMP8 to elucidate its previously shown tumour-protective role in oral tongue squamous cell carcinoma (OTSCC). MMP8 overexpressing (+) HSC-3 cells, previously demonstrated to have reduced migration and invasion, showed enhanced cell-cell adhesion. By analysing the secretomes of MMP8 + and control cells with terminal amine isotopic labelling of substrates (TAILS) coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS), we identified 36 potential substrates of MMP8, including FXYD domain-containing ion transport regulator 5 (FXYD5). An anti-adhesive glycoprotein FXYD5 has been previously shown to predict poor survival in OTSCC. Cleavage of FXYD5 by MMP8 was confirmed using recombinant proteins. Furthermore, we detected a loss of FXYD5 levels on cell membrane of MMP8 + cells, which was rescued by inhibition of the proteolytic activity of MMP8. Silencing (si) FXYD5 increased the cell-cell adhesion of control but not that of MMP8 + cells. siFXYD5 diminished the viability and motility of HSC-3 cells independent of MMP8 and similar effects were seen in another tongue cancer cell line, SCC-25. FXYD5 is a novel substrate of MMP8 and reducing FXYD5 levels either with siRNA or cleavage by MMP8 increases cell adhesion leading to reduced motility. FXYD5 being a known prognostic factor in OTSCC, our findings strengthen its potential as a therapeutic target.

Pápulas umbilicadas en los codos en una paciente con una vasculitis sistémica / Umbilicated Papules on the Elbows of a Woman With Systemic Vasculitis

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The Human Odontoblast Cell Layer and Dental Pulp Proteomes and N-Terminomes.

The proteome and N-terminome of the human odontoblast cell layer were identified for the first time by shotgun proteomic and terminal amine isotopic labeling of substrates (TAILS) N-terminomic analyses, respectively, and compared with that of human dental pulp stroma from 26 third molar teeth. After reverse-phase liquid chromatography-tandem mass spectrometry, >170,000 spectra from the shotgun and TAILS analyses were matched by 4 search engines to 4,888 and 12,063 peptides in the odontoblast cell layer and pulp stroma, respectively. Within these peptide groups, 1,543 and 5,841 protein N-termini, as well as 895 and 2,423 unique proteins, were identified with a false discovery rate of ≤1%. Thus, the human dental pulp proteome was expanded by 974 proteins not previously identified among the 4,123 proteins in our 2015 dental pulp study. Further, 222 proteins of the odontoblast cell layer were not found in the pulp stroma, suggesting many of these proteins are synthesized only by odontoblasts. When comparing the proteomes of older and younger donors, differences were more apparent in the odontoblast cell layer than in the dental pulp stroma. In the odontoblast cell layer proteome, we found proteomic evidence for dentin sialophosphoprotein, which is cleaved into dentin sialoprotein and dentin phosphoprotein. By exploring the proteome of the odontoblast cell layer and expanding the known dental pulp proteome, we found distinct proteome differences compared with each other and with dentin. Moreover, between 61% and 66% of proteins also occurred as proteoforms commencing with a neo-N-terminus not annotated in UniProt. Hence, TAILS increased proteome coverage and revealed considerable proteolytic processing, by identifying stable proteoforms in these dynamic dental tissues. All mass spectrometry raw data have been deposited to ProteomeXchange with the identifier , with the accompanying metadata at Mendeley Data ( https://data.mendeley.com/datasets/b57zfh6wmy/1 ).

Melanomas sobre tatuajes: una asociación casual con implicaciones prácticas / Melanomas Arising on Tattoos: A Casual Association with Practical Implications

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Linfoma B difuso de células grandes sistémico con afectación cutánea secundaria / Diffuse Systemic Large B-Cell Lymphoma With Secondary Skin Involvement

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Concentration of antifungal agents within host cell membranes: a new paradigm governing the efficacy of prophylaxis.

Posaconazole prophylaxis has proven highly effective in preventing invasive fungal infections, despite relatively low serum concentrations. However, high tissue levels of this agent have been reported in treated patients. We therefore hypothesized that the intracellular levels of antifungal agents are an important factor in determining the success of fungal prophylaxis. To examine the effect of host cell-associated antifungals on the growth of medically important molds, we exposed cells to antifungal agents and removed the extracellular drug prior to infection. Epithelial cells loaded with posaconazole and its parent molecule itraconazole, but not other antifungals, were able to inhibit fungal growth for at least 48 h and were protected from damage caused by infection. Cell-associated posaconazole levels were 40- to 50-fold higher than extracellular levels, and the drug was predominantly detected in cellular membranes. Fungistatic levels of posaconazole persisted within epithelial cells for up to 48 h. Therefore, the concentration of posaconazole in mammalian host cell membranes mediates its efficacy in prophylactic regimens and likely explains the observed discrepancy between serum antifungal levels and efficacy.

Bile secretory function in the obese Zucker rat: evidence of cholestasis and altered canalicular transport function.

BACKGROUND: Obese Zucker rats (ZR) have been used as an experimental model for non-alcoholic fatty liver disease and are particularly susceptible to various types of liver injury. Bile secretory function has not been assessed in ZR. AIM: To study bile secretion and expression of the main hepatobiliary transporters in ZR. METHODS: Bile flow and biliary secretion of lipids and glutathione were determined in eight and 14 week old obese ZR and their lean controls. Protein mass and mRNA of the Na(+)/taurocholate cotransporting polypeptide (Ntcp), the bile salt export pump (Bsep), and the multidrug resistant associated protein 2 (Mrp2) were assessed by western and northern blot, respectively. The effects of administration of a tumour necrosis factor alpha inactivator (etanercept) and an insulin sensitiser (rosiglitazone) were assessed in obese ZR while leptin was given to non-obese rats to study its effect on Mrp2 expression. RESULTS: ZR exhibited increased body weight and hyperlipidaemia. Only 14 week old obese ZR has fatty liver. Decreased bile flow and biliary lipid and glutathione secretion as well as reduced hepatic transport of both taurocholate and bromosulphthalein were found in obese ZR. Hepatic Mrp2 protein mass was markedly reduced (-70%) in obese rats while Ntcp and Bsep protein levels were similar to lean rats. Downregulation of Mrp2 seems to involve both transcriptional and post-transcriptional mechanisms probably related to insulin and leptin resistance. CONCLUSIONS: Obese ZR exhibit an impaired bile secretory function with significant functional and molecular alterations consistent with mild cholestasis. A defective hepatobiliary transport capacity may be a contributory factor in rendering the obese ZR more susceptible to liver injury.

Differential expression of canalicular membrane Ca2+/Mg(2+)-ecto-ATPase in estrogen-induced and obstructive cholestasis in the rat.

Extracellular adenosine triphosphate (ATP) may regulate hepatocyte and cholangiocyte functions, and under some conditions it may have deleterious effects on bile secretion and cause cholestasis. The canalicular membrane enzyme Ca2+/Mg2+-ecto-ATPase (ecto-ATPase) hydrolyzes ATP/adenosine diphosphate (ATP/ADP) and regulates hepatic extracellular ATP concentration. Changes in liver ecto-ATPase in estrogen-induced cholestasis were examined in male rats receiving 17alpha-ethinylestradiol (E groups) for 1, 3, or 5 days (5 mg/kg/day, sc) and compared with changes in rats subjected to obstructive cholestasis (O groups) for 1, 3, or 8 days. Activity of ecto-ATPase, protein mass in canalicular membranes and bile (estimated by Western blotting), steady state mRNA levels (by Northern blotting), and cellular and acinar distributions of the enzyme (histochemistry and immunocytochemistry) were assessed in these groups. Activity of ecto-ATPase, protein mass in isolated canalicular membranes, and enzyme mRNA levels were significantly increased in E group rats as compared with controls. In contrast, these parameters were markedly decreased in O group rats, and the enzyme protein was undetectable in bile. The ecto-ATPase histochemical reaction was markedly increased in the canalicular membrane of E group rats, extending from acinar zone 2 to zone 1, whereas it decreased in the O group. The ecto-ATPase immunocytochemical reaction was present in the canalicular membrane and pericanalicular vesicles in control and E group hepatocytes, but it decreased in obstructive cholestasis and was localized only to the canalicular membrane. Thus, significant changes in liver ecto-ATPase were apparent in 17alpha-ethinylestradiol-induced cholestasis that were opposite to those observed in obstructive cholestasis. Assuming that the alterations observed in obstructive cholestasis are the result of the cholestatic phenomenon, we conclude that changes in ecto-ATPase in 17alpha-ethinylestradiol-treated rats might be either primary events or part of an adaptive response in 17alpha-ethinylestradiol-induced cholestasis.

Effects of diosgenin, a plant-derived steroid, on bile secretion and hepatocellular cholestasis induced by estrogens in the rat.

Increased biliary secretion of cholesterol and lipid vesicles (unilamellae and multilamellae) induced by diosgenin (D), a plant-derived steroid, has cytoprotective effects in the rat liver subjected to obstructive cholestasis. In this study, our aims were to investigate the following: 1) the effects of D on the bile secretory process and on the cholestasis induced by estradiol-17beta-(beta-D-glucuronide) (E17G) or 17 alpha-ethynylestradiol (E) administration; 2) whether the potentially protective effects of D are related to D-induced increase of biliary cholesterol and lipid lamellae; and 3) whether D has other effects capable of modifying specific bile secretory processes or preventing the cholestatic effects of estrogens. Rats were fed a standard ground chow (control group) or chow containing D for 6 days. E17G was administered i.v. to control and D-fed rats and bile flow, bile salt output, and alkaline phosphatase excretion were examined. 17alpha-E was administered from days 4 to 6 to rats fed standard chow or chow plus D for 6 days and different functional parameters of the bile secretory process as well as the ultrastructure of hepatocytes and histochemistry of alkaline phosphatase and Mg2+-adenosine triphosphatase (ATPase) were examined. D-treatment markedly increased cholesterol and lamellar structures in bile and attenuated the acute cholestatic effects of E17G. D-feeding prevented the decrease of taurocholate maximum secretory rate and the increase of biliary alkaline phosphatase and Ca2+,Mg2+-EctoATPase (EctoATPase) excretion, as well as the increase of cholesterol/ phospholipids ratio, alkaline phosphatase activity, and EctoATPase content in canalicular plasma membranes induced by E. D-feeding did not prevent E-induced decrease of basal bile flow, bile salt, cholesterol, and phospholipid secretory rates nor the decrease of Na+,K+-ATPase activity and Na+-taurocholate cotransporting polypeptide (Ntcp) content in isolated sinusoidal membranes. Cholestatic alterations of canalicular domain were apparent in E-treated rats. D administration was also associated with changes of ultrastructure and histochemistry of hepatocytes. E-induced alterations in ultrastructure and acinar distribution and intensity of histochemical reaction of both enzymes were partially prevented by D-feeding. We conclude that D administration, in addition to inducing a marked increase of biliary cholesterol and lipid lamellar structures output, was associated to changes in hepatocyte morphology and plasma membrane composition, enzymes activity, and histochemistry. D-feeding attenuated the acute cholestatic effects of E17G. D-induced increase of bile cholesterol and lipid lamellae content was not apparent when D-fed rats received E. Despite this fact, D administration prevented some cholestatic effects of E, probably through different metabolic effects and/or direct membrane effects, not related to increased lipid lamellae excretion.

Adaptive regulation of hepatic bile salt transport: role of bile salt hydrophobicity and microtubule-dependent vesicular pathway.

BACKGROUND/AIMS: The hepatic transport of bile salts can be regulated by changes in bile salt pool size and/or in the flux of bile salts through the liver. Prolonged bile salt pool depletion is associated with down-regulation of maximum taurocholate transport and decreased canalicular membrane specific bile salt binding sites. This study was undertaken to investigate: a) whether adaptive down-regulation of maximum hepatic bile salt transport occurs to the same extent for bile acids of different hydrophobicity; and b) the role of microtubule-dependent vesicular pathway in the adaptive changes of bile salt transport capacity. METHODS: Male rats were subjected to 24-h or 48-h external biliary diversion to induce bile salt pool depletion. Basal bile flow, bile salt secretion and lipid secretion, maximum secretory rate of three bile salts of different hydrophobicity (tauroursodeoxycholate, taurocholate and taurochenodeoxycholate) and changes in the biliary excretion of two markers of the microtubule-dependent vesicular pathway (horseradish peroxidase and polyethyleneglycol molecular weight-900) were measured in control and bile salt-depleted rats. Taurocholate-stimulated horseradish peroxidase biliary excretion was also assessed in order to define whether the restoration of bile salt flux across the hepatocytes increased the excretion of this marker in bile salt-depleted rats. RESULTS: The reduction in the maximum secretory rate of the three bile salts under study observed after prolonged biliary diversion was clearly related to their hydrophobicity, with greater reduction for taurochenodeoxycholate and smaller reduction for tauroursodeoxycholate, compared with taurocholate. The biliary excretion of vesicular transport markers was significantly reduced in bile salt-depleted rats. However, when stimulated by taurocholate, biliary excretion of horseradish peroxidase was similar to controls. CONCLUSIONS: The magnitude of the decrease of the hepatic bile salt maximum transport capacity seen after bile salt pool depletion is directly related to the hydrophobicity of the bile salt infused. A functionally depressed vesicular transport pathway appears to be also a contributing factor to this phenomenon.

Consideracion del riesgo geologico en la evaluacion del impacto ambiental del dique derivador Los Molinos, Jujuy, Argentina

En los estudios de evaluacion del impacto ambiental, uno de los elementos a ser considerados debe ser el riesgo geologico. Se analiza el impacto ambiental del dique derivador Los Molinos sobre el medio fisico, lo que esta determinado por el cambio en el equilibrio del sistema hidrico, considerando para ello la dinamica de la cuenca del rio Grande

Modulation of hepatic content and biliary excretion of P-glycoproteins in hepatocellular and obstructive cholestasis in the rat.

BACKGROUND/AIMS: Release into bile of canalicular membrane enzymes, such as alkaline phosphatase and gamma-glutamyl transpeptidase, is significantly increased in rats subjected to experimental models of hepatocellular or obstructive cholestasis. This effect appears to be related to a greater susceptibility of these membrane intrinsic proteins to the solubilizing effects of secreted bile acids. It is not known whether canalicular membrane transport proteins, such as P-glycoprotein isoforms, involved in ATP-dependent xenobiotic biliary excretion and phospholipid secretion, are excreted into bile and whether this process is modified in cholestasis. The aims of this work have been to investigate in the rat: a) whether P-glycoproteins are normally excreted into bile, b) whether their excretion is modified in two experimental models of cholestasis, i.e., hepatocellular cholestasis induced by ethynylestradiol and obstructive cholestasis, and c) whether observed changes correlate with bile acid and phospholipid secretion and enzyme release into bile and with relative P-glycoprotein content in hepatic tissue and isolated and purified canalicular membranes. METHODS: P-glycoproteins in bile and hepatic tissue were identified and quantitated by Western-blotting and immunohistochemistry using the C219 MAb. Changes in total mdr mRNA were analyzed by Northern-blotting. RESULTS: Like canalicular membrane enzymes, P-glycoproteins are normally excreted into bile. Ethynylestradiol-induced cholestasis was associated with a 4.9-fold increase in P-glycoprotein excretion compared with controls while, in contrast, the excretion of the carrier decreased markedly in obstructive cholestasis to 2% of control values. P-glycoprotein excretion per nmol of secreted bile acids increased 4.4-fold in ethynylestradiol-induced cholestasis but decreased to 2% of control values in obstructive cholestasis. Total mdr mRNA levels in hepatic tissue were markedly increased (3.4-fold) in rats subjected to obstructive cholestasis and moderately increased (1.6-fold) in the ethynylestradiol group, compared with controls. P-glycoprotein content in isolated canalicular membranes was slightly decreased by 15% in ethynylestradiol-induced cholestasis, while it increased 4.7-fold in obstructive cholestasis. Immunohistochemistry of rat livers showed that P-glycoprotein reaction at the canalicular domain of hepatocytes at acinar zone 1 was decreased in ethynylestradiol-treated rats and markedly increased in obstructive cholestasis. CONCLUSIONS: Ethynylestradiol-induced cholestasis is associated with increased P-glycoprotein biliary excretion and decreased hepatic content. In contrast, obstructive cholestasis results in decreased P-glycoprotein biliary excretion and increased hepatic content. These results suggest that biliary P-glycoprotein excretion might be a modulating factor in canalicular membrane P-glycoprotein content. Increased P-glycoprotein release into bile in ethynylestradiol-treated rats is apparently not a consequence of cholestasis, but it might be a primary event and play a pathogenetic role in ethynylestradiol-induced cholestasis.

Enhanced biliary excretion of canalicular membrane enzymes in ethynylestradiol-induced cholestasis. Effects of ursodeoxycholic acid administration.

Cholestasis is associated with a marked increase in the release of canalicular membrane enzymes into bile. This phenomenon has been related to an increased lability of these canalicular membrane integral proteins to the solubilizing effects of secreted bile salts. To further characterize the effects of oral ursodeoxycholic acid (UDCA) administration on ethynylestradiol (EE)-induced cholestasis, the influence of this bile acid on changes in biliary excretion of membrane-bound enzymes was investigated. Bile flow, basal bile salt and biliary lipid secretory rates, the maximum secretory rate of taurocholate (TC SRm), and the biliary excretion of the canalicular membrane-bound ectoenzymes alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGT) were measured in rats after EE and/or UDCA administration. The activities of ALP, GGT and Na+,K(+)-ATPase in purified isolated canalicular and sinusoidal membrane fractions and the ultrastructure of hepatic acinus, including histochemical studies of ALP distribution, were also examined. EE significantly reduced bile flow, bile salt and biliary lipid secretory rates, and TC SRm, and caused dilatation and loss of microvilli at the canalicular pole of hepatocytes. Biliary excretion of ALP increased 2-fold, whereas biliary excretion of GGT was unchanged. The relationship between biliary excretion of ALP or GGT and bile salt secretion (units of enzyme activity secreted per nanomole of bile salt) was greater in EE-treated rats compared with controls (2.1- and 1.5-fold greater for ALP and GGT, respectively), indicating that in EE-induced cholestasis more enzyme was released into bile per nanomole of bile salt. Na+,K(+)-ATPase activity in sinusoidal membrane fraction was reduced significantly, whereas ALP activity increased in both membrane fractions in EE-treated rats. The histochemical distribution of ALP in the acinus showed a strong reaction in acinar zone 3 and at both the canalicular and sinusoidal membranes. Oral administration of UDCA prevented EE-induced bile secretory failure by normalizing bile flow, bile salt and biliary phospholipid secretory rates, and TC SRm. UDCA also prevented the EE-induced changes in the biliary excretion of enzymes. On the contrary, UDCA did not modify either the enzyme activity in isolated membrane fractions or the morphological or ALP histochemical changes associated with EE administration. These data indicate that in EE-induced cholestasis changes occur at the canalicular membrane, enabling this portion of the plasma membrane to be more susceptible to the solubilizing effect of bile salt, and that oral administration of UDCA prevents bile secretory failure and changes in the biliary excretion of ALP and GGT in EE-treated rats.