Mannitol

To identify genes of potential importance to cold, salt, and drought tolerance, global expression profiling was performed on Arabidopsis plants subjected to stress treatments of 4°C, 100 mm NaCl, or 200 mm mannitol, respectively. RNA samples were collected separately from leaves and roots after 3-and 27-h stress treatments. Profiling was conducted with a GeneChip microarray with probe sets for approximately 8,100 genes. Combined results from all three stresses identified 2,409 genes with a greater than 2-fold change over control. This suggests that about 30% of the transcriptome is sensitive to regulation by common stress conditions. The majority of changes were stimulus specific. At the 3-h time point, less than 5% (118 genes) of the changes were observed as shared by all three stress responses. By 27 h, the number of shared responses was reduced more than 10-fold (Ͻ 0.5%), consistent with a progression toward more stimulus-specific responses. Roots and leaves displayed very different changes. For example, less than 14% of the cold-specific changes were shared between root and leaves at both 3 and 27 h. The gene with the largest induction under all three stress treatments was At5g52310 (LTI/COR78), with induction levels in roots greater than 250-fold for cold, 40-fold for mannitol, and 57-fold for NaCl. A stress response was observed for 306 (68%) of the known circadian controlled genes, supporting the hypothesis that an important function of the circadian clock is to "anticipate" predictable stresses such as cold nights. Although these results identify hundreds of potentially important transcriptome changes, the biochemical functions of many stress-regulated genes remain unknown. ; fax 858 -784 -2862.

The role played by the gut in nonalcoholic fatty liver disease (NAFLD) is still a matter of debate, although animal and human studies suggest that gut-derived endotoxin may be important. We investigated intestinal permeability in patients with NAFLD and evaluated the correlations between this phenomenon and the stage of the disease, the integrity of tight junctions within the small intestine, and prevalence of small intestinal bacterial overgrowth (SIBO). We examined 35 consecutive patients with biopsy-proven NAFLD, 27 with untreated celiac disease (as a model of intestinal hyperpermeability) and 24 healthy volunteers. We assessed the presence of SIBO by glucose breath testing (GBT), intestinal permeability by means of urinary excretion of 51 Cr-ethylene diamine tetraacetate ( 51 Cr-EDTA) test, and the integrity of tight junctions within the gut by immunohistochemical analysis of zona occludens-1 (ZO-1) expression in duodenal biopsy specimens. Patients with NAFLD had significantly increased gut permeability (compared with healthy subjects; P < 0.001) and a higher prevalence of SIBO, although both were lower than in the untreated celiac patients. In patients with NAFLD, both gut permeability and the prevalence of SIBO correlated with the severity of steatosis but not with presence of NASH. Conclusions: Our results provide the first evidence that NAFLD in humans is associated with increased gut permeability and that this abnormality is related to the increased prevalence of SIBO in these patients. The increased permeability appears to be caused by disruption of intercellular tight junctions in the intestine, and it may play an important role in the pathogenesis of hepatic fat deposition.

The hPepT1-mediated transport properties of a series of 11 synthesized -and γ-peptides have been studied in Caco-2 cells. The results show that several of the compounds interact with the peptide transporter, but only two -dipeptides act as substrates and are transported across the cell monolayers. These two are lessefficient substrates than R-peptides. Larger derivatives than -dipeptides do not act as hPepT1 substrates, but instead, they appear to be substrates for P-glycoprotein efflux. ionization; HOBt, 1-hydroxy-1H-benzotriazole hydrate; TFA, trifluoroacetic acid. Three-letter amino-acid abbreviations are used for homologated amino acid derivatives: hXaa ( n , homoamino acid) 1 and γhhXaa (γ m,n , doubly homologated amino acid), where n indicates the position of the proteinogenic side chain.

Although epithelia, which often are in intimate contact with lymphoid cells, may bear receptors for various cytokines, it is unclear whether cytokines directly effect epithelial function. We examine the effects of the cytokine interferon (IFN) on barrier function of cultured monolayers of the Tg4 human intestinal epithelial cell line.-yIFN, in concentrations and expor sures required to show its other biological effects, directly affects such monolayers. Monolayer resistance is substantially diminished by yIFN. Such effects were not due to cytotoxicity as judged morphologically and by LDH assays. Solute fluxes and dual Na'-mannitol flux analysis indicate that the resistance decrease is due to an effect of yIFN on tight junction permeability. The effects of yIFN on monolayer barrier function were not duplicated by the cytokines interleukin 1, interleukin 2, or tumor necrosis factor. We speculate that such products of activation of lymphoid cells might influence barrier function of intestinal, and perhaps other epithelia in disease states.

Drought stress is one of the major limitations to crop productivity. To develop crop plants with enhanced tolerance of drought stress, a basic understanding of physiological, biochemical and gene regulatory networks is essential. Various functional genomics tools have helped to advance our understanding of stress signal perception and transduction, and of the associated molecular regulatory network. These tools have revealed several stress-inducible genes and various transcription factors that regulate the drought-stress-inducible systems. Translational genomics of these candidate genes using model plants provided encouraging results, but the field testing of transgenic crop plants for better performance and yield is still minimal. Better understanding of the specific roles of various metabolites in crop stress tolerance will give rise to a strategy for the metabolic engineering of crop tolerance of drought.

Background: Pyruvate transport is vital for growth and survival of T. brucei bloodstream form. Results: TbPT0 is a unique monocarboxylate transporter with preference for pyruvate and essential for bloodstream form viability. Conclusion: TbPT0 represents the first molecular identification of essential pyruvate transporters in T. brucei. Significance: Unique T. brucei pyruvate transporters provide a potential drug target against the bloodstream form parasites. . 2 The abbreviations used are: BF, bloodstream form; MCT, monocarboxylate transporter; PF, procyclic form; YNB, yeast nitrogen base medium; TM, transmembrane domain; SM, single marker.

This work presents a new dry powder system consisting of microencapsulated protein-loaded chitosan nanoparticles (CS NPs). The developed system was evaluated in vivo in rats in order to investigate its potential to transport insulin (INS), a model protein, to the deep lung, where it is absorbed into systemic circulation. The INS-loaded CS NPs were prepared by ionotropic gelation and characterized for morphology, size, zeta potential, association efficiency and loading capacity. Afterwards, the NPs were co-spray dried with mannitol resulting in a dry powder with adequate aerodynamic properties for deposition in deep lungs. The assessment of the plasmatic glucose levels following intratracheal administration to rats revealed that the microencapsulated INS-loaded CS NPs induced a more pronounced and prolonged hypoglycemic effect compared to the controls. Accordingly, the developed system constitutes a promising alternative to systemically deliver therapeutic macromolecules to the lungs, but it can also be used to provide a local effect.

Purpose: It is generally accepted that blood-brain barrier (BBB) failure occurs as a result of CNS diseases, including epilepsy. However, evidences also suggest that BBB failure may be an etiological factor contributing to the development of seizures.

Toxin A of Clostridium difficile causes severe inflammatory enterocolitis in man and animals that appears to be mediated in part by acute inflammatory cells that migrate into the toxin A-exposed mucosa. To determine the direct effects of toxin A on intestinal epithelial permeability and structure in the absence of other modulating factors, we used cultured monolayers of a human intestinal epithelial cell line (T84). A toxin A concentration of 7 X 10-1 ,ug/ml (3 X i0-' M) nearly abolished monolayer transepithelial resistance within 6-8 h. This marked permeability defect occurred while the monolayers were still confluent. Dual sodium-mannitol flux studies localized the permeability defect to the intercellular tight junction. Cytotoxicity assays and morphological evaluation using Nomarski optics and electron microscopy failed to demonstrate any evidence of cell damage at the time the maximum resistance response was observed. Fluorescent staining for F actin, however, revealed a marked decrease in fluorescent intensity in toxin-treated monolayers versus controls. These data show that toxin A can directly affect the barrier function of this model intestinal epithelium and initially does so by selectively enhancing tight junction permeability. Furthermore, cytoskeletal structure is markedly altered over the same time course, although the integrity of individual cells is maintained. Because the cytoskeleton of intestinal epithelial cells is known to be capable of regulating tight junction permeability, we speculate that the above effects of toxin A on epithelial barrier function result from alterations of the cytoskeleton.

Background: Pfs25 and Pvs25, surface proteins of mosquito stage of the malaria parasites P. falciparum and P. vivax, respectively, are leading candidates for vaccines preventing malaria transmission by mosquitoes. This single blinded, dose escalating, controlled Phase 1 study assessed the safety and immunogenicity of recombinant Pfs25 and Pvs25 formulated with Montanide ISA 51, a water-in-oil emulsion.

Purpose. The objective of this study was a systematic characterization and evaluation of cell culture models based on mixtures of Caco-2/HT29-MTX co-cultures for their use in screening for drug absorption and intestinal permeability in comparison to the properties of the respective mono-cultures. Methods. Co-cultures of Caco-2 cells (absorptive-type) and HT29-MTX cells (goblet-type) were set up. Three different cocultures (initial seeding ratios Caco-2/HT29-MTX: 90/10, 70/30, and 50/50) were grown on permeable filter supports, and monolayers were used for permeability studies with model compounds for paracellular absorption (atenolol, furosemide, H334/75, mannitol, terbutaline), transcellular absorption (antipyrine, ketoprofen, metoprolol, piroxicam), carrier-mediated absorption (D-glucose, Gly-Pro, and L-phenylalanine) as well as substrates for carrier-mediated secretion via P-glycoprotein (cimetidine and talinolol). Electrophysiological and microscopic controls were performed to characterize the cell cultures. Results. For compounds undergoing passive intestinal absorption permeabilities were generally higher in co-cultures than in Caco-2 monolayers, yielding highest values in pure HT29-MTX monolayers. This difference was most obvious for compounds transported via the paracellular pathway, where HT29-MTX cells may be up to 30 times more permeable than Caco-2 cells, whereas for lipophilic and highly permeable compounds, the difference in permeability values was less obvious. For drugs undergoing intestinal secretion mediated by P-glycoprotein, co-cultivation of Caco-2 cells with HT29-MTX cells led to increased apical to basolateral permeability which was decreased in the opposite direction, consistent with the fact that HT29-MTX cells do not express P-glycoprotein. When a carrier-mediated absorption mechanism is involved, the permeabilities observed were lower than the values reported for human small intestine and co-cultivation of HT29-MTX cells with Caco-2 cells resulted in even lower values as compared to the plain Caco-2 cultures. Conclusions. Co-cultures of HT29-MTX and Caco-2 cells offer the opportunity of modifying the permeability barrier of the cell monolayers both with respect to paracellular resistance and secretory transport via P-gp. Thus, in special cases, they allow more flexibility in adapting the in vitro system to the in vivo situation as compared to the monocultures. Another advantage is the obvious robustness of the method with respect to the reproducibility of the results. A problem remaining, however, is the quantitative expression of carriers involved in intestinal uptake of many nutrients and drugs.

Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant ( sre ) and salobreño ( sañ ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1 , sre1-2 , sañ3-1 , and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD-or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K m value for xanthoxin of 19 M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin → abscisic aldehyde → ABA transition as the last steps of the major ABA biosynthetic pathway.

Toxin A but not toxin B, appears to mediate intestinal damage in animal models of Clostridium difficile enteritis. The purpose of this study was to investigate the electrophysiologic and morphologic effects of purified C. difficile toxins A and B on human colonic mucosa in Ussing chambers. Luminal exposure of tissues to 16-65 nM of toxin A and 0.2-29 nM of toxin B for 5 h caused dose-dependent epithelial damage. Potential difference, short-circuit current and resistance decreased by 76, 58, and 46%, respectively, with 32 nM of toxin A and by 76, 55, and 47%, respectively, with 3 nM of toxin B, when compared with baseline (P < 0.05).

To evaluate the clinical benefit of increasing the osmotic load of the hypertonic solution administered for the treatment of refractory intracranial hypertension episodes in patients with severe head injury. Prospective, randomized study. A trauma center in a university hospital. Twenty consecutive patients with head trauma and persistent coma who required infusions of an osmotic agent to treat episodes of intracranial hypertension resistant to well-conducted standard modes of therapy were studied. Intracranial hypertension was considered refractory when it persisted despite deep sedation, optimal hemodynamic status, and, in some patients, drainage of cerebral spinal fluid. Patients were randomly assigned to receive isovolume infusions of either 7.5% hypertonic saline solution (2400 mOsm/kg/H(2)O) or 20% mannitol (1160 mOsm/kg/H(2)O). The patients were given 2 mL/kg (body weight) of either solution, i.e., 361 +/- 13 mOsm of saline or 175 +/- 12 mOsm of mannitol per injection. The ma...

A novel triple co-culture model of the human airway barrier was designed to simulate the cellular part of the air-blood barrier of the respiratory tract represented by macrophages, epithelial cells, and dendritic cells. When epithelial cells (A549 cells) were grown on filter inserts with pores of 3.0 m in diameter in a two-chamber system, they formed monolayers with polarization into apical and basolateral domains. The epithelial cell cultures were then supplemented with human blood monocyte-derived macrophages and dendritic cells on the apical and basal aspect, respectively. The single-cell cultures as well as the triple co-cultures were characterized in terms of a number of typical features, for example, morphology of cell types, integrity of epithelial layer, and expression of specific cell surface markers (CD14 for macrophages and CD86 for dendritic cells). The interplay of epithelial cells with macrophages and dendritic cells during the uptake of polystyrene particles (1 m in diameter) was investigated with confocal laser scanning and conventional transmission electron microscopy. Particles were found in all three cell types, although dendritic cells were not directly exposed to the particles. More investigations are needed to understand the translocation pathway

There is controversy over whether children in developing countries can catch up on their growth rates after bouts of diarrhoea. A factor influencing catch-up growth is the extent and duration of mucosal injury. To explore the relation between intestinal disease and growth performance, a non-invasive test of intestinal integrity, the lactulose:mannitol permeability test, was done regularly on children aged 2-15 months, whose growth was monitored over a mean of 7.5 months. The study revealed persistent abnormalities in the small bowel mucosa of 2-15 month old Gambian infants and a negative correlation between these abnormalities and growth. Up to 43% of observed growth faltering can be explained on the basis of these long-term intestinal lesions.

Background: The combination of bicarbonate and mannitol (BIC/MAN) is commonly used to prevent renal failure (RF) in patients with rhabdomyolysis despite the absence of sufficient evidence validating its use. The purpose of this study was to determine whether BIC/ MAN is effective in preventing RF in patients with rhabdomyolysis caused by trauma.

Electrical circuit analysis was used to study the structural development of occluding junctions (OJs) in cultured monolayers composed to T84 cells. The magnitude of the increments in transepithelial resistance predicted by such analysis was compared with the magnitude of the measured increments in resistance. Confluent sheets of epithelial cells were formed after cells were plated at high density on collagen-coated filters. Using Claude&#39;s OJ strand count-resistance hypothesis (1978, J. Membr. Biol. 39:219-232), electrical circuit analysis of histograms describing OJ strand count distribution at different time points after plating predicted that junctional resistance should rise in a proportion of 1:21:50 from 18 h to 2 d to 5 d. This reasonably paralleled the degree of rise in transepithelial resistance over this period, which was 1:29:59. The ability to predict the observed resistance rise was eliminated if only mean strand counts were analyzed or if electrical circuit analysis...

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8 ؉ T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4 ؉ Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8 ؉ T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8 ؉ T cells, indicated that elicitation of NY-ESO-1-specific CD8 ؉ T cell responses by crosspriming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines.

Recently, drug nanosuspensions have shown a potential for ophthalmic delivery. In this study, a hydrocortisone (HC) nanosuspension (NS) was developed using microfluidic nanoprecipitation as a recent, simple and cost-effective bottom-up technique of drug nanonization. For comparison, a second HC NS was prepared by top-down wet milling procedures. The produced nanosuspensions were characterized for particle size, shape and zeta potential. HC nanosuspensions of approximately 300 nm particle size were produced by adjusting experimental conditions of the two processing techniques. Results of X-ray diffraction and differential scanning calorimetry revealed that HC maintained the crystalline structure upon milling, while predominant amorphous particles were generated after precipitation. Ocular bioavailability of HC nanosuspensions was assessed in albino rabbits using HC solution as a control. A sustained drug action was maintained up to 9 h for the nanosuspensions compared to 5 h for the drug solution. The precipitated and milled NS achieved comparable AUC0–9h values of 28.06 ± 4.08 and 30.95 ± 2.2, respectively, that were significantly (P < 0.05) higher than that of HC solution (15.86 ± 2.7). After 2 months storage at room temperature, the milled HC NS showed good stability with no discernable changes in particle size, whereas the particle size of the precipitated HC NS increased to 440 nm.Changes in intraocular pressure (IOP) of Albino rabbits after administration of Hc solution and nanosuspensions produced by milling and precipitation.

We describe a model to study the effects of polymorphonuclear leukocyte (PMN) transmigration on the intestinal epithelial barrier. Human PMN were induced to transmigrate across high resistance monolayers of a cultured human intestinal epithelial cell line (T84 cells) by chemotactic gradients produced by formyl methionyl leucyl phenylalanine (FMLP). With maximal transmigration monolayer resistance decreased by 48 +/-12.6% in 15 min and by 83 +/-1.6% in 60 min. This response was dependent on the size of the FMLP gradient and the density of PMN transmigration. The decrease in resistance correlated with number of PMN migrating across monolayers, and was accompanied by increases in flux of paracellular tracers. Macromolecular tracer studies localized the leak sites to foci at which PMN impaled the epithelium. Removal of the chemotactic gradient led to restoration of baseline resistance within 18 h. PMN transmigration across intestinal epithelial monolayers occurs via intercellular occluding junctions and may be associated with a reversible increase in epithelial permeability.

Calcium plays a pivotal role in plant responses to several stimuli, including pathogens, abiotic stresses, and hormones. However, the molecular mechanisms underlying calcium functions are poorly understood. It is hypothesized that calcium serves as second messenger and, in many cases, requires intracellular protein sensors to transduce the signal further downstream in the pathways. The calcineurin B-like proteins (CBLs) represent a unique family of calcium sensors in plant cells. Here, we report our analysis of the CBL9 member of this gene family. Expression of CBL9 was inducible by multiple stress signals and abscisic acid (ABA) in young seedlings. When CBL9 gene function was disrupted in Arabidopsis thaliana plants, the responses to ABA were drastically altered. The mutant plants became hypersensitive to ABA in the early developmental stages, including seed germination and post-germination seedling growth. In addition, seed germination in the mutant also showed increased sensitivity to inhibition by osmotic stress conditions produced by high concentrations of salt and mannitol. Further analyses indicated that increased stress sensitivity in the mutant may be a result of both ABA hypersensitivity and increased accumulation of ABA under the stress conditions. The cbl9 mutant plants showed enhanced expression of genes involved in ABA signaling, such as ABA-INSENSITIVE 4 and 5. This study has identified a calcium sensor as a common element in the ABA signaling and stress-induced ABA biosynthesis pathways.

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis," was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).

Increased gut permeability (leaky gut) and endotoxin-mediated Kupffer cell activation are proposed as the mechanisms of alcoholic liver injury. Although ethanol feeding is shown to sensitize the liver for injury induced by parental administration of lipopolysaccharide (LPS), how enteral LPS loading affects alcoholic liver injury is yet to be tested. The present study provides direct evidence for enhanced entrance to portal circulation of LPS enterally administered to the intragastric ethanol infusion model. Portal and systemic blood endotoxin levels increased to 43.0 ؎ 4.1 and 6.2 ؎ 4.3 pg/mL at 2 hours following enteral LPS administration (5 mg/kg) in alcohol-fed animals, while no such increases were observed in pair-fed controls. However, endotoxin levels in systemic blood of alcohol-fed rats were reduced to 0 to 1.5 pg/mL 16 hours after LPS administration. Weekly enteral administration of LPS to the model for 9 weeks exacerbated an increase in plasma alanine transaminase (ALT) levels (227 ؎ 75 vs. 140 ؎ 70; P < .01), mononuclear infiltration (25 ؎ 22 vs. 6.4 ؎ 4.4/10 mm 2 ; P ‫؍‬ .02), sinusoidal congestion, and spotty necrosis, and induced diffuse coagulative necrosis and centrilobular fibrosis in some animals. Reverse-transcription polymerase chain reaction (RT-PCR) analysis confirmed the LPS effect at the tissue level by demonstrating accentuated induction of tumor necrosis factor ␣ (TNF-␣) and Cox-2 mRNA. In conclusion, enteral LPS administration potentiates alcoholic liver necrosis, inflammation, and fibrosis despite efficient endotoxin clearance by the liver and mild systemic endotoxemia that occurs episodically following enteral LPS challenge.

Post-infectious irritable bowel syndrome is a common clinical phenomenon of uncertain aetiology. To test the association between intestinal permeability and irritable bowel syndrome symptoms 2 years after a large waterborne outbreak of bacterial gastroenteritis. Consecutive adults with Rome I irritable bowel syndrome and controls without irritable bowel syndrome attending a community clinic were enrolled. Intestinal permeability was measured as the ratio of fractional urinary excretions of lactulose and mannitol, and compared among cases vs. controls and predictors of abnormal intestinal permeability were assessed. A total of 218 subjects (132 irritable bowel syndrome cases and 86 non-irritable bowel syndrome controls) completed the study protocol. About 27 (12%) had been diagnosed with the irritable bowel syndrome before the outbreak and 115 (53%) had been ill during the outbreak. Lactulose-mannitol ratios were increased among cases vs. controls (Mann-Whitney mean rank 118.8 vs. 95.3, P = 0.007), and cases were more likely to have a ratio &amp;gt;0.020 (P = 0.007). Among cases, those with increased intestinal permeability were more likely to report increased stool frequency. Both irritable bowel syndrome symptoms and male gender, but not diarrhoeal illness during the outbreak, were significant predictors of abnormal permeability. Irritable bowel syndrome symptoms are associated with a subtle increase in intestinal permeability irrespective of prior gastroenteritis. This may improve understanding of the aetiology of both sporadic and post-infectious irritable bowel syndrome.

Fifty-nine patients were treated in a prospective, randomized comparison of pentobarbital and mannitol for the control of intracranial hypertension resulting from head injury. Patients with elevated intracranial pressure (ICP) after evacuation of intracranial hematomas were randomized to one of two treatment groups; mannitol initially or pentobarbital initially, followed by the second drug as required by further elevation of ICP. Similarly, patients with raised ICP but without hematomas requiring evacuation were randomly assigned to two treatment groups in an identical paradigm. Those with ICP elevation and no hematoma treated with pentobarbital as initial therapy had a 77% mortality compared to a 41% mortality for those with mannitol as initial treatment. Patients with evacuated hematomas had mortalities of 40% and 43% (no significant difference) for pentobarbital and mannitol respectively. In both no-hematoma and hematoma streams pentobarbital was less effective than mannitol for ...

When subjected to stress, plants reprogram their growth by largely unknown mechanisms. To provide insights into this process, the growth of Arabidopsis (Arabidopsis thaliana) leaves that develop under mild osmotic stress was studied. Early during leaf development, cell number and size were reduced by stress, but growth was remarkably adaptable, as division and expansion rates were identical to controls within a few days of leaf initiation. To investigate the molecular basis of the observed adaptability, leaves with only proliferating, exclusively expanding, and mature cells were analyzed by transcriptomics and targeted metabolomics. The stress response measured in growing and mature leaves was largely distinct; several hundred transcripts and multiple metabolites responded exclusively in the proliferating and/or expanding leaves. Only a few genes were differentially expressed across the three stages. Data analysis showed that proliferation and expansion were regulated by common regulatory circuits, involving ethylene and gibberellins but not abscisic acid. The role of ethylene was supported by the analysis of ethylene-insensitive mutants. Exclusively in proliferating cells, stress induced genes of the socalled "mitochondrial dysfunction regulon," comprising alternative oxidase. Up-regulation for eight of these genes was confirmed with promoter:b-glucuronidase reporter lines. Furthermore, mitochondria of stress-treated dividing cells were morphologically distinct from control ones, and growth of plants overexpressing the alternative oxidase gene was more tolerant to osmotic and drought stresses. Taken together, our data underline the value of analyzing stress responses in development and demonstrate the importance of mitochondrial respiration for sustaining cell proliferation under osmotic stress conditions.

The active transport of a variety of nonelectrolytes by animal cells appears to be dependent upon extracellular Na (1). For example, mucosal strips from rabbit ileum will accumulate L-alanine in an unbound, osmotically active form when incubated in a solution containing 140 m u Na. Removal of Na from the incubation medium has two effects on this process: (a) the rate of net alanine uptake is markedly reduced; and (b) the ability of the tissue to achieve an intracellular alanine concentration greater than that in the incubation medium is abolished (2). While these observations certainly implicate Na in the alanine transport process, they shed little light on the mechanism of the Na-amino acid interaction. Thus, inhibition of accumulation could result from a decreased influx into the tissue, an increased efflux out of the tissue, or a combination of both. Further, the preparations employed in studies of intestinal accumulation of nonelectrolytes do not distinguish between solute movements across the mucosal and across the serosal membranes of the cells.

Although approximately 500,000 patients suffer from a stroke each year in the United States, treatment of these patients to date has consisted primarily of prevention, supportive measures, and rehabilitation. The modification of experimental cerebral infarction by new pharmacologic agents, along with encouraging results from the restoration of blood flow to areas of focal ischemia in both laboratory and clinical trials, suggests that a more aggressive approach might be considered in selected patients with acute stroke.

Background: The ideal identification of Staphylococcus aureus clinical isolates requires a battery of tests and this is costly in resource limited settings. In many developing countries, the tube coagulase test is usually confirmatory for S. aureus and is routinely done using either human or sheep plasma. This study evaluated Mannitol salt agar and the deoxyribonuclease (DNase) test for improving the efficiency of the tube coagulase test in resource limited settings. The efficiency of human and sheep plasma with tube coagulase tests was also evaluated. Methods: One hundred and eighty Gram positive, Catalase positive cocci occurring in pairs, short chains or clusters were subjected to growth on Mannitol salt agar, deoxyribonuclease and tube coagulase tests. Of these, isolates that were positive for at least two of the three tests (n = 60) were used to evaluate the performance of the tube coagulase test for identification of S. aureus, using PCR-amplification of the nuc gene as a gold standard.

Coffee consumption is associated with a lower risk of type 2 diabetes. We tested the hypothesis that this is mediated by incretin hormones by measuring the acute effects of decaffeinated coffee and coffee components on GLP-1 and GIP concentrations. A randomized cross-over trial of the effects of 12 g decaffeinated coffee, 1 g chlorogenic acid, 500 mg trigonelline, and placebo on total and intact GLP-1 and GIP concentrations during an oral glucose tolerance test took place in fifteen overweight men. No treatment significantly affected the overall GLP-1 or GIP secretion pattern following an OGTT relative to placebo. Decaffeinated coffee slightly increased total GLP-1 concentration 30 minutes after ingestion (before the OGTT) relative to placebo (2.7 pmol/L, p = 0.03), but this change did not correspond with changes in glucose or insulin secretion. These findings do not support the hypothesis that coffee acutely improves glucose tolerance through effects on the secretion of incretin hormones. Chronic effects of coffee and its major components still need to be investigated.

Bergström, C. A. S., 2003. Computational and Experimental Models for the Prediction of Intestinal Drug Solubility and Absorption. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 295. 66 pp. Uppsala. ISBN 91-554-5747-9.

A B S T R A C T The possibility that neutrophils produce the hydroxyl radical (OH-) was studied by examining the ability of these cells to support the release of ethylene from methional, a reaction in which it has been shown that OH-, but not O2or H202, may serve as the oxidizing agent. When neutrophils were exposed to opsonized zymosan in the presence of 0.35 mM methional, ethylene was released in quantities amounting to 44.6+±3.6 pmol/106 cells/40 min. Ethylene production required the presence of neutrophils, opsonized zymosan, and methional, indicating that it was formed from methional by stimulated but not resting neutrophils. Ethylene was not produced by zymosantreated cells from patients with chronic granulomatous disease, confirming the requirement for respiratory burst activity in this process. Ethylene production was suppressed by benzoic acid, an OHscavenger. Superoxide dismutase (3 ,g/ml) reduced ethylene production to 21% of control levels, but catalase had no significant effect in this system. These findings indicate that stimulated neutrophils produce a highly reactive oxidizing radical, possibly OH-, which releases ethylene from methional, and that the O2generated during the respiratory burst is involved in the production of this reactive species.

Weakly bound cell wall proteins of Arabidopsis thaliana were identified using a proteomic and bioinformatic approach. An efficient protocol of extraction based on vacuum-infiltration of the tissues was developed. Several salts and a chelating agent were compared for their ability to extract cell wall proteins without releasing cytoplasmic contaminants. Of the 93 proteins that were identified, a large proportion (60 %) was released by calcium chloride. From bioinformatics analysis, it may be predicted that most of them (87 out of 93) had a signal peptide, whereas only six originated from the cytoplasm. Among the putative apoplastic proteins, a high proportion (67 out of 87) had a basic pI. Numerous glycoside hydrolases, and proteins with interacting domains were identified, in agreement with the expected role of the extracellular matrix in polysaccharide metabolism and recognition phenomena. Ten proteinases were also found as well as six proteins with unknown functions. Comparison of the cell wall proteome of rosettes with the previously published cell wall proteome of cell suspension cultures showed a high level of cell specificity, especially for the different members of several large multigenic families.

Quantitative trait loci (QTL) mapping was used to identify loci controlling various aspects of seed longevity during storage and germination. Similar locations for QTLs controlling different traits might be an indication for a common genetic control of such traits. For this analysis we used a new recombinant inbred line population derived from a cross between the accessions Landsberg erecta (Ler) and Shakdara (Sha). A set of 114 F9 recombinant inbred lines was genotyped with 65 polymerase chain reaction-based markers and the phenotypic marker erecta. The traits analyzed were dormancy, speed of germination, seed sugar content, seed germination after a controlled deterioration test, hydrogen peroxide (H 2 O 2 ) treatment, and on abscisic acid. Furthermore, the effects of heat stress, salt (NaCl) stress, osmotic (mannitol) stress, and natural aging were analyzed. For all traits one or more QTLs were identified, with some QTLs for different traits colocating. The relevance of colocation for mechanisms underlying the various traits is discussed.

Hyperosmotic stress is known to significantly enhance net uptake of inorganic ions into plant cells. Direct evidence for cell turgor recovery via such a mechanism, however, is still lacking. In the present study, we performed concurrent measurements of net ion fluxes (with the noninvasive microelectrode ion flux estimation technique) and cell turgor changes (with the pressure-probe technique) to provide direct evidence that inorganic ion uptake regulates turgor in osmotically stressed Arabidopsis epidermal root cells. Immediately after onset of hyperosmotic stress (100/100 mm mannitol/sorbitol treatment), the cell turgor dropped from 0.65 to about 0.25 MPa. Turgor recovery started within 2 to 10 min after the treatment and was accompanied by a significant (30-80 nmol m Ϫ2 s Ϫ1 ) increase in uptake of K ϩ , Cl Ϫ , and Na ϩ by root cells. In most cells, almost complete (Ͼ90% of initial values) recovery of the cell turgor was observed within 40 to 50 min after stress onset. In another set of experiments, we combined the voltage-clamp and the microelectrode ion flux estimation techniques to show that this process is, in part, mediated by voltage-gated K ϩ transporters at the cell plasma membrane. The possible physiological significance of these findings is discussed. fax 416 -736 -5698.

In evaluating drugs that enter or are excluded from the brain, novel pharmaceutical strategies are needed. For this reason, we have developed a humanized Dynamic In vitro Blood-Brain Barrier model (hDIV-BBB) based on a novel human brain vascular endothelial cell line (HCMEC/D3), which closely mimics the BBB in vivo. In this system, HCMEC/D3 was grown in the lumen of hollow microporous fibers and exposed to a physiological pulsatile flow. Comparison with well-established humanized DIV-BBB models (based on human brain and non-brain vascular endothelial cells co-cultured with abluminal astrocytes) demonstrated that HCMEC/D3 cells cultured under flow conditions maintain in vitro physiological permeability barrier properties of the BBB in situ even in the absence of abluminal astrocytes. Measurements of glucose metabolism demonstrated that HCMEC/D3 cells retain an aerobic metabolic pathway. Permeability to sucrose and two relevant central nervous system drugs showed that the HCMEC/D3 cells grown under dynamic conditions closely mimic the physiological permeability properties of the BBB in situ (slope = 0.93). Osmotic disruption of the BBB was also successfully achieved. Peak BBB opening in the DIV-BBB lasted from 20 to 30 mins and was completely reversible. Furthermore, the sequence of flow cessation/ reperfusion in the presence of leukocytes led to BBB failure as demonstrated by a biphasic decrease in transendothelial electrical resistance. Additionally, BBB failure was paralleled by the intraluminal release of proinflammatory factors (interleukin-6 and interleukin-1b) and matrix metalloproteinase-9 (MMP-9). Pretreatment with ibuprofen (0.125 mmol/L) prevented BBB failure by decreasing the inflammatory response after flow cessation/reperfusion.