Trehalose, sucrose and raffinose are novel activators of autophagy in human keratinocytes through an mTOR-independent pathway.

Trehalose is a natural disaccharide that is found in a diverse range of organisms but not in mammals. Autophagy is a process which mediates the sequestration, lysosomal delivery and degradation of proteins and organelles. Studies have shown that trehalose exerts beneficial effects through inducing autophagy in mammalian cells. However, whether trehalose or other saccharides can activate autophagy in keratinocytes is unknown. Here, we found that trehalose treatment increased the LC3-I to LC3-II conversion, acridine orange-stained vacuoles and GFP-LC3B (LC3B protein tagged with green fluorescent protein) puncta in the HaCaT human keratinocyte cell line, indicating autophagy induction. Trehalose-induced autophagy was also observed in primary keratinocytes and the A431 epidermal cancer cell line. mTOR signalling was not affected by trehalose treatment, suggesting that trehalose induced autophagy through an mTOR-independent pathway. mTOR-independent autophagy induction was also observed in HaCaT and HeLa cells treated with sucrose or raffinose but not in glucose, maltose or sorbitol treated HaCaT cells, indicating that autophagy induction was not a general property of saccharides. Finally, although trehalose treatment had an inhibitory effect on cell proliferation, it had a cytoprotective effect on cells exposed to UVB radiation. Our study provides new insight into the saccharide-mediated regulation of autophagy in keratinocytes.

Transport behavior of hairless mouse skin during constant current DC iontophoresis I: baseline studies.

The fluxes of charged and nonionic molecules across hairless mouse skin (HMS) were induced by direct current iontophoresis and used to characterize the transport pathways of the epidermal membrane. Experimental data were used to determine permeability coefficients from which the effective pore radii (Rp) of the transport pathways were calculated. Permeants used in these experiments were nonionic permeants (urea, mannitol, and raffinose), monovalent cationic permeants (sodium, tetraethylammonium, and tetraphenylphosphonium ions), and monovalent anionic permeants (chloride, salicylate, and taurocholate ions). The Rp estimates obtained by the anionic permeant pairs were 49, 22, and 20 Å for the chloride/salicylate (Cl:SA), chloride/taurocholate (Cl:TC), and salicylate/taurocholate (SA:TC) pairs, respectively; with the cationic permeant pairs, the Rp values obtained were 19, 30, and 24 Å for the sodium/tetraethylammonium (Na:TEA), sodium/tetraphenylphosphonium (Na:TPP), and the tetraethylammonium/tetraphenylphosphonium (TEA:TPP) pairs, respectively. Rp estimates for HMS obtained from nonionic permeant experiments ranged from 6.7 to 13.4 Å. When plotted versus their respective diffusion coefficients, all of the permeability coefficients for the cationic permeants were greater than those of the anionic permeants. Additionally, the magnitudes of permeability coefficients determined in the current study with HMS were of the same order of magnitude as those previously determined in our laboratory using human epidermal membrane under similar iontophoresis conditions.

The recovery rate at the human terminal ileum of an orally administered non-digestive oligosaccharide (raffinose).

We clarified how raffinose, one of the non-digestive oligosaccharides, reaches the large intestine. Seven healthy male volunteers were given a test meal containing 10.0 g raffinose. A double-lumen tube was placed in the terminal ileum, and the ileal contents were aspirated through the tube. The amounts of raffinose were orally administered and collected from the terminal ileum and were compared with each other. The result was that the mean+/-standard error percentage of the amount of ingested raffinose collected in the terminal ileum was 97.1+/-2.4%. Furthermore, the average times taken for 20%, 40%, 60% and 80% of raffinose to reach the terminal ileum were 2.0+/-0.6 h, 2.6+/-0.7 h, 3.6+/-0.7 h and 4.9+/-0.7 h, respectively. In conclusion, approximately 100% of ingested raffinose was recovered in the terminal ileum in the present study. This corresponds with the present generally accepted definition of a dietary fibre.

Systematic studies on the paracellular permeation of model permeants and oligonucleotides in the rat small intestine with chenodeoxycholate as enhancer.

The objective of this study was to mechanistically and quantitatively analyze chenodeoxycholate-enhanced paracellular transport of polar permeants and oligonucleotides in the rat jejunum and ileum. Micellar chenodeoxycholate solutions were used to perturbate the tight junctions. Supporting studies included assessment of the aqueous boundary layer (ABL) with ABL-controlled permeants, measurements of the permeability coefficients and fluxes of the bile acid in dilute and micellar concentrations, and determinations of pore sizes with paracellular probes (urea, mannitol, and raffinose). The paracellular permeability coefficients, P(para), of two model oligonucleotides (ON3 and ON6; 12- and 24-mers with 11 and 23 negative charges, respectively) were determined. The enhanced permeabilities paralleled the increased fluxes of micellar bile salt solutions into mesenteric blood and the opening of the tight junctions as compared to controls. As the pore radius increased from 0.7 nm to a maximum of 2.4 nm in the jejunum and ileum, the absorption of ON3 was enhanced up to sixfold in the jejunum and about 14-fold in the ileum with P(para) values between 0.5 x 10(-6) and 6 x 10(-6) cm/s, whereas ON6 was enhanced up to twofold in the jejunum and fivefold in the ileum with permeabilities between 0.3 x 10(-6) and 2 x 10(-6) cm/s.

Global transcriptional analysis of Streptococcus mutans sugar transporters using microarrays.

The transport of carbohydrates by Streptococcus mutans is accomplished by the phosphoenolpyruvate-phosphotransferase system (PTS) and ATP-binding cassette (ABC) transporters. To undertake a global transcriptional analysis of all S. mutans sugar transporters simultaneously, we used a whole-genome expression microarray. Global transcription profiles of S. mutans UA159 were determined for several monosaccharides (glucose, fructose, galactose, and mannose), disaccharides (sucrose, lactose, maltose, and trehalose), a beta-glucoside (cellobiose), oligosaccharides (raffinose, stachyose, and maltotriose), and a sugar alcohol (mannitol). The results revealed that PTSs were responsible for transport of monosaccharides, disaccharides, beta-glucosides, and sugar alcohol. Six PTSs were transcribed only if a specific sugar was present in the growth medium; thus, they were regulated at the transcriptional level. These included transporters for fructose, lactose, cellobiose, and trehalose and two transporters for mannitol. Three PTSs were repressed under all conditions tested. Interestingly, five PTSs were always highly expressed regardless of the sugar source used, presumably suggesting their availability for immediate uptake of most common dietary sugars (glucose, fructose, maltose, and sucrose). The ABC transporters were found to be specific for oligosaccharides, raffinose, stachyose, and isomaltosaccharides. Compared to the PTSs, the ABC transporters showed higher transcription under several tested conditions, suggesting that they might be transporting multiple substrates.

Ex vivo application of carbon monoxide in University of Wisconsin solution to prevent intestinal cold ischemia/reperfusion injury.

Carbon monoxide (CO), a byproduct of heme catalysis, was shown to have potent cytoprotective and anti-inflammatory effects. In vivo recipient CO inhalation at low concentrations prevented ischemia/reperfusion (I/R) injury associated with small intestinal transplantation (SITx). This study examined whether ex vivo delivery of CO in University of Wisconsin (UW) solution could ameliorate intestinal I/R injury. Orthotopic syngenic SITx was performed in Lewis rats after 6 h cold preservation in control UW or UW that was bubbled with CO gas (0.1-5%) (CO-UW). Recipient survival with intestinal grafts preserved in 5%, but not 0.1%, CO-UW improved to 86.7% (13/15) from 53% (9/17) with control UW. At 3 h after SITx, grafts stored in 5% CO-UW showed improved intestinal barrier function, less mucosal denudation and reduced inflammatory mediator upregulation compared to those in control UW. Preservation in CO-UW associated with reduced vascular resistance (end preservation), increased graft cyclic guanosine monophosphate levels (1 h), and improved graft blood flow (1 h). Protective effects of CO-UW were reversed by ODQ, an inhibitor of soluble guanylyl cyclase. In vitro culture experiment also showed better preservation of vascular endothelial cells with CO-UW. The study suggests that ex vivo CO delivery into UW solution would be a simple and innovative therapeutic strategy to prevent transplant-induced I/R injury.

The intravascular persistence and methemoglobin formation of Hemolink (hemoglobin raffimer) in dogs.

Hemoglobin raffimer (HEMOLINK, Hemosol Inc, Mississauga, Canada) is an o-raffinose cross-linked, purified human hemoglobin-based oxygen therapeutic that is currently being evaluated in late stage clinical trials. It is composed of several molecular weight (MW) species, comprising principally of stabilized tetramers (34-42%) and oligomers (54-62%). The objective of this study was to determine the in vivo circulating half-life (T1/2) of hemoglobin raffimer (Hb raffimer) and of its individual MW components in dogs subjected to a topload infusion of 25% of the estimated blood volume (18 mL/kg). Sampling was done over a 64-hour period that was expected to be equivalent to approximately two-and-half to three half-lives. Methemoglobin (MetHb) levels were also measured at intervals over the same period. The mean circulating half-life of Hb raffimer was 25.4 +/- 3.9 hours. The T1/2 for the individual MW components (determined by size exclusion chromatography) of Hb raffimer was 11 +/- 2 hours for the cross-linked tetramer and 35 +/- 7 hours for the fraction of oligomers. The apparent volume of distribution for Hb raffimer was estimated at 78 mL/kg. There was no difference in the apparent volumes of distribution of the tetrameric and oligomeric components of Hb raffimer. Throughout the course of the experiment (in which MetHb could be measured), plasma MetHb concentration, expressed as a percentage of the total plasma hemoglobin concentration, remained at 10% or less, and the mass concentration of MetHb in plasma remained at about 1 g/L. Thus, in the dog subjected to an estimated 25% topload infusion, the T1/2 of the infused Hb raffimer is approximately one day with the intravascular retention of the individual Hb raffimer components being dependent on the MW. Furthermore, oxidation of Hb raffimer to MetHb is limited under these conditions.

Hepatic uptake of solutes from the preservation solution during hypothermic storage: a (1)H NMR study in rat liver.

The hepatic uptake of histidine and carnosine (histidyl-alanine), used as buffer agents in four preservation solutions, was studied during 24-h hypothermic storage of rat livers by use of (1)H nuclear magnetic resonance (NMR) spectroscopy. Results demonstrated that there was a progressive, concentration-linked passive diffusion of histidine into liver tissues throughout the storage period. A similar inward diffusion of carnosine was also noted. Of the carbohydrate osmotic buffers in the preservation solutions, mannitol permeated the liver tissues to a greater degree and more rapidly than raffinose after the flushing with equivalent concentrations and storage at hypothermia. In general, many solutes from preservation solutions will increasingly penetrate the hepatic inter- and intracellular spaces during extended hypothermic preservation and (1)H NMR spectroscopy is one technique that can assist in the identification of these changes.

Experimental and computational screening models for the prediction of intestinal drug absorption.

The aim of this study was to devise experimental protocols and computational models for the prediction of intestinal drug permeability. Both the required experimental and computational effort and the accuracy and quality of the resulting predictions were considered. In vitro intestinal Caco-2 cell monolayer permeabilities were determined both in a highly accurate experimental setting (Pc) and in a faster, but less accurate, mode (Papp). Computational models were built using four different principles for generation of molecular descriptors (atom counts, molecular mechanics calculations, fragmental, and quantum mechanics approaches) and were evaluated for their ability to predict intestinal membrane permeability. A theoretical deconvolution of the polar molecular surface area (PSA) was also performed to facilitate the interpretation of this composite descriptor and allow the calculation of PSA in a simplified and fast mode. The results indicate that it is possible to predict intestinal drug permeability from rather simple models with little or no loss of accuracy. A new, fast computational model, based on partitioned molecular surface areas, that predicts intestinal drug permeability with an accuracy comparable to that of time-consuming quantum mechanics calculations is presented.

A phase I study of oxidized raffinose cross-linked human hemoglobin.

OBJECTIVE: To evaluate the safety of oxidized-raffinose cross-linked human hemoglobin, Hemolink, in normal healthy volunteers. DESIGN: Randomized, placebo-controlled, double-blind study. SETTING: Clinical research facility of a contract research organization. PATIENTS: Forty-two healthy adult male volunteers of which 33 received Hemolink. INTERVENTIONS: Oxidized-raffinose cross-linked and polymerized hemoglobin as a 10% (w/v) solution, in doses of 0.025-0.6 g/kg or an equivalent volume of lactated Ringer's solution, was infused intravenously on day 1, and subjects were monitored for 3 days in the clinical facility with < or =6 wks follow-up. Major organ function was assessed pre- and postinfusion, by hemodynamic, electrocardiographic, pulmonary function, and clinical chemistry measurements. MEASUREMENTS AND MAIN RESULTS: Doses of 1.7-42 g of hemoglobin were administered with no serious adverse events noted. Abdominal pain of moderate to severe intensity was seen in some subjects at doses >0.4 g/kg and was alleviated with smooth muscle relaxants. There was a dose-dependent increase in mean arterial pressure with a plateau of approximately 14% above baseline at 0.1 g/kg. There was a concomitant reduction in heart rate, with no electrocardiographic abnormalities found. Respiratory function was not affected. There was a dose-dependent increase in serum bilirubin with values above the upper limit of normal at doses of > or =0.4 g/kg. Small increases in aspartate aminotransferase and alanine aminotransferase were noted in some patients, whereas alkaline phosphatase and gamma-glutamyltransferase remained in the normal range. Serum amylase concentrations were normal in 31 of 33 patients receiving Hemolink, whereas lipase was within the normal range in 21 of 33 patients. LDH was increased in a dose-dependent fashion. Two patients had increased creatine kinase concentrations, with a normal creatine kinase-MB mass fraction. All hematologic variables were within the normal range. The half-life of the oligomeric (>64 kDa) fraction of Hemolink was 18-20 hrs. CONCLUSION: Oxidized-raffinose cross-linked hemoglobin, Hemolink, at doses < or =0.6 g/kg were well tolerated in healthy volunteers with no evidence of organ dysfunction. Further investigation of its potential use in surgical and trauma settings appears warranted.

Effect of temperature on hepatic and renal uptake of components from University of Wisconsin solution.

In rat liver and kidney preservation, hepatic and renal uptake of 3H-adenosine, 3H-glutathione, and 3H-raffinose from University of Wisconsin solution and diffusion to the interstitial space were measured. At 4 degrees C only 0.38+/-0.47% and 2+/-0.92% of the total 3H-adenosine remained in the kidney and in the liver, respectively, but at 37 degrees C the amount remaining was 1+/-1% and 12+/-3% (P<0.001). Hepatic and renal uptake of the impermeant 3H-raffinose was unaffected by temperature. During flush out, interstitial accumulation of adenosine was significantly higher in livers than in kidneys and decreased during 24-h cold storage. Glutathione accumulation in the interstitial space was two orders of magnitude lower than 3H-adenosine accumulation and comparable to the impermeant raffinose. In summary, the bioavailability of components of preservation solutions at 4 degrees C is lower than at physiological temperatures, so that the application of cytoprotectants at 37 degrees C to organ donors, rather than simple addition to the cold storage solution, might improve cold storage preservation of livers and kidneys.

Hydraulic and diffusional permeabilities of isolated outer medullary descending vasa recta from the rat.

Water permeates many microvessel walls via a pathway shared with small hydrophilic solutes and also via an exclusive water pathway. In outer medullary descending vasa recta (OMDVR), the relationship between diffusional permeabilities to water and sodium indicates the existence of an exclusive water pathway and suggests that of a shared pathway. We investigated the latter possibility by estimating hydraulic permeability (Lp) and diffusional permeability to [3H]raffinose (P(raf)) in isolated, perfused OMDVR. The product of hydraulic permeability and osmotic reflexion coefficient of albumin (Lp sigma a) was 1.56 +/- 0.19 x 10(-6) cm.s-1.mmHg-1 (n = 28), calculated from transmural volume fluxes induced by perfusate-to-bath differences in albumin oncotic pressure (delta IIa). P(raf) in the same vessels was 40.1 +/- 7.5 x 10(-5) cm/s when delta IIa was zero. In separate experiments, sigma a was at least 0.89 +/- 0.10 (n = 17). Lp sigma a correlates with P(raf), indicating that OMDVR contain a shared pathway for convection driven by delta IIa and for diffusion of small hydrophilic solutes.

Determination of the magnitude of the water-exclusive pathway in cat skeletal muscle microvasculature.

OBJECTIVE: To measure the magnitude of the water-exclusive pathway in cat skeletal muscle. METHODS: The osmotic reflection coefficient (sigma d) was measured for sucrose, raffinose and cyancobalamine using osmotic transient techniques in the isolated, perfused cat hindlimb preparation at sufficiently high perfusate flows (60-80 ml/min-1 100 g-1) so that solute diffusion was not a factor. Microvascular filtration coefficient values required for the sigma d determination were measured using the capillary filtration coefficient (CFC) technique at these high flows. With these sigma d data and macromolecular reflection coefficient data from a previous study, discrete pore-modeling techniques were used to estimate the magnitude of the water movement through the water-exclusive pathway. RESULTS: CFC values increased significantly at very high flows ( > 80 ml/ min-1 100 g-1), but these values were unchanged from control at the lower flows used to measure sigma d. The sigma d values for sucrose and raffinose were 0.41 +/- 0.03 SE and 0.42 +/- 0.03 SE, respectively, in 12 limbs. In the same limbs, the sigma d for cyancobalamine was 0.52 +/- 0.03 SE, which was significantly (p < 0.05) larger, consistent with a larger Stokes-Einstein radius for this molecule. A 3-pathway model (small and large pores and a water-exclusive pathway) was fit to the data. The result was that 41 +/- 4% (95% confidence interval) of total water flow makes use of the water-exclusive pathway in this preparation. CONCLUSIONS: The very high fraction of water flow through the water-exclusive pathway in cat skeletal muscle suggests that this pathway is of major importance in microvascular water movement under normal conditions. Failure to take this finding into account can lead to inaccuracies in the estimation of parameters for pathways which carry solute.

Diffusive transport of solute in the rat medullary microcirculation.

Outer medullary descending vasa recta (OMDVR) permeability to sodium (PNa) is much lower than to urea (Purea). Based on these findings, we hypothesized that sodium and urea diffuse across the OMDFR wall by separate routes. To further test this, we simultaneously perfused OMDVR with 22Na and 36Cl, [14C]urea, [3H]raffinose, or tritiated water. The permeability of OMDVR to 22Na and [3H]raffinose was found to increase markedly and reversibly with perfusion rate. PNa was highly correlated with the permeability to Cl (PCl) and to [3H]raffinose (Praf) (R = 0.90 and 0.95, respectively) but not with Purea (R = 0.23). Praf was also correlated with inulin permeability (PIn) (R = 0.93). The intercepts for the regressions of PNa with PCl and Praf and for Praf with PIn were zero. In contrast, OMDVR with low PNa retained very high diffusional water permeability (PD) and Purea, a finding consistent with separate routes for permeation of those tracers. We previously established that thiourea is a competitive inhibitor of OMDVR urea transport. In the presence of 100 mM thiourea, OMDVR PNa and Purea were correlated (R = 0.71) but retained an intercept much > 0. We conclude that Na, Cl, raffinose, and inulin are likely to traverse the OMDVR wall through a common pathway, whereas specific mechanisms exist to regulate the permeation by urea and water.

In vitro perfusion of chinchilla thin limb segments: segmentation and osmotic water permeability.

The thin limb segments of the long loop of Henle are thought to play important roles in the urinary concentrating mechanism. In this study, we present new approaches to the identification, dissection, and in vitro perfusion of individual thin limb segments from all levels of the chinchilla renal medulla, including the deepest portions of the papilla. We have applied these techniques to the investigation of the osmotic water permeability along the chinchilla long loop of Henle. The results demonstrate that the osmotic water permeability of the thin descending limb is not uniformly high along its length, as previously thought, but that the distal 20% of the long-loop descending limb has a very low water permeability (approximately 50 microns/s). The transition to the low water permeability region of the thin descending limb is accompanied by a relatively abrupt change in morphology (increased cellularity and decreased diameter) that is readily perceptible in the perfused segments and even in the dissection dish. In contrast, the upper part of the chinchilla long-loop thin descending limb had an extremely high osmotic water permeability (greater than 2,000 microns/s) as observed in other species. Thin ascending limbs from deep in the inner medulla had water permeabilities that were indistinguishable from zero, as previously found in thin ascending limbs from near the inner-outer medullary junction. The presence of a low-water-permeability portion of the long-loop thin descending limb in chinchilla may have important implications with regard to the inner medullary concentrating process. A relatively low osmotic water permeability (397 microns/s) was also found in the deep inner medullary portion of the thin descending limb from the rat.

Effects of adrenergic agonists and antagonists on autophagic activity in isolated rat liver cells.

The effect of various adrenergic agonists on autophagic sequestration--measured as the transfer of electroinjected [3H]raffinose from cytosol to vacuoles of the autophagic pathway--was investigated. Epinephrine and other agonists with alpha-effects inhibited sequestration through a specific alpha 1-adrenergic, i.e. prazosin-sensitive, mechanism. The beta-adrenergic agonist isoproterenol also inhibited sequestration, but by a non-beta-specific (propranolol-insensitive) mechanism. All sequestration-inhibitory agents suppressed overall autophagic-lysosomal proteolysis. The inhibitory action of the adrenergic agonists on protein metabolism was not specific to the autophagic pathway since protein synthesis was suppressed as well. However, intracellular levels of ATP were not adversely affected, ruling out the possibility that the agonists might be generally cytotoxic.

Changes in jejunal permeability and passive permeation of sugars in intestinal biopsies in coeliac disease and Crohn's disease.

1. The relative effects of changes in mucosal surface area and mucosal permeability on the passive uptakes of mannitol and raffinose have been studied in vitro using jejunal biopsies from 48 controls, 32 patients with coeliac disease and 11 patients with Crohn's disease. Total mucosal permeation was corrected for surface area measured morphometrically to provide an index of mucosal permeability. 2. In untreated coeliac disease, permeation of mannitol was reduced by 35% (P = 0.006) and that of raffinose was increased by 66% (P = 0.0095) compared with controls, whereas mucosal permeability to mannitol was increased twofold (P = 0.009) and to raffinose fivefold (P = 0.0001). Mucosal permeability was similar for each sugar. 3. In treated coeliac disease, permeation and permeability for mannitol were normal, but remained elevated for raffinose by 23% (P = 0.036) and 41% (P = 0.024), respectively. 4. In Crohn's disease, permeation of mannitol was reduced by 21%, but that of raffinose and mucosal permeability to both sugars were normal. 5. These findings suggest that surface area is quantitatively more important than mucosal permeability in determining the total permeation of mannitol, while the converse is true for raffinose. The findings are compatible with paracellular uptake of raffinose, but with both paracellular and transcellular uptake of mannitol. Both pathways are affected in coeliac disease, whereas only transcellular uptake is affected in Crohn's disease.